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WO2018138139A1 - Method for assessing cell surface receptors of blood cells - Google Patents

Method for assessing cell surface receptors of blood cells Download PDF

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Publication number
WO2018138139A1
WO2018138139A1 PCT/EP2018/051713 EP2018051713W WO2018138139A1 WO 2018138139 A1 WO2018138139 A1 WO 2018138139A1 EP 2018051713 W EP2018051713 W EP 2018051713W WO 2018138139 A1 WO2018138139 A1 WO 2018138139A1
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WO
WIPO (PCT)
Prior art keywords
cells
cell surface
sample
filter
marker
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Application number
PCT/EP2018/051713
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French (fr)
Inventor
Erling Sundrehagen
Original Assignee
Gentian As
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Publication of WO2018138139A1 publication Critical patent/WO2018138139A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/045—Connecting closures to device or container whereby the whole cover is slidable
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/049—Valves integrated in closure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0644—Valves, specific forms thereof with moving parts rotary valves

Definitions

  • the present invention relates to a novel method for rapid assessment of one or more subclasses of blood cells of interest (BCol), as for example CD4+ cells and CD8+ cells, in a liquid whole blood sample or a sample derived therefrom; a method of determining the cell count for such cells; a method for determining the CD4/CD8 ratio; a method for determining the quantity of such receptors in a sample; as well as a vertical flow assay device for performing such assessment.
  • BCol blood cells of interest
  • Whole blood is a term used for human blood from a standard blood donation or blood sampling.
  • the blood is typically combined with an anticoagulant during the collection process, but is generally otherwise unprocessed.
  • Whole blood comprises the blood plasma, red blood cells (erythrocytes) and white blood cells (leucocytes) and platelets.
  • Heparin, citrate and EDTA are commonly used anticoagulation agents added to hinder coagulation in blood samples for laboratory analytical use.
  • CD4+ T helper cells are white blood cells that are an essential part of the human immune system. They are often referred to as CD4 cells, T-helper cells or T4 cells, and are a subpopulation of lymphocytes.
  • helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells.
  • CD4 cells send the signal and CD8 cells destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following immune suppression prior to a transplant, the body is left vulnerable to a wide range of infections that it otherwise would have been able to fight.
  • the blood cells comprise often cell surface receptors (membrane receptors, often in the form of transmembrane receptors). These molecules are specialized integral membrane proteins that take part in communication between the cell and the outside world. Extracellular signaling molecules (usually hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules) attach to the receptor, triggering changes in the function of the cell. This process is called signal transduction: The binding initi- ates a chemical change on the intracellular side of the membrane. In this way the receptors play a unique and important role in cellular communications and signal trans- duction.
  • extracellular signaling molecules usually hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules
  • CD4 transmembrane receptors
  • the receptors called CD4 are glycoproteins found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells.
  • CD4 receptors were discovered in the late 1970s and were originally known as leu-3 and T4 (after the OKT4 monoclonal antibody that reacted with it) before being named CD4 in 1984.
  • the CD4 protein is encoded by the CD4 gene.
  • CD4 count measures the number of T cells expressing CD4. While CD4 counts are not a direct HIV test - e.g. they do not check the presence of viral DNA, or specific antibodies against HIV - they are used to assess the immune system of a patient.
  • CD4 counts patients often undergo treatments when the CD4 counts reach a level of 350 cells/ ⁇ - in Europe but usually around 500 cells/ ⁇ - in the US; people with less than 200 cells/ ⁇ - are at high risk of contracting AIDS defined illnesses.
  • the newest National Institute of Health guidelines recommend treatment of any HIV-positive individuals, regardless of CD4 count. Medical professionals also refer to CD4 tests to determine efficacy of treatment.
  • T helper cells carry surface and cytoplasmic CD4 receptors.
  • Filion et al. reported that all monocytes are CD4 positive. The number of monocytes in whole blood is generally high. A method to determine the number of CD4 receptors associated with T helper cells therefore needs to encompass a step or a part of the method sorting away monocytes also carrying CD4 receptors. Other blood cells carrying CD4 (like macrophages) are contained in low, and in this context neglectable proportions in the blood. Flow cytometry is a powerful tool for identifying and enumerating cells.
  • the flow cytom- eter detects and counts individual cells passing in a stream through a laser beam. By examining large numbers of cells, flow cytometry can give quantitative data on the percentage of cells bearing different molecules, such as surface immunoglobulin, which characterizes B cells, the T-cell receptor-associated molecules known as CD3, and the CD4 and CD8 co-receptor proteins that distinguish the major T-cell subsets.
  • surface immunoglobulin which characterizes B cells
  • CD3 the T-cell receptor-associated molecules known as CD3
  • CD4 and CD8 co-receptor proteins that distinguish the major T-cell subsets.
  • Individual cells within a mixed population are tagged with specific antibodies labelled with fluorescent dyes, or for example, by specific antibodies followed by labelled antiimmunoglobulin antibodies.
  • the suspended mixture of labelled cells is then forced through an aperture, creating a fine stream of liquid containing cells spaced singly at intervals.
  • Sensitive photomulti- plier tubes detect both the scattered light, which gives information on the size and granularity of the cell, and the fluorescence emissions, which give information on the binding of the labelled antibodies and hence on the expression of cell-surface proteins by each cell.
  • the data may be displayed in the form of a two-dimensional scatter diagram or as a contour diagram, where the fluorescence of one dye-labelled antibody is plotted against that of a second, with the result that a population of cells labelling with one antibody can be further subdivided on the basis of its reactivity with the second antibody.
  • CD4 counts are measured in laboratories using said flow cytometry technology. Expensive and sophisticated equipment is needed, as well as highly trained personnel, a clean water supply and cold chain storage for reagents is generally required, necessitating the test to be carried out in centralized locations. Delays between testing and obtaining results can also lead to a significant 'loss to follow up' of patients and often they do not return to receive life-saving treatment.
  • a puncture depth of 1.8 mm with a blade-type lancet was used to achieve sufficient capillary blood flow.
  • the PIMA cartridge collected the blood in a 25 ⁇ _ receptacle. Of this initial volume, 5 ⁇ _ of blood was drawn into the PIMA cartridge and further used for cytometric analysis. The cartridge was capped and inserted immediately into the PIMA analyzer to run the test. During the analysis process, the blood was automatically mixed with freeze-dried fluo- rescently labeled antibodies (anti-CD3 and anti-CD4) contained in the cartridge and transferred to a detection chamber where images were taken of the labeled cells to calculate the number of CD4 cells per ⁇ _ of blood.”
  • the PIMA system was a good progress for near-patient testing, but still the PIMA system is based on a sophisticated instrument comprising a complex cassette which is expensive in production.
  • Immunoassays are another particularly useful form of assay that exploit the specificity, strength and diversity of antibody-antigen reactions to analyze samples and detect specific components therein.
  • a wide range of immunoassay techniques is available, such as those described in "The Immunoassay Handbook” Nature Publishing Group, 2001 .
  • a wide range of methods for the detection of antibodies to specific antigens is also known.
  • the enzyme-linked immunosorbent assay (ELISA) or the radio-immunoassay (RIA) is routinely used in laboratories.
  • Arrays and high-throughput screening methods are also employed. These methods generally require a high level of skill in laboratory techniques.
  • dynabeads coated with anti-CD4 antibodies are used to bind CD4+ T-lymphocytes.
  • Monocytes, that express CD14 and CD4 are ex- eluded from fresh blood samples sample using beads coated with anti-CD14 antibodies.
  • T regulatory-1 cells induce lgG4 production by B cells: role of IL-10
  • Satoguina JS Weyand E, Larbi J, Hoerauf A, in J Immunol (2005) 174:4718-4726.
  • the isolated CD4 T-lymphocytes are lysed, stained with acridine orange and stained nuclei are enumerated by fluorescence microscopy.
  • a "TRAx CD4" test kit is described in Paxton et al., Clin. Diagn. Lab. Immunol., 2(1 ):104-1 14, 1995. This kit is an ELISA based method to measure total CD4 in whole blood samples. The antibodies used did not distinguish between cell-bound and soluble CD4 (see Lyamuya et al., J. Imm Methods, 195:103-1 12, 1996).
  • WO 2006/1 15866 describes an immunochromatographic device for measuring CD4 antigens. However, again there is no disclosure in this document of a capture reagent capable of distinguishing between cell-bound and soluble CD4 lacking a cytoplasmic domain in sample from a subject. Further, the device described in WO 2006/1 15866 depends upon the flow of sample over a series of numbered capture areas to capture CD4 by saturating consecutive capture areas on a test strip to subsequently provide a visual indication of the concentration of CD4 cells in the sample.
  • the method is used for evaluating in a blood sample from a subject the level of T-cell associated CD4 comprising a cytoplasmic (cytosolic) and an extracellular (ecto) domain or the level of CD4 T-cells, the method comprising:
  • Said method comprises: a) applying the test sample to a sample portion of an immunochromatographic device wherein the sample portion is operably connected to a capture portion of the device and wherein components of the test sample flow from the sam- pie portion to and through the capture potion which comprises an antibody or antigen-binding fragment thereof that binds to the cytoplasmic domain of CD4 such that only CD4 comprising a cytoplasmic domain and not soluble CD4 that does not comprise a cytoplasmic domain binds to the antibody or frag- ment thereof to form a captured CD4;
  • the device which is a disposable, near-patient test device for the determination of CD4, comprising both the part of the CD4 receptor exposed on the surface and the intracellular part of the CD4 receptor of the cells. In this way, co-measurement of soluble parts of the CD4 receptor present in the blood plasma and not bound to the white blood cells is avoided. Furthermore, a magnetic separation of monocytes is an integral part of the test device. The test is easy-to-use and only requires a finger-prick blood sample to perform the test. It is a test device that is well suited for testing where sophisticated laboratory equipment is not available.
  • lateral flow technology the flow of reagents and sample is parallel to the surface of the device, typically a filtration device, with reagents - often in dry form - is connected to or placed or immobilized within the filter.
  • Numerous such test devices have been made, both for qualitative, semi-quantitative and quantitative measurement of high number of analytes.
  • Anal Bioanal Chem (2009) 393:569-582, Geertruida A. Posthuma-Trumpie & Jakob Korf & Aart van Amerongen provide a review entitled "Lat- eral flow immunoassay: its strengths, weaknesses, opportunities and threats.”
  • an alternative technology to lateral flow technology can be a vertical flow technology.
  • CRP C-reactive protein
  • the vertical flow assay is characterized by a sample volume of 5 ⁇ _, an assay time of 2 minutes, sample material of whole blood, serum or plasma, measuring range: 8 - 200 mg/L for whole blood samples and 5 - 120 mg/L for serum and plasma samples.
  • the present invention very surprisingly made it possible to apply for the assessment of specific blood cells an even more simplified vertical flow principle, and, specifically, without any use of immobilized specific binder in the filter.
  • the present invention em- ploys the use of colorimetric, or luminencene, i.p. fluorometrie or chemiluminescence, measurement of the color or luminescent, i.p.fluorimetric or chemiluminescent signals, developed on the surface of a filter, which is well known in vertical flow immunoassays, however antibody immobilization in the filter is not necessary.
  • Said color or luminescence may either originate from a colored or luminenscent binding reagent (as for ex- ample comrising an antibody) or from a coloured or luminescent substrate of an enzyme marker attached to a per se non-colored or non-luminescent binding reagent.
  • the present invention relates in one embodiment to a method for the assessment of the amount of receptor molecules of a specific class of receptors bound to a "specific class" or "specific group” of cells (also designated as blood cells of interest (BCol)) in a sample of whole blood or a sample derived from whole blood.
  • said class of receptor molecules is the class of CD4 receptors, and typically, the class (also designated herein as subclass, subset or subpopulation) of cells carrying said receptor molecules are T-lymphocytes.
  • the assay method of the present invention in deviation from the teaching of PCT/EP2016/067639, is characterized by mixing in a "first step" the said sample or an aliquot of the said sample with a "first liquid” comprising antibodies against receptor molecules of said specific class of receptors bound to a "specific class” or “specific group” of cells (also designated as blood cells of interest (BCol)) and also comprising antibodies binding to other structures on the surface of "other cells” different from said above-mentioned specific group (or class) of cells but carrying said receptors (said "other cells” are also designated as disturbing blood cells (DBC)), forming particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in said specific group of cells (BCol).
  • the said antibodies directed against said "other cells” in said "first liquid” have a specific affinity for CD14, which is very abundant on monocytes (and monocytes also carry CD4 receptors, and thus would otherwise disturb the assay).
  • the method of the present invention is further characterized by said "first liquid” carrying antibodies towards said “other cells” forming clusters of said “other cells” or the antibodies are polymerized or immobilized on particles or polymers or other large molecules facilitating the formation of particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in "said specific group" of cells.
  • the antibodies towards said “other cells” of said “first liquid” have a specific affinity for receptor CD14, thus enabling the formation of particles or aggregates or clusters of particles or cells comprising the monocytes of the said sample with a size significantly larger than the size of the cells in said "specific group” of cells.
  • the said "first liquid” is selected to rapidly lyse the erythrocytes of the sample, and for this purpose may have a low ionic strength, and a high enough volume to maintain the said low ionic strength after being mixed with the sample to be analyzed. Hypotonic lysis of erythrocytes without lysis of leucocytes is described by Cunha et al in Anal.
  • Lysis of the erythrocytes is very common in vertical flow immunoassays of whole blood samples.
  • Other suitable solvent systems allowing lysis of erythrocytes without lysis of leucocytes are commercially available, as for example the whole blood lysis buffer ACK by Thermo Fisher Scientific Inc or the FACS Lysing Solution from BD.
  • the "second step” of the method of the present invention is a filtration step where the said particles or clusters or aggregates, with significantly larger size than the "specific group" of cells (i.e. BCol) to be analyzed which are either present in free form or are already reacted with said antibodies against receptor molecules on the surface of said specific group of cells, are filtered away with a first filter letting the "specific group” of cells (in free or bound form) to be analyzed through the filter.
  • BCol the "specific group” of cells
  • cells comprising CD14 receptors including the monocytes of the sample, having formed clusters by reacting with antibodies with specific reactivity towards CD14 receptors - optionally with antibodies being conjugated to polymers or immobilized on particles - are in this way filtered away using a filter letting T cells (and T cells carrying CD4 receptors) (either in free form or already reacted with anti-CD4 antibody) through the filter.
  • the said "first filter” must have a pore size which enables the passage of the "specific group" of cells to be analyzed.
  • the said "specific group" of cells is constituted by T-lymphocytes (either in free form or already reacted with anti-receptor antibody), hence - in the said embodiment - the said "first filter” must have a pore-size enabling said T-lymphocyte species to pass through the filter.
  • Filter materials having a low unspecific binding of cells and having a rather narrow distribution of pore size is preferred.
  • a nylon web filter is a very good option, since it's pore-size is well defined, and it's unspecific binding is rather low.
  • T-lymphocytes will easily pass through a 30 ⁇ filter, however aggregates of monocytes, especially when aggregated on rather large particles, e.g. 15 ⁇ to 50 ⁇ in diameter, particles carrying anti-CD14 receptor antibodies (and optionally also anti-CD4 receptor antibodies), will be withheld by such filters.
  • the said "first filter” may also consist of glass, glass fibre, poly- propylene, polyethylene, fluoropolymer, cellulose, nitrocellulose, polyamide and blends thereof.
  • a blocking treatment against unspecific binding of proteins and cells is preferred.
  • T-lymphocytes either in free form or already reacted with anti- receptor antibody
  • a pore size of the filter of 10 to 50 ⁇ is suitable, however a preferred pore size is 12 to 40 ⁇ , and even more preferred is a pore size of 15- to 30 ⁇ .
  • the method of the present invention comprises passing the remaining mixture through a "second filter” retaining the said "specific group” of cells in said sample (either in free form or already reacted with anti-receptor antibody), however letting receptor molecules (either in free form or already reacted with anti-receptor antibody) in solution pass through the filter.
  • the pore size of this "second filter” is, therefore, smaller than the pore size of the first filter.
  • the said “second filter” may consist of glass, glass fibre, poly- propylene, polyethylene, fluoropolymer, cellulose, nylon, nitrocellulose, polyamide and blends thereof.
  • a blocking treatment against unspecific binding of proteins and cells is preferred.
  • the said "specific group” of cells is T-lymphocytes
  • a suitable pore size of said membrane for capturing the T-cells are membranes with an average pore size 1 -15 ⁇ , preferentially 2-10 ⁇ , and even more preferred 3-8 ⁇ , allowing smaller particulate materials from the sample materials after the hypotonic solution to pass through the membrane.
  • the said "specific group” of cells is constituted by other cells, other pore sizes are preferred.
  • the said "second filter” may then optionally be washed by a washing buffer or a wash- ing solution.
  • the said "specific group" of cells is constituted by the lymphocytes, including the T-lymphocytes, often called the CD4+ T-cells.
  • the BCol retained on said "second filter" are assessed.
  • said label may be selected from an enzyme or colored or fluorescent particle.
  • Figure 1 shows a vertical flow assay device which comprises an upper cover sheet
  • FIG. 1 shows a perspective view on another variant of a vertical flow assay device embodiment of the invention comprising an upper rotatable casing element (1 ) and a lower casing element (2) and a sample feed opening (3) and a reading opening (4).
  • Figure 3 shows the device of Figure 2 insertable into a corresponding opening
  • Figure 4 shows a cross section of the assay device according to the Figure 2.
  • Figure 5 shows a top view of another embodiment of the assay device of the invention a sample provided with two pairs of feed openings and reading openings (3, 4 and 3', 4').
  • Figure 6 shows the results for monocyte removal in blood incubated in ACK lysing buffer with 2,5x10 5 anti CD14-pluribeads/ml and 0, 10, 20, or 30 g/ml ALP-anti-CD4.
  • Figure 7 shows the results for monocyte removal in blood incubated in ACK lysing buffer with 7,5x10 5 anti CD14-pluribeads/ml and 0, 10, 20, or 30 g/ml
  • Figure 8 shows the results for monocyte removal in blood incubated in ACK lysing buffer with 7,5x10 5 anti CD14-pluribeads/ml and 0 or 10 ⁇ / ⁇ ALP-anti- CD4 filtered using different mesh size of nylon net filter.
  • Figure 9 shows a modification of a vertical flow assay device according to Figure 2.
  • the assay device comprises an upper casing element (31 ) and a lower casing element (32).
  • the upper element (31 ) has a sample feed opening (33), a washing opening (34), and a reading opening (35).
  • the lower casing element (32) comprises a wedge (40), consisting of a flat piece of essentially non- transparent material and acting as an optical shield of filter which is rotat- ably mounted on the inner side of the lower casing element (32).
  • Figure 10 shows a perspective exploded view of the device of Figure 9 from the bottom. shows a cross-section of the assay device of Figure 9 along an axis indi cated by the letter A (see also Section A indicated in Figure 9) immediate ly before assembly of upper (31 ) and lower (329 casing element:
  • Figure 12 shows a) the chemiluminescence image of five nitrocellulose membranes with increasing concentration of CD4 cells, b) An average intensity of the chemiluminescence signal for the five concentrations is shown.
  • a blot im- aging system (Azure Biosystems c600) has been used.
  • Figure 13 shows a) the fluorescence image of five nitrocellulose membranes with increasing concentration of CD4 cells, b) An average intensity of the fluorescence signal for the five concentrations is shown.
  • a blot imaging sys- tern (Azure Biosystems c600) has been used.
  • a “whole blood” sample as used in the assay method according to the invention is a sample derived from a mammal, in particular a human being. Any “whole blood sample” may be used. Said samples may be used "as is”, i.e. without any pre-treatment, directly as taken from the blood donor, or may be pre-treated prior to the assay.
  • whole blood in this context means a non-modified sample of whole blood or a sample where an anticoagulant has been added to the sample or a sample derived from whole blood, e.g. by adding a buffer or another liquid.
  • suitable samples are native, untreated whole blood and pre-treated whole-blood, blood, like EDTA blood, citrate blood, heparin blood.
  • the originally obtained samples may be further modified by dilution. Fractionation of whole blood to remove constituents which might disturb the assay is normally not required. Dilution may be performed by mixing the original sample with a suitable sample liquid, like a suitable buffer, in order to adjust the concentration of the constituents, as for example of the analyte.
  • the sample may also be pre-treated by hemolysis, as for example selective hemolysis of erythrocytes.
  • modified samples exemplify samples "derived from" the original whole blood sample collected or isolated from the body of the mammal.
  • An “analyte” to be assayed according to the invention is a cell marker, in particular a cell surface marker, more particularly CD4 or CD8.
  • CD4 cluster of differentiation 4
  • CD4+ T-helper cells are white blood cells that are an essential part of the human immune system. They are often referred to as CD4 cells, T-helper cells or T4 cells.
  • helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells, which then destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following im- mune suppression prior to a transplant, the body is left vulnerable to a wide range of infections that it would otherwise have been able to fight.
  • CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co- receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocom- patibility complex (MHC) molecule, but is specific for the class I MHC protein. There are two isoforms of the protein, alpha and beta, each encoded by a different gene. The CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells.
  • CD14 (cluster of differentiation 14), also known as CD14, is a human gene. The protein encoded by this gene is a component of the innate immune system.
  • CD14 exists in two forms, one anchored to the membrane by a glycosylphosphatidylinositol tail (mCD14), the other a soluble form (sCD14). Soluble CD14 either appears after shedding of mCD14 (48 kDa) or is directly secreted from intracellular vesicles (56 kDa). CD14 is expressed mainly by macrophages and (at 10-times lesser extent) by neutrophils. It is also expressed by dendritic cells and monocytes.
  • a "Blood cell of Interest" (BCol) as referred to herein belongs to a class or population or, more particular, to a sub-class or sub-population of cells typically present in a whole blood sample to be assessed according to the invention.
  • Such (sub)- classes or (sub)- populations are distinguishable from each other in the test environment (whole blood sample) on the basis of a particular cell surface marker or a pattern of such markers which may be analyzed by means of corresponding antibody molecules specific for said marker or pattern of markers.
  • a "sub-class", “sub-set” or “sub-population” of cells refers to a group of blood cells which are functionally and antigenically related. Examples thereof are (CD4+) T-Helper cells or (CD8+) cytotoxic T cells.
  • T-lymphocytes T-lymphocytes and B- lymphocytes.
  • Free describes cells (like CD4+ cells) or antigen (like non-cell surface bound forms of a cell surface receptor molecule, like for example CD4 receptor) as contained in the sample to be analyzed which are not or essentially not reacted with a reagent, like an antibody, as applied in an assay method of the invention.
  • “Bound” describes cells (like CD4+ cells) or antigen (like non-cell surface bound forms of a cell surface receptor molecule, like for example CD4) as contained in the sample to be analyzed which are quantitatively or at least essentially quantitatively reacted with a reagent, like an antibody, as applied in an assay method of the invention.
  • Disposinguishable in the context of the present invention means that the particular marker is either “specific” for said particular BCol, i.e. is not detectable in any other body cell; or is “subclass-specific” and therefore not detectable in another cell popula- tion of the blood sample to be analyzed; or is "non-specific” as it is detectable on other blood cells which are present in the whole blood sample as well, however, which are either present in a very low proportion, and does not negatively affect or falsify the assay result, or are removed from the sample before the assessment of the BCol is performed.
  • Specific for" a class, population, sub-class or sub-population of cells in the context of the present invention therefore, has to be understood broadly if not otherwise stated.
  • “Assessing” or “assessment” is intended to include both quantitative and qualitative determination in the sense of obtaining an absolute value for the amount or concentration of the analyte, present in the sample, and also obtaining an index, ratio, percentage, visual or other value indicative of the level of analyte in the sample. Assessment may be direct or indirect and the chemical species actually detected need not of course be the analyte itself but may for example be a derivative thereof.
  • the “accuracy” of an analytical method of the present invention is the method's ability to accurately determine the concentration of the analyte in a sample, compared to the concentration as determined by an even more reliable reference method.
  • the “precision” of an analytical method of the present invention is the variation in the results when the concentration of the analyte in a sample is determined repeatedly.
  • a "robustness" of an assay according to the present invention is the method's ability to tolerate interfering substances and variations in assay conditions without influencing the resulting value of the analyte concentration determination.
  • inert protein as used in the context of the invention is a protein of any origin (for example, human or non-human mammalian, microbial) which does not disturb the assay method of the invention; in particular, it should have substantially no or no detecta- ble affinity for the analyte to be analyzed and/or for the antibodies as used in the assay method of the invention.
  • particle size or “particle diameter” is if not otherwise stated herein defined as “mean particle size” or “mean particle diameter”.
  • the particles of the pre- sent invention in particular the nanoparticles and immunoparticles derived therefrom by coupling of antibodies thereto, a characterized by a narrow, in particular an "essentially monomodal” or “monomodal” particle size distribution.
  • Particle size determination may be performed in a manner known per se, as for example by applying particle size distribution measurements on a Malvern Mastersizer instrument.
  • the meas- urement may be performed in 0.1 M NaOH.
  • mean particle size values stated herein are either D(0.5) or D(4.3) values which may slightly differ but which, nevertheless are in the indicated parameter range, D(0.5) represents the mean particle size in ⁇ at which 50% of the distribution is smaller and 50% of the size distribution is larger. D(4,3) represents the volume mean diameter. Mean particle sizes may also be determined microscopically, as for example by transmission electron microscopy (TEM).
  • TEM transmission electron microscopy
  • Antibody relates to any class of "immunoglobulin molecule" (like IgA, D, E G, M, W, Y) and any isotype, including without limitation lgA1 , lgA2, lgG1 , lgG2, lgG3 and lgG4. Said term refers, in particular, to a functional (i.e. having the ability to bind to an antigen) monoclonal or polyclonal antibody (Ab) or fragment antibody (fAb) capable of binding to a particular antigen.
  • a functional i.e. having the ability to bind to an antigen
  • Ab monoclonal or polyclonal antibody
  • fAb fragment antibody
  • Said Abs and fAbs are selected from chemically or en- zymatically produced molecules or may be produced non-recombinantly or recombi- nantly by prokaryotic or eukaryotic microorganism or cell lines, or may be produced by higher organisms, like mammalian, preferably non-human mammalian species, or non- mammalian species, preferably avian species, or plants.
  • Said fAbs may be selected from the group consisting of: monovalent antibodies (consisting of one heavy and one light chain), Fab, F(ab') 2 (or Fab 2 ), Fab 3 , scFv, bis-scFv, minibody, diabody, triabody, tetrabody, tandab; and single antibody domains, like V H and V L domains, and fragments thereof; wherein polyvalent fragments thereof may bind to different or, preferably, the same antigenic determinant of the same antigen, like in particular CD4 or CD8.
  • Luminescence is herein understood as the emission of light by a substance not resulting from heat and is thus a form of cold-body radiation. It can, in particular, be caused by chemical or biochemical reactions or electrical energy. It encompasses in the context of the present invention "chemiluminescence”, i.e. the emission of light as a result of a chemical reaction; and “photoluminescence” i.e. a result of the absorption of photons. Photoluminescence encompasses "fluorescence”, i.e. a type of photoluminescence that is the result of singlet-singlet electronic relaxation (typical lifetime: nanoseconds); and “phosphorescence”, i.e. a type of photoluminescence that is the result of triplet-singlet electronic relaxation (typical lifetime: milliseconds to hours), (see also definition of luminescence provided by Wikipedia).
  • Luminescent (like fluorescent or chemiluminescent) substrates are preferably used herein as substrates which, as a result of chemical or biochemical (i.p. enzymatic) reaction show luminescence (like fluorescence or chemiluminescence).
  • labelled antibody refers to an antibody molecule as defined above with a label incorporated that provides for the identification of the antibody (preferably after binding to the respective antigen).
  • the label is a "detectable marker", e.g., obtained by the incorporation of a radio-labelled amino acid or by the attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected in a manner known per se, as for example by optical or colorimetric methods or by luminescence, i.p. fluorescence or chemiluminescence, detection, depending on the type of substrate applied).
  • marked avidin e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected in a manner known per se, as for example by optical or colorimetric methods or by luminescence, i.p. fluorescence or chemiluminescence, detection, depending on the type of substrate applied.
  • luminescence i.p. fluorescence or chemiluminescence, detection, depending on the type of substrate applied.
  • radioisotopes or radionuclides e.g., 3 H 14 C , 35 S, 90 Y, "Tc, 111 ln, 125 l, 131 l, 177 Lu, Ho, or Sm
  • fluorescent labels e.g., FITC, rhodamine, lanthanide phosphors, Europium chelates
  • enzymatic labels e.g., horseradish peroxidase, luciferase, alkaline phosphatase
  • a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags
  • gadolinium chelates such as gadolinium chelates
  • luminescent particles like fluorescent particles
  • a binding molecule like an antibody
  • the enzymatic label may be used to generate a suitable colored or luminescent, i.p. fluorescent or chemiluminescent, signal.
  • Alkaline phosphatase substrates suitable for chemiluminescence or fluorescence detection are commercially available from different suppliers (as for example CDP-StarTM Substrate or CSPD ® substrate from Thermofisher Scientific; or DuoLuX from Vector Laboratories).
  • the term "epitope” or "antigenic determinant” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody.
  • an antibody is said to "specifically" bind an antigen when it at least preferentially or exclusively recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • Present on the surface of a cell means that said molecule (like cell surface marker) is either bound to the cell surface or is integral part of the cell membrane and extends beyond the cell membrane into the extra-cellular space and optionally also into the in- tra-cellular space (i.e. the cytoplasm).
  • Specific for in the context of a reaction comprising the binding of a binding agent (like an antibody) to a target (like in particular an antigen, like CD4 or CD8), defines the ability of the binding agent to specifically recognize and bind said particular intended target while showing no cross-reactivity with a different target (in particular antigen) which might also be present in the sample to be analyzed.
  • a binding agent like an antibody
  • a target like in particular an antigen, like CD4 or CD8
  • Hemolysed or “Hemolysis” defines, that the red blood cells (RBCs) as contained in a whole blood sample do undergo a hemolytic cell disruption during, and preferably prior to the analytical assessment according to the present invention. Unless otherwise stated it refers, to any suitable cell lysis system, like for example to a hypotonic lysing system, for the lysis of erythrocytes without lysis of leucocytes (as for example described by Cunha et al in Anal. Methods, 2014,6, 1377-1383, entitled “Kinetics of hypotonic lysis of human erythrocytes”).
  • Agglutinate and aggregation are used as synonyms herein. These terms describe the clumping of particles e.g. when an antibody or other molecule binds multiple particles and joins them, creating a large complex. Agglutination occurs if an antigen is mixed with its corresponding antibody (also called isoagglutinin). The term also describes the clumping of cells such as red blood cells or monocytes in the presence of an antibody or complement or other molecules like lectins.
  • a “non-aggregating" or “non-cross-linking” antibody does not aggregate the antigen, Either said antibody is mono-functional, i.e. contains one single antigen binding site. Alternatively the antibody may be bi- or polyfunctional, however, due to other processes, as for example steric hindrance, is unable to bind more than one antigen, Another cause may be that the antigen binding determinant on a larger antigen, like a cell, is singularly present, so that said antigen is bound by not more than one antibody mole- cule.
  • a “vertical flow assay” or “vertical flow immune assay” is characterized by the vertical flow of a fluid through the assay device.
  • the assay device comprises a multiplicity (i.e. at least two or more particularly three) layers either of identical or, preferably, of different functionality, as for example with respect to selective permeability (size exclusion) or different absorption characteristics for liquids, stacked one upon the other.
  • Such functional layers may be selected from grids, filter membranes and absorbent layers.
  • an "absorbent layer” comprises a suitable natural or synthetic material which has the ability to physically absorb the liquid phase (including constituents dissolved or suspended therein) of the sample to be analyzed, the washing liquids added during the assay method as well as the liquid phase of the liquid reagent medium (solution or dispersion of required reagents in a liquid phase) added into the device as well as unre- acted constituents of said reagent medium.
  • the size (volume) of said absorbent layer depends on the total volume of liquid to be absorbed and the absorption capacity of the absorbent material and should preferably exceed the volume of the liquid to be absorbed.
  • the term "upper” refers to the side of the device at which the sample to be analyzed (as for example an optionally pre-treated blood sample) is added and enters the device.
  • first configuration of a device may also be designated as “sample addition configuration”.
  • the "second configuration" of a device may also be designated as “reagent addition configuration” or "read-out configuration” or “reading configuration”.
  • the "first opening" of a device may also be designated as “sample addition opening” or “sample feed opening”.
  • sample addition opening or “sample feed opening”.
  • sample feed opening In said opening the optionally pre-treated blood sample is added and washed into the first filter layer, so that cell agglomerates optionally formed in said sample are retained by said filter.
  • the "second opening” of a device may also be designated as “reagent addition opening”, “reading opening” or “read-out opening”.
  • a detectable signal formed upon addition of a reagent specific for the analyte (as for example cells or cell surface markers to be assessed) may be detected and read out from said opening.
  • Multiplex detection relates to the simultaneous detection of different analytes (like antigens) in the same sample, and preferably in the same assay device, at the same or different spots.
  • multiplexing is easily achieved by spotting the same sample at one or more predetermined locations and/or patterns on the assay device.
  • multiplexing can also be coupled with analytical probes (as for example antibodies) carrying distinguishable labels, as for example coupled to nanoparticles of different color. If different spots are applied for different analytes the presence of a particular antigen is easily detectable by the appearance of the corresponding label (like color or luminescence, like i.p. fluorescence and chemiluminescence) signal.
  • a mixed label like color or luminescence, like i.p., fluorescence and chemiluminescence
  • the composition of the mixed label like color or luminescence, like, i.p. fluorescence or chemiluminescence
  • a suitable manner as for example, spectroscopically.
  • the term "essentially” designates values from about 80 to about 100%, in particular 80 to about 99,9 %, preferably about 95 to 99,5%, more preferably 98 to 99%.
  • features, parameters and ranges thereof of different degree of preference including general, not explicitly preferred features, parameters and ranges thereof). Unless otherwise stated, any combination of such two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description.
  • the present invention relates to the following particular embodiments
  • said sample may additionally comprise (or is suspected to comprise) disturbing blood cells (DBC), which carry at least one of said first cell surface markers (M1 ) as non-specific marker and would thus disturb the assessment of the said subclass of BCol also carrying at least one of said markers (M1 ), and/or wherein said sample may additionally comprise (or is suspected to comprise) at least one free (as for example dissolved), non-cell surface bound form, like a (for example soluble) extracellular fragment, of at least one, preferably of each of said first cell surface markers (M1 ), which method comprises
  • step (1 ) (2) removing from said sample as obtained in step (1 ) any disturbing blood cells (DBC), which also carry at least one of said first cell surface markers (M1 );
  • step (3) removing from said sample as obtained in step (2) any free, non-cell surface bound form of each of said first cell surface markers (M1 );
  • steps (2) and (3) may occur simultaneously or sequentially. They also may start simultaneously but may be terminated sequentially, for example if an additional processing step, like a further washing step is required to complete step (3).
  • said whole blood sample is blood from a mammalian, preferably human, individual, like a blood donor, or a patient suffering from a disease or suspected to suffer from a disease affecting the cellular profile or composition of the population of whole blood cells, in particular of at least one of said BCol. It can be obtained e.g. from venous collection through a needle, or from capillary blood collected after a finger stick by a sharp object.
  • the present method comprises the assessment of one single sub-class of BCol, and steps (1 ) to (4) are performed once.
  • said one single sub-class comprises CD4 + cells
  • the surface marker M1 is CD4.
  • the DBC comprise CD14 + cells which also carry the M1 marker CD4, in particular said DBC comprise CD14 + monocytes.
  • Said non-cell surface bound form of said first cell surface marker M1 is derived from CD4, i.e. comprises a soluble fragment thereof.
  • the present method comprises the multiplex assessment of two different sub-classes of BCol and steps (1 ) to (4) are performed separately for each subclass of cells.
  • the present method comprises the multiplex assessment of two different sub-classes of BCol (as for example CD4 + cells and CD8 + cells) and steps (1 ) to (4) are performed for a first subclass of BCol (as for example CD4 + cells) and at least steps (2) to (4) are separately performed for the second sub-class of cells (as for example CD8 + cells) if no other blood cells would disturb the assessment of said second sub-class of cells.
  • said two different sub-classes comprises CD4 + cells (the first subclass) and CD8 + cells (the second subclass) and the surface markers M1 to be assessed are CD4 (i.e. M1 a) and CD8 (i.e. M1 b).
  • the DBC comprise CD14 + cells, in particular CD14 + monocytes, which also carry said CD4 marker (M1 a).
  • Said non-cell surface bound form of said markers M1 a and M1 b is derived from CD4 and/or CD8, i.e. comprises a soluble, non-cell bound fragment of CD4 and/or CD8.
  • the present method comprises the multiplex assessment of two different sub-classes of BCol and steps (1 ) to (4) are performed only once.
  • the present method comprises the multiplex as- sessment of two different subclasses of BCol and steps (1 ), (2) and (3) are performed only once while step (4) is performed for each of said subclasses separately.
  • said two different sub-classes comprises CD4 + cells (the first sub-class) and CD8 + cells (the second subclass) and the surface markers M1 to be assessed are CD4 (i.e. M1 a) and CD8 (i.e. M1 b).
  • the DBC comprise CD14 + cells, in particular CD14 + monocytes, which also carry said CD4 marker (M1 a).
  • Said non-cell surface bound form of said markers M1 a and M1 b is derived from CD4 and/or CD8, i.e. comprises a soluble fragment of CD4 and/or CD8.
  • the assessment step is, in a preferred aspect, based on the assessment of color or luminescence, more particularly fluorescence or chemiluminescence, like a change of color or change of luminescence, more par- ticularly fluorescence or chemiluminescence.
  • the assay method of embodiment 1 which is a vertical flow assay method, in particular a vertical flow immunoassay.
  • step (1 ) comprises
  • (a) and (b) are either performed simultaneously or sequentially in any order.
  • (a) and (b) are performed simultaneously, and more preferably the corresponding reagents for (a) and (b) are contained in the same liquid reagent medium (like cell lysis buffer).
  • step (2) said DBCs are removed by filtration via a first filter (F1 ), in particular through a grid or net, as for example a Nylon net.
  • F1 first filter
  • DBCs are aggregated by means of immunoglobulin molecules, which bind to a second cell surface marker (M2) which is not present on the surface of said BCol (and thus may be identified as distinguishable marker), in particular wherein said second cell surface marker (M2) is distinguishable for, or may even be specific for said DBCs.
  • said DBC binding immunoglobulins are selected from free antibodies, polymeric antibodies or antibodies, bound to the surface of solid particles, in particular polymer particles.
  • step (3) said non- cell surface bound form of said first cell surface marker (M1 ) is removed by filtration by applying a second filter (F2) which is permeable for said non-cell surface bound form of said first cell surface marker (M1 ) free or bound to said reagent for assessing M1 , but which retains said BCol.
  • step (4) said assessment of step (4) is performed by means of immunoglobulin molecules, preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, (specifically) reactive with said first cell surface marker (M1 ), preferably an extracellular part of said marker.
  • BCol carrying a first cell surface marker (M1 ) and bound to said reagent for assessing said cell surface bound M1 ;
  • M1 non-cell surface bound form of cell surface markers
  • non-cell surface bound form of cell surface markers (M1 ); bound to said reagent for assessing said cell surface bound M1 .
  • filter (F1 ) is a grid or sieve, having a grid or mesh size in the range of 10 to 50, preferably 12 to 40, in particular 15 to 30 ⁇ .
  • BCol carrying a first cell surface marker (M1 ) and bound to said reagent for assessing said cell surface bound M1 ;
  • M1 non-cell surface bound form of cell surface markers
  • non-cell surface bound form of cell surface markers (M1 ); bound to said reagent for assessing said cell surface bound M1 .
  • filter (F2) is a membrane filter, having a pore size in the range of 1 to 15, preferably 2 to10, in particular 3 to 8 ⁇
  • BCol are selected from a sub-class of lymphocytes, in particular T-lymphocytes, and said DBCs are monocytes.
  • said first cell surface marker (M1 ) is a T-lymphocyte marker (M1 a), in particular the CD4 cell surface receptor molecule.
  • step (5) removing from said sample (optionally as obtained in step (5)) any free, non-cell surface bound form of said first cell surface markers (M1 b);
  • steps (5) and (6) may occur simultaneously or sequentially.
  • step (6) said non-cell surface bound form of said second cell surface marker (M1 b) is removed by filtration by applying a filter which is permeable for said non-cell surface bound form of said cell surface marker (M1 b) free or bound to said reagent for assessing (M1 b) but which retains said sub-class of BCol carrying (M1 b) and BCol carrying (M1 b) and bound to said reagent for assessing (M1 b).
  • step (7) is performed by means of immunoglobulin molecules, preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, reactive with said cell surface marker (M1 b), preferably an extracellular part of said marker.
  • immunoglobulin molecules preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, reactive with said cell surface marker (M1 b), preferably an extracellular part of said marker.
  • DBCs are CD14 + monocytes.
  • aggregation of DBCs in step (1 ) is performed by adding a first liquid comprising immunoglobulins, preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, said liquid being able to lyse erythrocytes contained in the sample.
  • labelled antibodies specifically reactive to said CD4 receptors of said specific sub-group of cells where said label is constituted by an enzyme or colored or luminescent, like i.p. chemiluminescent or fluorescent particle or metal colloid particle which, preferably, do not aggregate said sub-group of CD4 cells; and antibodies binding to other structures on the surface of other cells different from said specific sub-group of cells but carrying said CD4 receptors, forming particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in said specific sub-group of cells, and significantly larger than the size of the cells in said specific sub-group of cells bound to said labelled antibodies,
  • a substrate generating a colored or luminescent, like i.p. fluorescent or a chemiluminescent substance
  • steps (1 b) and (2) may occur simultaneously or sequentially. They also may start simultaneously but may be terminated sequentially, for example if an additional processing step, like a further washing step is required to complete step (2).
  • a preferably selective, preferably hypotonic, lysis of erythrocytes is performed to said blood sample prior to the assessment.
  • a hypotonic lysis of erythrocytes without lysis of leucocytes is performed.
  • Lysis may be performed in any of the above described embodiments at any suitable period of time before the respective assessment step is performed. Particularly, lysis may be performed as an initial measure, i.e. even before steps (1 ) of embodiment 1 or (1 a) in embodiment 35 are performed. Lysis may also be performed together with step (1 ) of embodiment 1 , or together with step (1 a) and/or step (1 b) of embodiment 35. More particularly, lysis may be performed between step (1 ) and step (2) of embodiment 1 , or between step (1 a) and step (1 b) of embodiment 35.
  • Lysis may also be performed for embodiment 3 simultaneously with part steps (a) and (b) or between part steps (a) and (b) or immediately after part steps (a) and (b) of embodiment 3 have been completed. Lysis may also be performed for embodiment 28 simultaneously with steps (5) and (6) or between steps (5) and (6) or immediately after steps (5) and 6) of embodiment 28 have been completed. Particular reference is made to the timing of the lysis step in the assay examples provided below.
  • a method for assessing the quantity of CD4 receptors located on the surfaces of CD4 + cells and optionally for assessing the quantity of CD8 receptors located on the surfaces of CD8 + cells in a sample of whole blood or a sample derived from blood comprises performing a method of one of the embodiments 1 to 38 and correlating the signal obtained for the assessment of the group of CD4 + cells with the quantity of cell-bound CD4 + receptor, and optionally correlat- ing the signal obtained for the assessment of the group of CD8 + cells with the quantity of cell-bound CD8 + receptor.
  • immunoglobulin molecules as applied in said method are antibodies, like monoclonal or polyclonal non-human, in particular non-rodent antibodies, like avian antibodies (in particular anti-CD4, anti-CD8 and anti-CD14 antibodies).
  • invention 42 which method comprises removing or blocking background signals caused by one or more constituents of the applied sample, which permeate filter (F2).
  • a vertical flow assay device for performing the method of any of the embodiments 1 to 41 , which device comprises an upper cover sheet (101 ) provided with at least one circular, preferably liquid, sample feed opening (102) and a lower absorbent layer (105) fixed to said upper cover sheet (1 );
  • a second filter (F2) (104) fixed between said upper cover sheet (101 ) and said lower absorbent layer (105), and separating said at least one feed opening (102) , and the circular filter (F1 ) (106) inserted therein from the absorbent layer (105).
  • said first circular filter (F1 ) (106) is fixed via a carrier ring (108) to an adhesive tape (107), said ring (108) having an outer diameter slightly smaller than the diameter of the sample feed opening (102), and having an inner diameter chosen to define a free circular space sufficient for quantitatively taking up a predetermined sample volume.
  • the testing compartment comprising an upper testing compartment inner surface (1 a) of the upper casing element (1 ) and a lower testing compartment in- ner surface (2a) of the lower casing element (2),
  • the upper casing element (1 ) being movable with respect to the lower casing element (2), thereby defining a first configuration and a second configuration of the assay device
  • the upper casing element (1 ) having a first opening (3) and a second open- ing (4), which both provide access from the outside to the testing compartment, the first opening (3) and the second opening (4) being arranged in such a manner that the position of the first opening (3) with respect to the lower casing element (2) at the first configuration is essentially the same as the position of the second opening (4) with respect to the lower casing element (2) at the second configuration.
  • the assay device according to embodiment 50 characterized in that the upper casing element (1 ) is rotatable with respect to the lower casing element (2).
  • Assay device characterized in that the stack of functional layers comprises an upper membrane layer (6) and a lower absorbent layer (7), which are arranged on top of each other and extend essentially in parallel to the upper testing compartment surface (1 a) and the lower testing compartment surface (2a).
  • Assay device according to one of the preceding embodiments 50 to 52, characterized in that at least the upper membrane layer (6) is fixed to the lower casing element (2).
  • Assay device characterized in that a movement limiter (21 ) is formed in the upper casing element (1 ) and another movement limiter (22) is formed in the lower casing element (2), wherein the movement limiters (21 , 22) are provided in such a manner that the upper casing element (1 ) is movable with respect to the lower casing element (2) between a first extreme position corresponding to the first configuration and a second extreme position corresponding to the second configuration.
  • Assay device characterized in that the upper casing element (1 ) has several first openings (3, 3') and second openings (4, 4'), every one of the first openings (3, 3') being associated with one second opening (4, 4'), wherein the first openings (3, 3') and the second openings (4, 4') are arranged in such a manner that the positions of the first openings (3, 3') with respect to the lower casing element (2) at the first configuration are essentially the same as the position of the associated second openings (4, 4') with respect to the lower casing element (2) at the second configuration. 56.
  • the device of one of the embodiments 46 to 55, wherein said filter (106, 5) has openings or pores retaining aggregated blood cells, in particular, aggregated CD14 + monocytes, and is permeable for non-aggregated blood cells, in particular CD4 + cells and optionally CD8 + cells.
  • the device of embodiment 56, wherein said filter (106, 5) is a net filter having a grid size in the range of 10 to 50 ⁇ , preferably 12 to 40 ⁇ , more preferably 15 to 30 m.
  • the device of one of the embodiments 46 to 57, wherein said second filter (104) or membrane element (6) has openings or pores retaining non-aggregated blood cells and is permeable to constituents soluble in said liquid sample.
  • the device of embodiment 58, wherein said second filter (104) or membrane element (6) has a pore size in the range of 1 to 15 ⁇ , preferably 2 to 10 ⁇ , more preferably 3 to 8 ⁇ .
  • the device of any of the embodiments 46 to 59, wherein said absorbent layer 105, 7) has an absorbing capacity sufficiently high to absorb any liquid constituents of sample and reagents and washing solutions added to the sample feed opening (102, 3, 3') during the course of the vertical flow assay.
  • Assay device comprising
  • the upper (31 ) and the lower casing element (32) being assembled in such a manner that a testing compartment is formed, which is suited to take up a stack of functional layers (43, 36, 37),
  • the testing compartment comprising an upper testing compartment inner surface (31 a) of the upper casing element (31 ) and a lower testing compartment inner surface (32b) of the lower casing element (32),
  • the upper casing element (31 ) being rotatable with respect to the lower casing element (32), thereby defining a first, a second and a third configuration, of the assay device,
  • the upper casing element (31 ) having a first opening (33), a second opening (34), and a third opening (35) each of which provide access from the outside to the testing compartment,
  • the first opening (33) the second opening (34) and the third opening (35) being arranged in such a manner that the position of the first opening (33) with respect to the lower casing element (32) at the first configuration is essentially the same as the position of the second opening (34) with respect to the lower casing element (32) at the second configuration, and the position of the second opening (34) with respect to the lower casing element (32) at the second configuration is essentially the same as the position of the third opening (34) with re- spect to the lower casing element (32) at the third configuration,
  • the assay device is provided with said stack of functional layers (43, 36, 37), which is taken up by the testing compartment, said stack comprising an upper membrane layer (36) and a lower absorbent layer (37), which are arranged on top of each other and extend essentially in parallel to the upper testing compartment surface (31 a) and the lower testing compartment surface (32a),
  • a wedge (40) is positioned between the membrane layer (36) and the absorbent layer (37).
  • testing compartment is provid- ed with a filter layer (43), which is arranged essentially in parallel to the upper membrane layer (36), wherein
  • the filter layer (43) is arranged in such a manner that it is positioned between the first opening (33) and the upper membrane layer (36) and
  • the filter layer (43) is attached to the upper testing compartment surface (31 a).
  • the reagents applied in the assay methods as herein described may be provided in a liquid form. It may also be of advantage to provide individual or all reagents in solid form, as for example in freeze-dried, lyophilized form. Said solid reagents may be provided in separate form or as one mixture or more mixtures of two or more solid constituents. Prior to use said reagents may then be di- luted with a suitable conventional liquid solvent, like distilled water, for reconstitution. The provision of reagents in solid form may improve the storage stability of the reagents.
  • individual or, preferably all, reagents required for performing the initial method step may be included in said one or more, preferably one solid preparations.
  • the present invention provides an assay kit, comprising an assay device as described herein above, and the assay reagents, in solid or liquid form as described above, and optionally washing solutions, required for performing the assay method.
  • immunoglobulins are preferably directed against an extracellular part (antigen binding domain) of one of said markers. If isoforms of one of said markers exist said immunoglobulins may be directed to individual or all isoforms to be found in /on the DBCs to be removed or BCols to be assessed according to the invention.
  • Polyclonal anti-human CD4, CD8 or CD14 antibodies can be prepared by methods well known in the art, such as those described for example by Chase, M. W., 1967, in “Methods of Immunology and Immunochemistry", ed. Williams, A. et al., M. W., pp. 197-209, Academic Press, New York. Briefly, animals of a suitable species (for example rabbits, goats, or sheep, or, preferably avian species, in particular poultry, like hens) are repetitively immunized with purified antigen in an appropriate adjuvant, for example Freund's complete or incomplete adjuvant.
  • an appropriate adjuvant for example Freund's complete or incomplete adjuvant.
  • polyclonal antibodies are purified by methods such as for example ammonium sulfate or ammonium chloride precipitation, anionic exchange chromatography, immunoaffinity chromatography, and/or affinity chromatography.
  • methods such as for example ammonium sulfate or ammonium chloride precipitation, anionic exchange chromatography, immunoaffinity chromatography, and/or affinity chromatography.
  • antibodies of high avidity may be preferred. Since polyclonal antibodies comprise many different antibody molecules, an affinity constant cannot be calculated, however high avidity and affinity was obtained by conventional polyclonal antibody techniques. Rabbit antibodies obtained by conventional methods were used, however even better results were obtained with sheep antibodies. Even better results were obtained when avian antibodies were used.
  • the avian antibodies may be according to the methods described in Larsson A, Baaloew R-M, Lindahl T, and Forsberg P-0 in Poultry Science 72:1807-1812, 1993. It is contemplated that the avians being genetically more distinct from humans are able to generate antibodies to- wards human CD4, CD8 or CD14 that have a higher avidity than polyclonal mammalian antibodies.
  • IgYs Polyclonal avian antibodies routinely are obtained from egg yolk (and are therefore designated IgYs).
  • Egg yolk contains large amounts of lipids making their further use problematic.
  • IgY can be isolated from egg yolk by using stepwise ammonium sulphate (for example 25 to 40 %) and polyethylene glycol (PEG) precipitation.
  • stepwise ammonium sulphate for example 25 to 40 %
  • PEG polyethylene glycol
  • IgY purification kits obtainable from Gallus Immuno- tech Inc, Cary, USA, or the Eggcellent Chicken IgY Purification Kit, obtainable from Pierce, Rockford, USA may also be employed considering the manufacturer's instruc- tions.
  • the avidity of polyclonal antibodies may be further increased by using antibodies that were purified by the use of antigen affinity purification methods, for example according to the teaching in "Affinity Purification of Proteins” downloaded from www.piercenet.com (April 2006) and incorporated by reference Affinity purification is described in more detail below.
  • a suitable human CD4, CD8 or CD14 affinity column For affinity purification of (for example avian) polyclonal anti-human CD4, CD8 or CD14 antibodies a suitable human CD4, CD8 or CD14 affinity column has to be prepared.
  • Purified human CD4, CD8 or CD14 is fixed by a standard protocol to a suitable solid support as for example are Sepharose or Affi-Gel, activated to covalently bind the anti- gen to the support (suitable activated solid supports are for example available from Pierce, Rockford, USA).
  • An affinity column is then prepared from said antigen-carrying resin. Successful affinity purification of antibody depends on effective presentation of the relevant epitopes on the antigen to binding sites of the antibody.
  • antigens are small and immobilized directly to a solid support surface by multiple chemical bonds, important epitopes may be blocked or sterically hindered, prohibiting effective antibody binding. Therefore, it is best to immobilize antigens using a unique functional group (e.g., sulfhydryl on a single terminal cysteine in a peptide) and to use an activated support whose reactive groups occur on spacer arms that are several atoms long. For larger antigens, especially those with multiple sites of immobilization, the spacer arm length becomes less important since the antigen itself serves as an effective spacer between the support matrix and the epitope.
  • a unique functional group e.g., sulfhydryl on a single terminal cysteine in a peptide
  • binding buffers are phosphate buffered saline (PBS) and Tris buffered saline (TBS) at pH 7.2 and 150 mM NaCI (premixed buffer packs are for example available from Pierce, Rockford, USA).
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • additional binding buffer is used to wash unbound material from the support.
  • the wash buffer may contain additional salt or detergent to disrupt any weak interactions.
  • purified antibodies are eluted from an affinity resin by altering the pH and/or ionic strength of the buffer (common elution buffers are for example available from Pierce, Rockford, USA).
  • Antibodies in general are resilient proteins that tolerate a range of pH from 2.5 to 1 1 .5 with minimal loss of activity, and this is by far the most common elution strategy. In some cases, an antibody-antigen interaction is not efficiently disrupted by pH changes or is damaged by the pH, requiring that an alternate strategy has to be employed.
  • Step 1 Wash the column (-1 ml resin bed) to remove residual protein before each use using 10 column volumes of the following sequence of buffers:
  • Step 2 Centrifuge 10 ml of crude antibody preparation to remove precipitates.
  • Step 3 Apply the crude antibody preparation to the column using a slow flow rate.
  • Step 4 Wash the column extensively with 10 ml of 0.1 M PBS, pH 7,2, 0.15 M NaCI
  • Step 5 Elute the antibody using 3 ml 0.15 M Ammonium Hydroxide, pH 10,5 ⁇ 0,2. Collect fractions into adequate tubes. Read the A 2 so of each fraction using an appropriate blank (i.e., 0.15 M Ammonium Hydroxide, pH 10,5 ⁇ 0,2).
  • Step 6 Pool the appropriate fractions. Get an A 2 so of the pools and let the antibodies maturate at room temperature for a maximum of 2 weeks. If the antibodies are to be use immediately after maturation, follow the coating procedure. If contrary, the antibodies should be dialyzed against PBS containing a preservative, such as NaN 3 or Proclin 950, and stored at 4°C.
  • a preservative such as NaN 3 or Proclin 950
  • Step 7 At the end, the column must be washed extensively with PBS containing a pre- servative, such as NaN 3 or Proclin 950.
  • a pre- servative such as NaN 3 or Proclin 950.
  • Polyclonal antibodies are often more preferred than monoclonal antibodies in particle- enhanced assays.
  • Polyclonal antibodies contrary to monoclonals, are inherently reactive to many different epitopes on the antigens (or analytes), and therefore more easily create cross-bindings and networks between the antigens molecules per se, and between the antigens and the particles to which the antibodies are immobilized.
  • monoclonal antibodies generally bind to one type of epitopes only, which makes it more difficult to form cross-bindings and networks.
  • the diagnostic industry often prefers, however, the use of monoclonal antibodies, because they are easier to standardized and to quality control to a predefined standard, especially over a product life-time of many years.
  • Monoclonal anti-human CD4, CD8 or CD14 antibodies also can be prepared by methods well known in the art, as for example those described by G. Kohler at al., 1975, Nature 256, 495, G. Galfre et al., 1981 , Meth. Enzymol. 73, 3-46, or R. Kennet, 1980, in: "Hybridomas: a new dimension in biological analysis", ed. R. Kennet et al., Plenum press, New York & London.
  • Spleen cells or peripheral blood cells from immunized mice or rats are fused with a myeloma cell line, using for instance the polyethylene fusion method. After fusion the cells are grown under suitable conditions, for example on culture plates and a selection of correctly fused cells is performed using for example the hypoxanthine/ aminopterin/ thymidine (HAT) selection method.
  • Antibody producing cell lines are identified by methods such as EIAs, RIAs or agglutination assays. After identification of the antibody producing cell line, the cells are repeatedly sub-cloned, as for example by the method of limited dilution, to guarantee that the new growing cell line derives from one single cell.
  • Chimeric anti-human CD4, CD8 or CD14 antibodies can be obtained by methods well known in the art such as that described by G. L. Boulianne et al., 1984, Nature 312, 643-645. The procedure can be briefly described as follows. The DNA of the antigen- binding site from a monoclonal antibody of one species or parts thereof are transferred to the DNA of the antibody framework of another antibody of a different species. This new construct is cloned into an expression vector, which is transferred to the corresponding expression system to produce the antibody. C.1.4 Recombinant antibodies
  • Recombinant anti-human CD4, CD8 or CD14 antibodies can be obtained without using animal vehicles by methods known in the art, such as those described by G. Winter et al., 1991 , Nature, 349, 293 or J. S. Huston et al., 1988, Proc. Ntl. Acad. Sci. USA, 85, 5879. Those methods involve the following steps: introduction of DNA (cDNA or synthetic DNA) coding for an antibody or fragments thereof into a host cell, for example E. coli, fungi, yeast, plants or eukaryotic cells, selection of antibodies with the desired specificity and affinity and expressing the antibody or fragment thereof in the corresponding expression system.
  • DNA cDNA or synthetic DNA
  • C.1.5 Antibody fragments (fAbs) Fragments as described herein above, like Fab-, and F(ab') 2 -fragments of polyclonal antibodies, monoclonal antibodies of any species (including chimeric antibodies and or recombinant antibodies) can be prepared by methods well known in the art, such as those described for example by A. Nissonoff et al., 1960, Arch Biochem Biophys, 89, 230, or R. P. Porter, 1959, Biochem J, 73, 1 19, or E. Harlow et al, 1988, in "Antibodies- -A Laboratory Manual", 626-631 , Cold Spring Harbour Press, New York, USA.
  • Such particles are either applied in the step of agglutinating DBCs are applied for the detection of the cell surface markers M1.
  • the material for preparing the particles as used in the invention may be any natural or synthetic, inorganic, organic, non-polymer or polymer material suitable for generating and performing particle-enhanced light scattering assays.
  • Such materials include for example selenium, carbon, gold; nitrides of carbon, silicon or germanium, for example Si 3 N 4 ; oxides of iron, titanium or silicion, for example Ti0 2 or Si0 2 ; and polymeric materials such as for example, polystyrene, polyvinyl chloride), epoxy resins, poly(vinylidene chloride), poly(alpha-naphtyl methacrylate), poly(vinylnaphtalene), or copolymers thereof, in particular copolymers of styrene and a copolymerizable eth- ylenically unsaturated compound, for example styrene-(meth)acrylate co-polymers.
  • Particles made of polymeric materials, as well as core-shell particles consisting of an inner core polymerized from styrene and an outer shell formed by copolymerization from styrene with a copolymerizable, ethylenically unsaturated compound, as described for example in U.S. Pat. No. 4,210,723, are also suitable.
  • Suitable polymeric particles for conjugation can be purchased from Bangs Particles Inc. or Interfacial Dynamics Inc, Merck SA, France, or other suitable sources.
  • the particles can be activated for binding to antibodies according to numerous methods, a thorough teaching of such coupling chemistry can be found, e.g. in TechNote 205, Rev. 003, for example March, 2002, "Covalent Coupling” (incorporated by reference) which can be downloaded from Bangs Laboratories, Inc.'s web-site.
  • coupling may be achieved by means of particles carrying on their surface carboxyl-, amino-, hydroxyl-, hydrazide- or chloromethyl groups.
  • the molecule to be coupled may either react direct- ly with such groups or by means of a suitable linker, as for example carbodiimides, glutaraldehyde, or cyanogen bromide.
  • the particles conjuggated with a suitable anti M1 -antibody may be further modified by the attachment of a detectable marker, like a fluorophore or a chromophore.
  • a detectable marker like a fluorophore or a chromophore.
  • Corresponding particles are commercially available or may be produces by suitable preparative methods well known in the art (see for example: Site-specific labelling of proteins using cyanine dye reporters described in CA 2493309 A1 ; Protein specific fluorescent microspheres for labelling a protein described in US 4326008 A; or Protein specific fluorescent microspheres for labelling a protein described in US 4326008 A).
  • Figure 1 is a vertical section of such a device illustrating in particular the sequence of different layers of filter and adsorbent materials required for performing the assay.
  • a central circular aperture 102 is provided in an upper square disc layer 101 .
  • a thin layer 103 of glue is provided in order to fix a circular piece of a filter 104 with a suitable pore size, to the lower side of said disc layer 101 , with its center in the middle of the central aperture 102 of said disc.
  • the glue layer 103 also fixes to the lower side of the said disc 101 a square absorbent pad 105 of about the same size as that of the disc 101 .
  • a disc of a suitable net filter 106 attached to a ring 108 is inserted into the central aperture and is removably fastened to the up- per side of disc 101 by means of an adhesive tape 107 fixed to the upper side of the ring 108.
  • tape 107 a central aperture is formed which allows adding the sample to be analyzed, and washing reagents on top of the net filter 106.
  • Filter 106 may be removed from the device after sample addition and washing is completed by pulling off the tape 107. Washing buffer and further reagents may then be added to the remaining "opened" device through aperture 102 directly onto filter 104.
  • the test result (as for example a color reaction, may be visually inspected and further analyzed through said aperture 102.
  • Figures 2, 3 and 4 illustrate another non-limiting embodiment of a vertical flow assay device.
  • the assay device comprises an upper casing element 1 and a lower casing element 2.
  • the upper casing element 1 has a first opening 3, in the depicted case a sample feed opening, and a second opening 4, in the depicted case a reading opening 4.
  • the upper 1 and the lower casing element 2 are assembled on top of each other.
  • the assembly comprising the upper 1 and lower casing element 2 has the shape of a flat round disc, i.e. the radius of the resulting assembly is larger than the thickness of the disc.
  • a card 10 is provided with a hole 10a, which is suited to take up the assembled assay device.
  • the shape of the hole 10a of the card 10 is formed in such a way that it is suited to interlock with at least one portion of the lower casing element 2.
  • the hole 10a may also comprise a notch, which is suited to hold the lower casing element 2 in place and to prevent it from a rotation with respect to the card 10.
  • an explanatory imprint may be provided on the card 10, e.g., instructions for the use of the assay device or information to facilitate the quantification of measurements using the assay device, as for example reference colored spots as explained above.
  • the upper 1 and lower casing element 2 comprise an upper 1 a and a lower testing compartment inner surface 2a, which are facing each other and extend essentially in parallel to each other.
  • the upper 1 and lower casing element 2 are furthermore formed in such a way that a testing compartment is formed between them.
  • the upper 1 a and lower testing compartment inner surface 2a form the top and bottom surfaces of a cylindrical testing compartment.
  • the testing compartment is provided with an upper membrane layer 6 and a lower absorbent layer 7, which are arranged on top of each other and extend essentially in par- allel to the upper 1 a and the lower testing compartment inner surface 2a.
  • the testing compartment is essentially filled out by the upper membrane layer 6 and the lower absorbent layer 7, i.e. said layers as inserted in form-locking manner.
  • the upper membrane layer 6 is spaced apart from the upper testing compartment inner surface 1 a while still being inserted in the lower testing compartment in form-locking manner.
  • a movable wedge comprising a flat piece of essentially non- transparent material acting as an (preferably) optical shield or filter
  • a movable wedge comprising a flat piece of essentially non- transparent material acting as an (preferably) optical shield or filter
  • said wedge is then positioned in the area under the second opening 4, thereby preventing background signals originating from material washed into the absorbent layer 7 to be detected through opening 4.
  • the second, lower testing chamber inner surface 2a is provided with a protrusion 2b that is suited to hold the lower absorbent layer 7 in place by restricting its mobility, in particular by inhibiting any mobility during the rotational movement of the assay device during the assay procedure, particularly by completely avoiding rotational movement inside the testing compartment.
  • the protrusion 2b further extends into the testing compartment and is suited to also hold the upper membrane layer 6 in place.
  • the lower absorbent layer and/or the upper membrane layer are kept in place alternatively or additionally by other attachment means, e.g., by glue.
  • the assembly further comprises a filter layer 5, which in the depicted embodiment is arranged inside a recession 5a of the upper testing compartment inner surface 1 a right below the first opening 3.
  • the filter layer 5 is attached to the upper casing element 1 , particularly to restrict its motion with respect to the upper casing element 1 .
  • the filter 5 is glued to the upper casing element 1 such that the first opening 3 is covered on the side facing the testing compartment.
  • the second opening 4 of the upper casing element 1 serves primarily as a reading opening 4, wherein the second opening offers direct optical access from outside through the upper casing element 1 to the testing compartment and an unobstructed view of the upper membrane layer 6.
  • the second opening is also used for the optional addition of reagent solutions and the addition of washing solutions on top of the membrane layer carrying the analyte (like particular blood cells) retained on the surface of said membrane layer 6.
  • the lower absorbent layer 7 comprises an absorbent material for taking up lower molecular substances and liquid which are not retained by the upper membrane 6.
  • the upper membrane layer 6 comprises a semi-permeable membrane retaining the analyte, in particular blood cells suspected to carrying the analyte in said cells, or preferably, on the cell surface, and retaining the analyte, in particular blood cells carrying the analyte on the cell surface, and bound to the detection reagent, like a labelled detection antibody.
  • the filter layer 5 comprises a semi-permeable membrane or preferably grid, permeable for non-agglutinated blood cells and smaller constituents of the sample, while retaining larger agglomerates of blood cell which have to be removed before the analytical detection reaction on the surface of the upper membrane is finally performed.
  • the assembly of the upper 1 and lower casing element 2 comprises an interlocking mechanism in which the upper casing element 1 takes up a portion of the lower casing element 2. Due to the round shape of the interlocking portions of the upper 1 and lower casing element 2, the upper 1 and lower casing element 2 may be rotated with respect to each other, wherein a rotational angle defines a position of the two casing elements 1 , 2 to each other. Latches 12 are provided on the interlocking portion of the lower casing element 2, which are suited to hold the assembly of the upper 1 and lower casing element 2 firmly in place and leave essentially only a rotational degree of freedom for motion of the casing elements 1 , 2 relative to each other.
  • latches 13 are provided on a portion of the lower casing element 2 interlocking with the hole 10a in the card 10 as shown in Figure 3.
  • the latches 12, 13 may be formed in different ways, as a person skilled in the art will appreciate.
  • corresponding grooves are formed in the upper casing element 1 corresponding to the latches 12 of the lower casing element. Similar structures may be formed in the card 10 in order to facilitate the interlocking action with the lower casing element 2.
  • FIG 5 a top view of another non-limiting embodiment of an assay device is depicted.
  • the configuration of the assay device is analogous to the structures described above with reference to Figures 2 to 4.
  • the lower casing element 2 is not depicted in Figure 5 except for the rotation stop 22.
  • the upper casing element 1 is provided with two rotation stops 21 , which engage with the rotation stop 22 of the lower casing element 2 in the first and second configuration, respectively.
  • the first configuration of the assay device is shown and the second configuration can be reached by rotating the upper casing element 1 anticlockwise with respect to the lower casing element 2 towards the second extreme rotation angle that is defined by the rotation stops 21 , 22.
  • a first pair 3, 4 and a second pair 3', 4' of first 3, 3' and second openings 4, 4' are formed in the upper casing element 1 , wherein the first openings 3, 3' are provided with ridges 3a, 3a' around their respective circumference. Also, the filter layers 5, 5' that extend across the first openings 3, 3' on the bottom side of the upper casing element 1 are shown by a hatching inside the first openings 3, 3'.
  • the pairs of openings 3, 4, 3', 4' are arranged in such a manner that, in the second configuration (after rotation), the positions of the second openings 4, 4' relative to the lower casing element, which is represented by its rotation stop 22, will be essentially the same as the positions of the first openings 3, 3' in the first configuration.
  • the label 1 1 is arranged on the top surface of the upper casing element 1 and the explanation imprint 1 1 b is visible to a user of the assay device.
  • circum- ferential imprints 4a, 4a' are provided in the label 1 1 around the second openings 4, 4'. Different hatching of the circumferential imprints 4a, 4a' illustrate the differences in col- oring that, e.g., help the user to easily differentiate the individual second openings 4, 4' from each other or give a reference color for the interpretation of a colorimetric read-out of the assay.
  • Figures 9 to 1 Another embodiment of the assay device according to the invention is shown in Figures 9 to 1 1.
  • Figures 9 and 10 show the assay device in a perspective exploded view from the top and from the bottom, respectively.
  • Figure 1 1 shows a cross-section of the assay device along an axis indicated by the letter "A” in Figures 9 and 1 1 .
  • the terms “upper” and “lower” relate to the directions in the drawings.
  • the assay device comprises an upper casing element 31 and a lower casing element 32.
  • the upper element 31 has a first opening 33, in the depicted case a sample feed opening 33, a second opening 34, in the depicted case a washing (or color or signal development) opening 34, and a third opening 35, in the depicted case a measurement (or reading) opening 35.
  • the sample feed opening 33 is provided with a ridge 33a, similar to the one 3a described in the previous embodiment ( Figure 5).
  • a filter layer 43 is arranged below the sample feed opening 33 on the inner surface 31 a of the upper casing element 31 , covering the sample feed opening 33 such that any sample passing through the opening 33 will have to pass the filter layer.
  • the filter layer 43 is not covering the second 34 or third opening 35 as to allow free access to parts of the device located below and/or unhindered sight from the top of the device through the second 34 and third opening 35.
  • the upper casing element 31 is provided with an upper groove 39 extend- ing in a circular arch around the center R of the casing element 31 and an upper guiding pin 38a on its inner surface 31 a.
  • the upper surface 32b of the lower casing element 32 is provided with a lower groove 38 extending in a circular arch around the center of the casing element 32 and a lower guiding pin 39a, which are configured such that they may en- gage with the upper guiding pin 38a and the upper groove 39, respectively.
  • the center of the casing elements 31 and 32 define the rotation axis of the assembled device during the rotational movement of the upper 31 and lower casing element 32 relatively to each other.
  • the upper guiding pin 38a engages with the lower groove 38 and may slide along it upon rotation of the upper 31 and lower casing element 32 relatively to each other.
  • the lower guiding pin 39a engages with and may slide along the upper groove 39 upon rotation.
  • the rotational movement of the upper 31 and lower casing element 32 relatively to each other is restricted and guided by the movement of the upper 38a and lower guiding pin 39a in the lower 38 and upper groove 39, respectively.
  • Different mechanisms to ensure a secure rotational movement may be utilized in further embodiments.
  • features of the previously described embodiments may be used to secure the assembly of the upper 31 and lower casing element 32, and/or stoppers may be provided to define certain rotational angles.
  • the lower casing element 32 comprises a recess, which is in the present case cuboid, suitable to act as a testing compartment similar to the one formed by the upper 1 and lower casing elements 2 of the previously described embodiment.
  • the recess has a lower testing compartment inner surface 32a, which in the present case is quadratic.
  • the compartment formed by the recess takes up the lower absorbent layer 37.
  • a membrane element 36 is arranged above the lower absorbent layer 37.
  • the membrane element 36 is formed as a stripe, which is fixed to the upper surface 32b of the lower casing element 32, spanning the whole width of the recess.
  • the membrane element 36 is configured such that it is held in a fixed position relatively to the lower casing element 32, even when the upper 31 and lower casing elements 32 are rotated relatively to each other.
  • the openings 33, 34, 35 are arranged within the upper casing element 31 such that a rotation relatively to the lower casing element 32 will lead to different configurations at defined rotation angles, here for example 90°, where one of the openings 33, 34, 35 will take the same position relatively to the lower casing element 32 as another opening 33, 34, 35 will take in another configuration.
  • one more (third) opening 35 is provided in addition to the first 33 and second opening 34.
  • three different configurations of the device are defined by a certain reference position relatively to the lower casing element 32, as for example a specific section of the membrane element 36, which may be taken by the first 33, second 34 and third opening 35, respectively.
  • sample feed configuration will be referred to as a first configuration (opening 33 is positioned immediately above the reference position on the membrane element 36) ;
  • washing configuration will be referred to as the second configuration (opening 34 is positioned immediately above the reference position on the membrane element 36);
  • measurement configuration will be re- ferred to as the third configuration (opening 35 is positioned immediately above the reference position on the membrane element 36).
  • the sample feed opening 33 is positioned above the membrane element 36 and the lower absorbent layer 37.
  • a filter layer 43 is fixed to the upper casing element 31 , spanning the area of the sample feed opening 33.
  • the upper casing element 31 is rotated, here by an angle of 90° with respect to the lower casing element 32 such that the washing opening 34 is positioned above the same position of the lower casing element 32 as the sample feed opening 33 had taken in the sample feed configuration.
  • the membrane element 36 is accessible through the washing opening 34, for example for adding a washing buffer, which may penetrate through the membrane element 36 by capillary suction towards the lower absorbent layer 37.
  • the present embodiment provides a "shutter" 40 that is shielding the signal from the lower absorbent layer 37 from the measurement opening 35.
  • the lower casing element 32 further comprises a wedge (which may also be designated as shutter) 40, comprising a flat piece of essentially non-transparent material.
  • the wedge 40 is rotatably mounted on the lower guiding pin 39a, which thereby also serves as a rotational axis 39a for the wedge 40.
  • a portion of the wedge 40 is arranged such that it is partially covering the lower groove 38 of the lower casing element 32.
  • the upper guiding pin 38a slides along the lower groove 38 and will, at certain rotation angles, reach the wedge 40 and pushes it out of the area of the lower groove 38.
  • the wedge 40 may be rotated around its rotational axis 39a.
  • two states are defined for the arrangement of the wedge 40: In the first (sample feed) and second (washing) configuration of the assay device, the wedge 40 is arranged in such a way that only a small portion of the wedge 40 is arranged between the membrane element 36 and the lower absorbent layer 37. In particular, the wedge 40 is not positioned in the area under the first 33 and second opening 34, respectively. At the same time, a portion of the wedge 40 is covering the lower groove 38.
  • the upper 31 and lower casing elements 32 of the assembled assay device are further rotated with respect to each other.
  • the upper guiding pin 38a will slide along the lower groove 38 and, after a certain rotational angle has been reached, pushes the wedge 40 out of its way.
  • the rotation of the wedge 40 is configured such that a portion of the wedge 40 is pushed in between the lower absorbent layer 37 and the membrane element 36.
  • the wedge 40 is spanning the whole area that is covered by the measurement opening 35 with respect to the membrane element 36.
  • This measurement configuration is shown in the cross-section of Figure 1 1.
  • any signal originating from the lower absorbent layer 37 is prevented from being transmitted through the membrane layer 36 and from being detected during the measurement through the measurement opening 35.
  • the wedge 40 may be made of different materials, for example polymer materials or plastics. Whatever material is used, it is in a preferred embodiment, essentially non- transparent for background signals that are to be expected from the lower absorbent layer 37, which might disturb the assessment of the analyte to be determined. It another embodiment the wedge 40 may also be reflective for the desired signal, stemming from the membrane element. For example, the wedge 40 may be white or at least reflective for a range of wavelengths that are emitted by a marker compound that is used in the assay. Therefore, the noise from washed-out markers in the lower absorbent layer 37 is reflected away from the measurement opening 35, while the desired signal from marker material on the membrane element 36 is reflected towards the measurement opening 35. The wedge 40 may therefore both prevent background noise and amplify the signal to be measured and also the signal to noise ratio.
  • the upper casing element 31 further comprises corner elements 42a, 42b, 42c, 42d that are arranged at its upper surface.
  • the upper 31 and the lower casing element 32 may be assembled on top of each other and the assembled assay device is suitable to be inserted into a reading device (not shown).
  • a suitable commercially available reading devices is for example the Optricon Cube reflectance reader manufactured by opTricon, Berlin, Germany.
  • the corner elements 42a, 42b, 42c, 42d are suita- ble to guide the assembly upon insertion of the cube onto the device of the invention and ensure a predefined insertion position of the assembly.
  • the assay device of the invention may comprise other structural features acting as equivalent guiding elements, as an alternative to or acting in combination with the corner elements 42a, 42b, 42c, 42d of the present embodiment.
  • This embodiment refers to the assessment of CD4 receptors on CD4 + lymphocytes, in particular T helper cells.
  • the labelled antibody is reactive to the CD4 receptor, and said label is constituted by an enzyme or colored or fluorescent particle. If an enzyme is used as label, a preferred embodiment is characterized by the subsequent exposure to the said filter of a substrate forming a colored substance, preferentially a precipitating colored substance, or a fluorescent or a chemiluminescent substance.
  • the correlation between color or fluorescence or chemiluminescence generated in the method of the present invention and the concentration of said class of receptor mole- cules can be performed as follows: There is a direct relationship between the amount of the said specific receptor molecules and the color to be measured, since the amount of colored particles bound relates to the amount of said specific receptor molecules present in the sample to be tested. This color is then detectable either visually with comparison to pre-evaluated, pre-calibrated and/or predetermined coloristic diagrams or by measurement of the amount of color by electronic color detectors either freely available on the market or the one developed for the present invention. Corresponding fluorescent or chemiluminescent methods can be used.
  • Measurement instruments used are easily calibrated and adjusted to colored substances or immunoparticles used, their color scheme and detection range needed. In calibration for detection instruments a known amount of analyte is used, giving a good ratio of background vs. signal, and will allow users to be provided with exact calculated readouts.
  • an enzyme including but not limited to peroxidase enzymes or alkaline phosphatase - is used in the place of colored or fluorescent substances, a color generating a chemiluminescence generating or a fluorescent generating substrate for said enzymes are used. Measurements of two components with different color deposited on a filter by measurement of reflectance at two and more wavelengths is well known to the skilled man of the art.
  • enzymatic color system generation for example based on color development or luminescence, like i.p., fluorescence or chemiluminescence
  • kinetic measurements can be employed, and the measurement can be performed using a "video" mode.
  • the HSL (hue, saturation and lightness) scheme provides a de- vice-independent way to describe color. Especially instructive is http://www.handprint.com/LS/CVS/color.html on the internet. (July 2015).
  • reference colored spots are placed or fastened in close proximity to the membrane with immobilized antibodies or bother binding molecules or fragments thereof, preferentially on the holder of the assay membrane.
  • these reference spots are measured as well.
  • the measurement of said reference spot can - by the software of the measurement instrument, be used to compensate for instrument- to-instrument and other hardware variations, to increase the overall accuracy of the assay.
  • These reference spots may define color scale for each color in the analytical measurement.
  • the instrument e.g. the camera on a mobile telephone take a picture or a series of pictures of the surface to be measured, and also the reference spots on the device.
  • Different software programs can convert the pixels measured into numeric values and define color rooms in different numeric system. Very common is the RGB (Red Green Blue) color room.
  • the RGB color model is an additive color model in which red, green, and blue light are added together in various ways to reproduce a broad array of colors. The name of the model comes from the initials of the three additive primary colors, red, green, and blue. (Wikipedia 16 July 2016)
  • HSL and HSV are the two most common cylindrical-coordinate representations of points in an RGB color model. The two representations rearrange the geometry of RGB in an attempt to be more intuitive and perceptually relevant than the cartesian (cube) representation.
  • HSL and HSV are used today in color pickers, in image editing software, and less commonly in image analysis and computer vision.
  • GIMP GIMP /gimp/ (GNU Image Manipulation Program) is a free and open-source raster graphics editor used for image retouching and editing, free-form drawing, resizing, cropping, photo-montages, converting between different image formats, and more specialized tasks. See www.gimp.org, where all aspects are explained. The result is reported in the number of CD4-lymphocyt.es and CD8-lymphocyt.es per volume unit and/or as a ratio between the two numbers.
  • the CD4 assessment may be performed with a simple device as depicted in Figure 1 , as follows:
  • a whole blood sample was mixed with dilution buffer adapted to cell lysis, as for example, but not limited to hypotonic lysis, of erythrocytes as contained in the sample while not lysing the leucocytes.
  • the dilution buffer also contains anti CD14 antibody in a form suitable to agglomerate CD14 monocytes, and it con- tains anti-human CD4 receptor antibodies with detectable marker (like enzyme, colored particles) for example, but not necessarily limited to, in a form which do not or essentially not agglomerate CD4 cells.
  • the corresponding substrate was transferred to the hole 102 of the filtration device and was sucked into the filter 104.
  • the reading was compared to a calibration curve stored in the software generated by calibration samples with known content of CD4+ lymphocytes, analyzed in identical experiments, and the content of CD4+ lymphocytes was calculated.
  • luminescent like i.p. fluorescent and chemiluminescent marker molecules can be used.
  • the CD4 assessment may be performed with a more advanced device as depicted in Figures 2, 3 and 4 as follows:
  • a whole blood sample was mixed with dilution buffer adapted to hypotonic lysis of erythrocytes as contained in the sample while not lysing the leucocytes.
  • the dilution buffer also contains anti CD14 antibody in a form suitable to agglomerate CD14 monocytes, and it contains anti-human CD4 receptor antibodies with detectable marker (like enzyme, colored particles) for example, but not necessarily limited to, in a form which do not or essentially not agglomerate CD4 cells.
  • step 7 The reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated. If the detectable marker is for example a colored particle, then step 5 and the color development according to step 6 is not of course not necessary.
  • the CD4 assessment as described above for the more advanced device as depicted in Figures 2, 3 and 4, may in analogy also be performed with a device depicted in Figure 5 where two blood samples may be assessed simultaneously.
  • the angle of rotation of the uppercasing element 1 is in this case about 90 °.
  • CD4 and CD8 assessment may be performed in analogy to the assessment of CD4, as described above by applying a device of Figure 1 or a device as depicted in Figures 2 to 4 comprised the following steps:
  • Steps 4 and 6 may be performed in identical manner.
  • Step 1 a suspension anti-CD4 antibodies and anti-CD8 antibodies conjugated each conjugated to different, distinguishable markers, as for example latex particles of different color (like to red carboxylated latex and blue carboxylated latex) was provided.
  • the color on the filter 104 or 6 was measured reflectometrically using a standard Apple i-Phone telephone and its inbuilt flash-light.
  • two for example red (a weak and a strong) and two for example blue color dots (a weak and a strong) are also placed on top of the device was depictured.
  • the BGR file obtained (see above) was used (by converting the files to gray scale) the place and the limits of the dots were decided.
  • the GIMP program (see above), all the pixels were transformed to HSV color values.
  • the maximum and the minimum responses with respect to the two blue color dots defined the blue color room, and the maximum and the minimum responses with respect to the two red color dots defined the red color room.
  • the HSV value from the test spot (with both red and blue articles) was then interpolated into the red and the blue HSV color rooms, and HSV values for all pixels were calculated and normalized.
  • the obtained normalized values were then compared to the values obtained with the calibrating samples of known CD4 and CD8 positive lymphocytes (who had also been analyzed with for example a conventional Becton Dickinson Excalibur Flow Cytometry system), which had been stored in the calibrating file of the computer in the i-Phone system, and the results were reported on the display and in the electronic output.
  • the result is reported in numbers per volume unit CD4-lymphocyt.es, CD8-lymphocyt.es per volume unit and as a ratio between the two.
  • the CD4 and CD8 assessment as described above for the more advanced device as depicted in Figures 2, 3 and 4, may in analogy also be performed with a device depicted in Figure 5 where two blood samples, one for CD4 and the other for CD8, may be assessed simultaneously in different pairs of openings (3,4 and 3', 4').
  • the angle of rotation of the uppercasing element 1 is in this case about 90 °.
  • dilution buffer adapted to hypotonic lysis of erythrocytes as contained in the sample while not lysing the leucocytes.
  • the dilution buffer also contains anti CD14 antibody in a form suitable to agglomerate CD14 monocytes.
  • One aliquot of the sample also contained anti-human CD4 receptor antibodies with detectable marker (like im- munoparticles of a first color or enzyme).
  • the other aliquot of the sample also contained anti-human CD8 receptor antibodies with detectable marker (like im- munoparticles of a second color or enzyme) 2.
  • the nylon mesh filter 5 was removed by performing twisting the upper casing element 1 of the device (angle of rotation about 90 °) so that openings 4, 4' are now exactly in the previous position of openings 3, 3' relative to the filter 6, i.e. the section of the filter where said CD4+ helper cells are adsorbed on the filter.
  • Whatman nitrocellulose filter (catalogue no 7193-002 for 3 ⁇ pore size, cat no. 7195- 004 for 5 ⁇ pore size, and 104001 12 for 8 ⁇ pore size.)were soaked for 30 minutes in SuperBlock T20 (TBS) Blocking Buffer (Thermo Scientific, prod. No. 37536). This blocking procedure was performed to avoid unspecific binding of protein and cells to the filters when later used in the vertical filtration devices.
  • TBS SuperBlock T20
  • a 20 ⁇ nylon net filter (Millipore prod. No. NY3002500) can be supported in the periphery by a ring of polystyrene or another stiffer material, since the nylon net filter is a fluffy material.
  • the said stiffer material should be glued, e.g. by Clearsol Cas- co glue, or melted to the nylon net filter to hinder liquids from leaking in between the ring and the nylon net filter (see filter (106) and ring (108) in Fig.1 as described in Preparation Example 7, below).
  • FCA Freund's complete adjuvants
  • Polyclonal anti human CD14 antibodies can be prepared by methods well known in the art, such as those described for example by Chase, M. W., 1967,. in “Methods of Immunology and Immunochemistry", ed. Williams, A. et al., M. W., pp. 197-209, Academic Press, New York. Briefly, animals of a suitable species (for example rabbits, goats, or sheep, or, preferably avian species, in particular poultry, like hens) are repetitively immunized with purified antigen in an appropriate adjuvant, for example Freund's com- plete or incomplete adjuvant.
  • an appropriate adjuvant for example Freund's com- plete or incomplete adjuvant.
  • polyclonal antibodies are purified by methods such as for example ammonium sulfate or ammonium chloride precipitation, anionic exchange chromatography, immunoaffinity chromatography, and/or affinity chromatography.
  • Polyclonal avian antibodies routinely are obtained from egg yolk (and are therefore designated IgYs).
  • Egg yolk contains large amounts of lipids making their further use problematic.
  • IgY can be isolated from egg yolk by using stepwise ammoni- um sulphate (for example 25 to 40 %) and polyethylene glycol (PEG) precipitation.
  • IgY purification kits obtainable from Gallus Immu- notch Inc, Cary, USA, or the Eggcellent Chicken IgY Purification Kit, obtainable from Pierce, Rockford, USA may also be employed considering the manufacturer's instruc- tions.
  • the avidity of polyclonal antibodies may be further increased by using antibodies that ware purified by the use of antigen affinity purification methods, for example according to the teaching in "Affinity Purification of Proteins” downloaded from www.piercenet.com (April 2006) and incorporated by reference Affinity purification is described in more detail below.
  • the total antibody fraction (the IgY fraction) from the egg yolk was isolated by ammonium chloride precipitation, in a conventional manner according to prior art methods of egg antibody isolation (see for example Larsson A, Baaloew R-M, Lindahl T, and Forsberg P-0 in Poultry Science 72:1807-1812, 1993).
  • the eluted specific anti- CD14 antibodies were dialyzed against phosphate buffered saline and concentrated to 3 mg/ml using an Amicon Centricon centrifugation filtration device with molecular weight cut-off of 30.000 Dalton.
  • This stock solution was then diluted 1 :3 in 30mM borate buffer (pH 9,1 -9,3 with 150mM sodium chlo- ride, 0,1 % Tween 20, 0,5 mg/ml PSA (porcine serum albumin) and 0,1 % ProClinTM 950 biocide).
  • Preparation Example 4 Polymerization of anti-CD14 antibodies 10 mg of anti CD14 IgY antibodies, prepared as described in Preparation Example 2 above, was added dropwise 1 ml of a PBS solution of 4 mg of dithiobis (sulfosuccin- imidyl propionate) (manufactured by Pierce Corp., hereinafter referred to as DTSSP) with stirring at room temperature. After stirring the mixed solution at 35°C for 30 minutes, the mixed solution was filtered through a Sepharose gel (manufactured by Pharmacia Fine Chemical Inc., Sephadex G25M column). This gave approximately 6 ml of the PBS solution containing IgY polymer (hereinafter referred to as IgYagg.
  • Commercially available anti-CD14-coated beads 32 ⁇ CD14 S-pluriBeads® anti- human (PluriSelect, prod. No. 19-01400-10), was added to said buffer solution at an amount (7,5 ⁇ 10 5 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the present invention.
  • an amount of anti-CD14 antibodies which will bind all monocytes of 20 ⁇ blood must be present in 400 ⁇ of the sample dilution buffer.
  • a titration of the amount necessary can be done by setting up a dilution series of said antibodies in 400 ⁇ samples of dilution buffer, and test by applying the method described in reference example 1 .
  • Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti-CD4) (EDU- 2 clone of monoclonal anti-human CD4 receptor, Diatec AS, Oslo, Norway) was added.
  • the amount ALP-anti-CD4 will be adjusted to result in sufficient binding to CD4+ lym- phocytes in the presence of free CD4 receptors and CD4 bound to monocytes.
  • concentration is between 5 and 30 ⁇ g ml.
  • Preparation Example 6 Washing buffer A solution of 0,14 M sodium chloride, 1 g/l of Tween 20 (Sigma), 0,01 M of 2-amino-2- hydroxymethyl-propane-1 ,3-diol (Sigma), 1 gram bovine serum albumin (Sigma) per liter and 1 g/l of Proclin 300, pH adjusted to 7.4 was prepared.
  • a vertical filtration device was formed around a 0,20 mm thick square polystyrene disc measuring 22 x 22 mm (101 ). In the center of the polystyrene disc a 5 mm circular hole (102) is punched out with a standard punching instrument.
  • a circular piece of nylon net filter, 15, 20 or 30 ⁇ " ⁇ ,(106) is attached by melting.
  • the nylon net filter a piece of a nitrocellulose (104) with a mean pore size of 3, 5 or 8 ⁇ , prepared according to Preparation Example 1 above, having a diameter of 10 mm, is placed, covering the circular hole of the polystyrene disc with the nylon net filter in between.
  • the said polystyrene disc was cov- ered by a CF7 absorbent pad with an area of about 360 mm 2 (105) (100% cotton linter material) from GE Health Care Life Sciences.
  • a 5 mm diameter disc of a nylon net filter (106) may be fastened to the polystyrene disc by the use of Clearseal Casco glue and a ring of polystyrene (108) and a 22 x 10 mm piece of adhesive tape (107), with a central aperture of 3 mm in the adhesive tape (107).
  • Reference Example 1 Blood Cell analysis by means of automated cell sorting
  • Anti-CD14-conjugated beads such as CD14 pluribeads or anti-CD14-polymer ⁇ Dynabeads CD14 (product no. 1 1 149D, Life Technologies)
  • Nylon net filter 15 ⁇ (pluriStrainer 15 ⁇ (Cell Strainer) product no. 43-50015-
  • Nylon net filter 20 ⁇ (pluriStrainer 20 ⁇ (Cell Strainer) product no. 43-50020- 03, Pluriselect)
  • Nylon net filter 30 ⁇ mounted in a filtration device (product no. NY3002500, Millipore)
  • PBS Phosphate buffered saline, Gibco® PBS tablets pH 7.4, product number 18912-014, Thermo Fisher Scientific or equivalent
  • DAPI-staining reagent NucBlueTM Live Cell Stain ReadyProbesTM Reagent, product number R37605, Thermo Fisher Scientific
  • sample preparation buffer prepared according to Preparation Example 5.
  • Control samples are prepared in ACK lysing buffer, and in ACK lysing buff- er with pluribeads only.
  • the buffers can also be prepared with other anti-CD14- conjugated beads or anti-CD14-polymer.
  • Another control sample were prepared in ACK lysing buffer and incubated with CD14-Dynabeads in an amount sufficient to bind most monocytes after removal by magnetic separation.
  • the samples were mixed on a rocking table during incubation. After incubation, the samples were filtrated through 15 ⁇ , 20 ⁇ or 30 ⁇ nylon net filters to remove particle bound monocytes.
  • the monocytes bound to dynabeads in the positive control are removed by magnetic separation. After monocyte elimination, 700 ⁇ is removed from each sample and prepared for Countess® II FL measurements according to the following protocol:
  • the samples were analysed for number of monocytes (Alexa Fluor® 647 Mouse Anti- Human CD14 stained cells) and total number of leucocytes (DAPI stained cells, DAPI stains all cells with a nucleus). All samples were analysed in triplicate using Countess® II FL automated cell counter. The settings were adjusted to minimize the background for each light source (bright field, CY 5 and DAPI). After capturing a picture the intensity settings were adjusted to encompass all visible cells. It was also determined that all cells measured as CY 5-positive (Alexa Fluor® 647 Mouse Anti-Human CD14 stained monocytes) had a DAPI-stained nucleus and was measured as DAPI-cells.
  • Assay Example 1 A vertical flow CD4 assay using Alkaline Phosphatase (ALP)- antiCD4 conjugates
  • the purpose of the experiments described in this example is to evaluate different parameters in the CDcard (as depicted in Figure 3 equipped with an assay device as depicted in Figure 5) for a simplified test setup of the present invention.
  • the main purpose is to verify that all steps of the simplified test method works.
  • suitable rang- es for mesh size of nylon filter, pore size of nitrocellulose filter and concentration of ALP-antiCD4 in lysis buffer will be determined. These parameters will be varied.
  • Anti- CD14 bead concentration, incubation time, blocking of nitrocellulose, absorbent size will be fixed parameters but may be further varied if necessary.
  • the results for monocyte removal will be evaluated using Countess II FL (see Reference Example 1 above)and the results for CD4-cell staining in the CD-card prototype.
  • Alkaline phosphatase enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti-CD4) (0,5 mg/ml in TBS with 50% glycerol, EDU-
  • Nylon net filter 15 ⁇ (pluriStrainer 15 ⁇ (Cell Strainer) product no. 43-50015- 03, Pluriselect)
  • Nylon net filter 20 ⁇ (pluriStrainer 20 ⁇ (Cell Strainer) product no. 43-50020- 03, Pluriselect)
  • Nylon net filter 30 ⁇ mounted in a filtration device (product no. NY3002500, Millipore)
  • the anti-CD14 pluribeads were washed once with ACK lysing buffer. All other components (Table 2) were added to the samples and incubation was performed on a rocking table for 30 minutes.
  • the samples were filtered through a 20 ⁇ nylon mesh filter (pluristrainer) and further prepared for analysis using Countess II FL (see Reference Example 1 )
  • Table 3 sample preparation for Pluribead concentration of 7,5x10 5 beads/ml and different mesh size for nylon net filter
  • the anti-CD14 pluribeads were washed once with ACK lysing buffer. All other components (Table 3) were added to the samples and incubation was performed on a rocking table for 30 minutes.
  • nitrocellulose filter was blocked with superblock T20 (TBS) for 30 minutes and dried in air at RT.
  • the blood was mixed with sample buffer including anti-CD14 pluribeads at a concentration of 7,5x10 5 beads/ml and ALP-anti-CD4 at concentration of 10, 20 or 30 ⁇ / ⁇ I ACK lysing buffer.
  • the samples were incubated for 60 minutes before CD card analy- sis.
  • the control plasma samples were prepared with the same concentration of ALP- anti-CD4 concentration but no pluribeads.
  • the plasma samples were not incubated in this experiment but prepared just before use.
  • the blood samples that were used are the same as in table 2 above but the samples were tested in CDcard after 60 minutes. The samples are described in Table 4. All samples are filtered in the test described in 0 and 2.5 and filtered again through the 20 ⁇ nylon filter melted on the CDcard. For all samples 5 ⁇ nitrocellulose filters were used.
  • Table 4 samples tested for CD4 staining using 7,5x10 5 beads/ml and different ALP- anti-CD4 concentrations in incubation buffer.
  • the prototype for CDcard with two separate sample application sites was used.
  • a 20 ⁇ nylon net filter is attached by melting underneath the sample appli- cation well. After sample application and washing, the top lid is rotated 90° to remove the nylon filter and allow the color development to be performed directly on the nitrocellulose filter.
  • the blood samples were always applied to the left and plasma control to the right. The sample information was written on the card and the sample no. indicated on the reading site.
  • CSPDTM Substrate (0.25 mM Ready-To-Use) with Nitro-BlockTM Enhancer 5. Read the signal for 10 minutes. Optionally, stop the reaction after the desired time by addition of e.g. 0, 1 M HCI.
  • sample buffer including anti-CD14 pluribeads at a concentration of 7,5x10 5 beads/ml and ALP-anti-CD4 at concentration of 10 ⁇ g ml in ACK lys- ing buffer.
  • the samples were incubated for 30 minutes and filtered through a 20 ⁇ nylon net filter.
  • the samples were applied to the CDcard test device with a nylon net filter (20 ⁇ ) and with varying pore sizes for the nitrocellulose filter (3, 5 or 8 ⁇ ).
  • the main purpose was to investigate the level of staining of CD4 cells as well as staining for plasma sample.
  • a higher staining for plasma samples with decreasing pore size would be a sign of antibody mediated aggregation of free CD4 that is stuck to the nitrocellulose filter and contributes to the staining.
  • the control plasma samples were prepared in an identical manner as the corresponding blood samples. The samples are described in Table 6. The samples were filtered through the nylon filter twice, before cell counting and in the CDcard test device.
  • Table 6 Samples tested in CD card using 7,5*10 5 beads/ml and, 10 ng/ml ALP-anti- CD4 in the incubation buffer, 20 ⁇ nylon net filter and varying the pore size of the nitrocellulose (NC) in the CDcard.
  • the assay performed with a device of Figure 1 comprised the following steps:
  • the sample was incubated for 30 minutes allowing lysis of erythrocytes, binding of anti-CD14 beads to monocytes and of ALP-anti-CD4 to CD4+ cells and free
  • CD4 receptor 30 minutes thereafter, 100 ⁇ of said mixture was applied on top of the nylon mesh filter in the filtration device according to Preparation Example 7 above, and is immediately sucked into the device.
  • Example 7 above, and was sucked into the device.
  • the reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated.
  • the said anti- human CD4 antibody can be coupled to a colored substance or a luminescent, i.p. fluo- rescent or chemiluminescent substance which can be read immediately after step 4, above.
  • Colored immunoparticles comprise antibodies or immunoreactive fragments thereof and particulate materials exhibiting a color or luminescence, like i.p. fluorescence.
  • the particulate material can be coupled to the said antibodies or fragments by physical absorption or covalent coupling, often with a spacer or bridging molecules.
  • the colored material may be constituted - but is not limited - to pigments in or on latex particles or polymer particles, which can be made from many different materials, or metal colloids like gold colloids or ferric colloids or carbon particles.
  • Fluorescent particles like Bangs Particles product code FCEU001/FC02F can also be used.
  • Such colored and fluorescent particles are described in the prior art and are well known by the skilled man of the art. They are typically purchased from suppliers like Merck France, Life technologies (US) and/or Bangs Laboratories (US). Polymer particles are supplied in all sizes and colors, also as fluorescent particles. The size and color intensity of the particles must be adjusted to the sensitivity and the capacity needed for the assay methods, as well as to the pore size of the membranes used in the product of the in- vention.
  • the present invention employs a membrane for capturing a specific group of cells, and the measurement of receptors associated with said groups of cells.
  • the said mem- brane has a pore size adapted to capture said group of cells, and allowing other smaller particles to pass through the said membrane.
  • the specific group of cells are T lymphocytes
  • a suitable pore size of said membrane for capturing the T-cells are membranes with an average pore size 1 -10 ⁇ , preferentially 3-9 ⁇ , and even more preferred 3-5 ⁇ , allowing smaller particulate materials contained in the sample material to pass through the membrane.
  • the present invention employs colored immunoparticles in a size generating a good signal from the colored immunoparticles, but small enough to pass through the said membrane which captures the said specific group of cells.
  • the present invention employs immunoparticles sized from 60 to 400 nm, more preferred from 80 to 300 nm, even more preferred 95 to 200 nm in diameter.
  • Preparation Example 8 Anti-CD4 antibodies conjugated to blue carboxylated latex
  • blue carboxylated latex particles from Mil- lipore, Europe (Prod. No. PSI 90-91 ), with a mean diameter of 1 17 nm, were employed.
  • 5 mg EDU-2 clone of monoclonal anti-human CD4 receptor antibodies, from Diatec AS, Norway, were dialyzed to 5 ml buffer (pH 9.5 in 5 mM borate, 7.5 mM sodium chloride). 23,4 mg of said carboxylated blue latex particles were washed by centrifugation and are suspended in 2 ml water.
  • EDC EDC
  • pH 9
  • This stock solution was then diluted 1 :3 in 30mM borate buffer (pH 9,1 -9,3 with 150mM so- dium chloride, 0,1 % Tween 20, 0,5 mg/ml PSA and 0,1 % ProClin 950).
  • the assay performed with a device of Figure 1 comprised the following steps:
  • a whole blood sample of 25 ⁇ was mixed with 500 ⁇ dilution buffer according to Preparation Example 5 above but prepared with anti-CD4-conjugated colored beads instead of ALP-anti-CD4 according to Preparation example 8 above.
  • the sample was incubated for 30 minutes. 2. 30 minutes thereafter, 150 ⁇ of said mixture was transferred to the nylon mesh filter of the filtration device according to Preparation Example 7 above, and is immediately sucked into the device (106).
  • Red carboxylated latex particles from Merck Estapor (Prod. No. 784 K1 -010) with a mean diameter of 190 nm, were employed.
  • 5 mg of UCHT-4 clone monoclonal anti- human CD8 receptor antibodies, from Diatec AS, Norway, were dialyzed to 5 ml buffer (pH 9.5, 5 mM borate, 7.5 mM sodium chloride).
  • 35 mg of said carboxylated red latex particles were washed by centrifugation and suspended in 2 ml water.
  • 0,8 mg EDC (Sigma, US) was dissolved into the particle suspension and the antibody solution was mixed with the latex suspension, and stirred for 5 hours.
  • the assay performed with a device of Figure 1 comprised the following steps:
  • a whole blood sample of 20 ⁇ was mixed with 400 ⁇ dilution buffer according to Preparation Example 5 above including both anti-CD4 conjugated blue beads and anti-CD8 conjugated red beads as described in Preparation Example 9 above. The sample is incubated for 30 minutes.
  • washing solution according to Preparation Example 6 above was transferred to the nylon mesh filter of the filtration device according to Preparation Example 7 above, and was sucked into the device.
  • the color on the nitrocellulose filter was measured reflec- tometrically using a standard Apple i-Phone telephone and its inbuilt flash-light. Simultaneously two red (a weak and a strong) and two blue color dots (a weak and a strong) placed on the device was depictured. For all five spots, the BGR file obtained (see above) was used (by converting the files to gray scale) the place and the limits of the dots were decided. By the GIMP program (see above), all the pixels were transformed to HSV color values. The maximum and the minimum responses with respect to the two blue color dots defined the blue color room, and the maximum and the minimum responses with respect to the two red color dots defined the red color room.
  • the obtained normalized values were then compared to the values obtained with the calibrating samples of known CD4 and CD8 positive lymphocytes (who had also been analyzed with the Becton Dickinson Excalibur Flow Cytometry system), which had been stored in the calibrating file of the computer in the i-Phone system, and the results were reported on the display and in the electronic output.
  • the result is reported in numbers of CD4-lymphocyt.es per volume unit, number of CD8-lymphocyt.es per volume unit and as a ratio between the two.
  • Preparation Example 10 A vertical flow assay device similar to that according to Figure 2.
  • a vertical filtration device according to Figures 2 and 4 was provided in shape from a 5 mm thick circular polystyrene capsule measuring 50 mm in diameter.
  • In the top part (2) of the polystyrene capsule there are two 3 mm circular holes, one deeper (3) for sample loading and one shallow hole (4) for reading the signal. Underneath the deeper circular hole (3), a circular piece of nylon net filter (5), 15, 20 or 30 ⁇ mesh size, is attached by melting.
  • the nylon net filter (5) Underneath the nylon net filter (5) a piece of a nitrocellulose membrane (6) with a mean pore size of 3, 5 or 8 ⁇ , prepared according to Preparation Example 1 above, having a diameter of about 10 mm, is placed, covering the circular hole (3) with the nylon net filter (5) in between.
  • CF7 absorbent pad (7) with an area of about 360 mm 2 (100% cotton linter material) from GE Health Care Life Sciences.
  • a 5 mm diameter disc of a nylon net filter (5) may be fastened to the inner surface of the top part of (2) of the polystyrene disc by the use of Clearseal Casco glue and a ring of polystyrene and a 22 x 10 mm piece of adhesive tape, with a central aperture of 3 mm in the adhesive tape ( corresponding to the aperture of the circular hole (3).
  • the nylon net filter (5) attached to the top part (2) of the polystyrene capsule can be removed from the nitrocellulose membrane (6) by turning the top part (2) of the capsule in an angle of 180 ° .
  • Preparation Example 11 A vertical flow assay device similar to that according to Figure 9 to 11.
  • a vertical filtration device according to Figures 9 to 1 1 was provided in the shape of a 5 mm thick circular polystyrene capsule measuring 50 mm in diameter.
  • the top part (31 ) of the polystyrene capsule there are three 3 mm circular holes (33), (34) and (35), one deeper for sample loading (33) and two shallower for color development (34, position 2 ) and reading the signal (35), distributed at equal distances to and at equal angels of 90 ° around the central rotational axis of the device.
  • the nylon net filter (42) Underneath the nylon net filter (42) a piece of a nitrocellulose with a mean pore size of 3, 5 or 8 ⁇ (36), prepared according to Preparation Example 1 above, is placed, covering the circular hole (33) with the nylon net filter (43) in between.
  • CF7 absorbent pad with an area of about 360 mm2 (37) (100% cotton linter material) from GE Health Care Life Sciences.
  • a thin piece or sheet of a non- transparent material (e.g. plastic or metal) (40) is included.
  • a non-transparent material e.g. plastic or metal
  • the non-transparent material is placed between nitrocellulose (36) and the absorbent (37) but not underneath the sample loading well (33), or the color development well (34).
  • the non- transparent material (40) is moved into a position between the nitrocellulose (36) and the absorbent (37) and at a position underneath the reading hole (35).
  • This movement is performed by a tap or pin (38a) located on the inner surface (31 a) of the top part (31 ) of the capsule forcing the non-transparent material (40) in place.
  • the non-transparent material is fixed on a pivot tap or pin (39a) located on the inner surface (32a) of the bottom part (32) of the capsule.
  • the non-transparent material (40) is now covering the absorbent underneath the reading hole (35) and blocking background signal originating from the absorbent (37).
  • a 5 mm diameter disc of a nylon net filter (43) may be fastened to the inner surface (31 a) of the top part (31 ) of the polystyrene disc by the use of Clearseal Casco glue and a ring of polystyrene and a 22 x 10 mm piece of adhesive tape, with a central aperture of 3 mm in the adhesive tape corresponding to the aperture of the circular hole (33).
  • Preparation example 12 Sample incubation buffer containing anti-CD14 conjugated beads and Alkaline Phosphate enzyme conjugated monoclonal mouse an- tihuman CD4 receptor antibodies Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti- CD4) can be used as sample incubation buffer.
  • TBS Tris buffer saline pH 7,4
  • ALP-anti- CD4 Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies
  • ALP-anti-CD4 Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (EDU- 2 clone of monoclonal anti-human CD4 receptor, Diatec AS, Oslo, Norway) was added.
  • the amount ALP-anti-CD4 will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes.
  • concentration is between 0,3 and 3,0 ⁇ g ml.
  • Preparation example 13 Sample incubation buffer containing anti-CD14 conjugated beads and anti-CD4 conjugated 80 nm gold particles
  • Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and anti-CD4-cojugated gold nanoparticles can be used as sample incubation buffer.
  • anti-CD14-coated beads 32 ⁇ CD14 S-pluriBeads® antihu- man (PluriSelect, prod. No. 19-01400-10), were added to said buffer solution at an amount (8,3*10 5 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the pre- sent invention. If, for example, 25 ⁇ of whole blood will be analyzed, and a volume of 75 ⁇ incubation buffer shall be used, an amount of anti-CD14 antibodies which will bind all monocytes of 25 ⁇ blood must be present in 75 ⁇ of the sample dilution buffer (more than 6,3x10 4 beads).
  • anti-CD4 conjugated gold nanoparticles according to Preparation Example 15 was added.
  • the amount of anti-CD4 conjugated gold nanoparticles will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes.
  • the concentration is between 1 x 10 10 and 1 x 10 12 particles/ml.
  • Preparation example 14 Sample incubation buffer containing anti-CD14 conju- gated beads and anti-CD4 conjugated 100 nm Europium particles
  • Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and anti-CD4-cojugated Europium nanoparticles can be used as sample incubation buffer.
  • Commercially available anti-CD14-coated beads 32 ⁇ CD14 S-pluriBeads® antihu- man (PluriSelect, prod. No. 19-01400-10), were added to said buffer solution at an amount (8,3x 105 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the present invention.
  • an amount of anti-CD14 antibodies which will bind all monocytes of 25 ⁇ blood must be present in 75 ⁇ of the sample dilution buffer (more than 6,3x 104 beads).
  • anti-CD4 conjugated Europium nanoparticles according to Preparation Example 16 were added.
  • the amount of anti-CD4 conjugated Europium nanoparticles will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes.
  • the concentration is between 1 x10 10 and 1 x10 12 particles/ml.
  • Preparation Example 15 Anti-CD4 antibodies conjugated to gold nanoparticles
  • 80 nm gold nanoparticles from Innova Biosciences Prod. No. SKU: 231 -0005
  • the manufacturer's instruction was followed for conjugation to EDU-2 clone of monoclonal anti-human CD4 receptor antibodies, from Diatec AS, Norway.
  • the antibody was diluted to 0,05 mg/ml in antibody diluent (provided in the conjugation kit).
  • 12 ⁇ antibody solution was mixed with 42 ⁇ reaction buffer (provided with the kit).
  • 45 ⁇ of the said solution was used to reconstitute the freeze-dried gold particles and the conjugation reaction was left for 15 minutes.
  • 5 ⁇ quencher (provided with the kit) was added to stop the conjugation reaction.
  • the particles were washed, and unconjugated antibody removed, by adding 500 ⁇ quenching solution diluted 1 :10 in ultrapure water.
  • the particles were centrifuged for 10 minutes at 9000 g and 500 ⁇ of the supernatant was withdrawn.
  • the particles were resuspended in the buffer before use.
  • Preparation Example 16 Anti-CD4 antibodies conjugated to 100 nm carboxylated Europium particles.
  • 100 nm Europium nanoparticles from Bangs laboratories Inc. (Prod. No. FCEU001 ) were conjugated with anti-human-CD4.
  • the manufacturer's instruction was followed for conjugation to EDU-2 clone of monoclonal anti-human CD4 receptor antibodies, from Diatec AS, Norway.
  • the particles (1 ml_, 10 mg/mL) were washed 2 times in 10ml_ of 0,1 M MES Buffer; pH 5.7 and then resuspended in 10ml_ of the same buffer.
  • 100mg of EDC were added to the particles and was allowed to react with the particles for 15 minutes at room temperature (18- 25 ° C), with continuous mixing.
  • the activated particles were washed 2 times in 10 ml coupling buffer (0,2 M sodium borate buffer pH 8,5 i.e. 0,2 M boric acid adjusted to pH 8,5 using NaOH). After washing the particles were resuspended in 5ml_ of coupling buffer.
  • the antibody was diluted to 5 ml using the same coupling buffer. 0.15 mg antibody result in maximum coverage on 10 mg of particles.
  • the antibody was used in 10 times excess, i.e. 1 ,5 mg.
  • the antibody solution and particle solution were combined, and the coupling reaction was performed for 2,5 h with constant mixing.
  • the coupling buffer was removed by filtration and 10 ml quenching solution (Tris buffer saline, Etha- nolamine 40 mM and Ovalbumin 10 g/L, pH 7,1 ) was added. The solution was stirred overnight.
  • Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and fluorescent Alexa Fluor®647 Mouse Anti-Human CD4 can be used as sample incubation buffer.
  • an amount of anti-CD14 antibodies which will bind all monocytes of 25 ⁇ blood must be present in 75 ⁇ of the sample dilution buffer (more than 6,3*10 4 beads).
  • fluorescent Alexa Fluor®647 Mouse Anti- Human CD4 (BD Biosciences Catalog No. 557707) was added. The amount of fluores- cent Alexa Fluor®647 Mouse Anti-Human will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes.
  • the added volume is between 5 and 50 ⁇ to 75 ⁇ incubation buffer.
  • the assay performed with a device of Figures 9 to 1 1 comprised the following steps:
  • the sample was incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and of ALP-anti-CD4 to CD4+ cells and free CD4 receptor.
  • the sample was mixed with FACSTM lysing solution (BD Biosciences, catalogue no. 349202) to a final volume of 500 ⁇ , and erythrocytes were lysed for 5 minutes
  • the sample volume can be 50- 150 ⁇ .
  • the nylon mesh filter was removed by turning the top part 90 ° 7. Thereafter, 50 ⁇ of Seramun Purple S-008-NBT liquid enzyme substrate Seramun GmbH was transferred to the color development hole of the polystyrene disc of the filtration device according to Preparation Example 1 1 above, and was sucked into the nitrocellulose filter.
  • the substrate volume can be 10-100 ⁇ .
  • the reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated.
  • Assay Example 6 A vertical flow CD4 assay using colloidal gold immunoparti- cles
  • the assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
  • a blood sample containing 100, 500, 1000 and 1500 CD4 cells/ ⁇ of 25 ⁇ is mixed with 75 ⁇ of a solution of gold particles conjugated with anti-CD4 according to Preparation Example 13. 2. The solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and Au-anti-CD4 to CD4+ cells and free CD4 receptors.
  • the solution is then mixed with BD FACSTM Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 ⁇ and erythrocytes are lysed for 5 minutes. 4. Thereafter, 100 ⁇ of said mixture is transferred to the nylon mesh filter of the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50-150 ⁇ . 5. Thereafter, 200 ⁇ of washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
  • BD FACSTM Lysing Solution BD Biosciences Catalogue No. 349202
  • the nylon mesh filter was removed by turning the top part 90 ° and then the top part was turned another 90 ° to the reading well and the non- transparent material was moved into position between the nitrocellulose and the absorbent.
  • the assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
  • a blood sample containing 100, 500, 1000 CD4 cells/ ⁇ of 25 ⁇ is mixed with 75 ⁇ of a solution of fluorescent particles conjugated with anti-CD4 according to Preparation Example 14.
  • the solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and Au-anti-CD4 to CD4+ cells and free CD4 receptors.
  • the solution is then mixed with BD FACSTM Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 ⁇ and erythrocytes are lysed for 5 minutes.
  • BD FACSTM Lysing Solution BD Biosciences Catalogue No. 349202
  • the sample volume can be 50-150 ⁇ .
  • washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
  • the nylon mesh filter was removed by turning the top part 90 ° and then the top part was turned another 90 ° to the reading well and the non- transparent material was moved into position between the nitrocellulose and the absorbent.
  • Assay Example 8 A vertical flow CD4 assay using enzyme immunoconjugates and fluorescent substrate (e.g. DuoLuxTM from Vector Laboratories)
  • the assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
  • a blood sample containing 100, 500, 1000 and 1500 CD4 cells/ ⁇ of 25 ⁇ is mixed with 75 ⁇ of a solution of anti-CD4 according to Preparation Example 12.
  • the solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and anti-CD4 to CD4+ cells and free CD4 receptors.
  • the solution is then mixed with BD FACSTM Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 ⁇ and erythrocytes are lysed for 5 minutes.
  • BD FACSTM Lysing Solution BD Biosciences Catalogue No. 349202
  • the sample volume can be 50-150 ⁇ .
  • washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
  • the nylon mesh filter was removed by turning the top part 90 ° . 7. Thereafter, 25 ⁇ of the fluorescent substrate is added to the color development hole of the polystyrene disc of the filtration device according to Preparation Example 1 1 above, and was sucked into the nitrocellulose filter.
  • the substrate volume can be 10-100 ⁇ .
  • a vertical flow CD4 assay using enzyme immunoconjugates luminescent substrate e.g. CDP-StarTM from ThermoFisher Scientific
  • the assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
  • a blood sample containing 100, 500, 1000 and 1500 CD4 cells/ ⁇ of 25 ⁇ is mixed with 75 ⁇ of a solution of anti-CD4 according to Preparation Example
  • the solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and anti-CD4 to CD4+ cells and free CD4 receptors.
  • the solution is then mixed with BD FACSTM Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 ⁇ and erythrocytes are lysed for 5 minutes.
  • BD FACSTM Lysing Solution BD Biosciences Catalogue No. 349202
  • the luminescent substrate e.g. CDP-StarTM from Ther- moFisher Scientific
  • the substrate volume can be 10-100 ⁇ .
  • the assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
  • a blood sample containing 100, 500, 1000 and 1500 CD4 cells/ ⁇ of 25 ⁇ is mixed with 75 ⁇ of a solution of fluorescent Alexa Fluor®647 Mouse Anti- Human CD4 according to Preparation Example 17.
  • the solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and Alexa Fluor®647 Mouse Anti-Human CD4 to CD4+ cells and free CD4 receptors.
  • the solution is then mixed with BD FACSTM Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 ⁇ and erythrocytes are lysed for 5 minutes. 4. Thereafter, 150 ⁇ of said mixture is transferred to the nylon mesh filter of the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device.
  • the sample volume can be 50-150 ⁇ .
  • washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device. 6. Thereafter, the nylon mesh filter was removed by turning the top part 90 ° and then the top part was turned another 90 ° to the reading well and the non- transparent material was moved into position between the nitrocellulose and the absorbent.

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Abstract

The present invention relates to a novel method for rapid assessment of one or more subclasses of blood cells of interest (BCoI), as for example CD4+ cells and CD8+ cells, in a liquid whole blood sample or a sample derived therefrom; a method of determining the cell count for such cells; a method for determining the CD4/CD8 ratio;a method for determining the quantity of such receptors in a sample;as well as a vertical flow assay device for performing such assessment.

Description

Method for Assessing Cell Surface Receptors of Blood Cells
The present invention relates to a novel method for rapid assessment of one or more subclasses of blood cells of interest (BCol), as for example CD4+ cells and CD8+ cells, in a liquid whole blood sample or a sample derived therefrom; a method of determining the cell count for such cells; a method for determining the CD4/CD8 ratio; a method for determining the quantity of such receptors in a sample; as well as a vertical flow assay device for performing such assessment. Background of the invention
Whole blood is a term used for human blood from a standard blood donation or blood sampling. The blood is typically combined with an anticoagulant during the collection process, but is generally otherwise unprocessed. Whole blood comprises the blood plasma, red blood cells (erythrocytes) and white blood cells (leucocytes) and platelets. Heparin, citrate and EDTA (Ethylene Diamine Tetra Acetic Acid) are commonly used anticoagulation agents added to hinder coagulation in blood samples for laboratory analytical use. CD4+ T helper cells are white blood cells that are an essential part of the human immune system. They are often referred to as CD4 cells, T-helper cells or T4 cells, and are a subpopulation of lymphocytes. They are called helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells. CD4 cells send the signal and CD8 cells destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following immune suppression prior to a transplant, the body is left vulnerable to a wide range of infections that it otherwise would have been able to fight.
The blood cells comprise often cell surface receptors (membrane receptors, often in the form of transmembrane receptors). These molecules are specialized integral membrane proteins that take part in communication between the cell and the outside world. Extracellular signaling molecules (usually hormones, neurotransmitters, cytokines, growth factors or cell recognition molecules) attach to the receptor, triggering changes in the function of the cell. This process is called signal transduction: The binding initi- ates a chemical change on the intracellular side of the membrane. In this way the receptors play a unique and important role in cellular communications and signal trans- duction. Many transmembrane receptors are composed of two or more protein subunits which operate collectively and may dissociate when ligands bind, fall off, or at another stage of their "activation" cycles. (Wikipedia citation July 24, 2014). The receptors called CD4 (cluster of differentiation 4) are glycoproteins found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. CD4 receptors were discovered in the late 1970s and were originally known as leu-3 and T4 (after the OKT4 monoclonal antibody that reacted with it) before being named CD4 in 1984. In humans, the CD4 protein is encoded by the CD4 gene. (Isobe M, Huebner K, Maddon PJ, Littman DR, Axel R, Croce CM (June 1986). "The gene encoding the T-cell surface protein T4 is located on human chromosome 12". Proc. Natl. Acad. Sci. U.S.A. 83 (12): 4399-4402, and Ansari-Lari MA, Muzny DM, Lu J, Lu F, Lilley CE, Spanos S, Malley T, Gibbs RA (April 1996). "A gene-rich cluster between the CD4 and triosephosphate isomerase genes at human chromosome 12p13". Ge- nome Res. 6 (4): 314-26.)
HIV infection leads to a progressive reduction in the number of T cells expressing CD4. Medical professionals refer to the "CD4 count" to decide when to begin treatment during HIV infection or how to regulate medication during disease. Normal blood values are usually expressed as the number of cells per microliter (μΙ_) (or cubic millimeter, mm3) of blood, with normal values for CD4 cells being 500-1200 cells/mm3. A CD4 count measures the number of T cells expressing CD4. While CD4 counts are not a direct HIV test - e.g. they do not check the presence of viral DNA, or specific antibodies against HIV - they are used to assess the immune system of a patient. Patients often undergo treatments when the CD4 counts reach a level of 350 cells/μΙ- in Europe but usually around 500 cells/μΙ- in the US; people with less than 200 cells/μΙ- are at high risk of contracting AIDS defined illnesses. The newest National Institute of Health guidelines recommend treatment of any HIV-positive individuals, regardless of CD4 count. Medical professionals also refer to CD4 tests to determine efficacy of treatment.
Not only T helper cells carry surface and cytoplasmic CD4 receptors. In J Immunol Methods. 1990 Dec 31 ; 135(1 -2):59-69, Filion et al. reported that all monocytes are CD4 positive. The number of monocytes in whole blood is generally high. A method to determine the number of CD4 receptors associated with T helper cells therefore needs to encompass a step or a part of the method sorting away monocytes also carrying CD4 receptors. Other blood cells carrying CD4 (like macrophages) are contained in low, and in this context neglectable proportions in the blood. Flow cytometry is a powerful tool for identifying and enumerating cells. The flow cytom- eter detects and counts individual cells passing in a stream through a laser beam. By examining large numbers of cells, flow cytometry can give quantitative data on the percentage of cells bearing different molecules, such as surface immunoglobulin, which characterizes B cells, the T-cell receptor-associated molecules known as CD3, and the CD4 and CD8 co-receptor proteins that distinguish the major T-cell subsets. Individual cells within a mixed population are tagged with specific antibodies labelled with fluorescent dyes, or for example, by specific antibodies followed by labelled antiimmunoglobulin antibodies. The suspended mixture of labelled cells is then forced through an aperture, creating a fine stream of liquid containing cells spaced singly at intervals. As each cell passes through a laser beam it scatters the laser light, and any dye molecules bound to the cell will be excited and will fluoresce. Sensitive photomulti- plier tubes detect both the scattered light, which gives information on the size and granularity of the cell, and the fluorescence emissions, which give information on the binding of the labelled antibodies and hence on the expression of cell-surface proteins by each cell. If two or more antibodies are used, each coupled to a different fluorescent dye, then the data may be displayed in the form of a two-dimensional scatter diagram or as a contour diagram, where the fluorescence of one dye-labelled antibody is plotted against that of a second, with the result that a population of cells labelling with one antibody can be further subdivided on the basis of its reactivity with the second antibody.
Typically, CD4 counts are measured in laboratories using said flow cytometry technology. Expensive and sophisticated equipment is needed, as well as highly trained personnel, a clean water supply and cold chain storage for reagents is generally required, necessitating the test to be carried out in centralized locations. Delays between testing and obtaining results can also lead to a significant 'loss to follow up' of patients and often they do not return to receive life-saving treatment.
In addition, the majority of non-reference laboratories and clinics in countries most affected by HIV cannot regularly monitor CD4 counts and access to testing can be difficult or even impossible in rural areas. To facilitate near-patient testing and reduce the need for centralized laboratories with very advanced and complicated flow cytometer instruments, the Alere Inc, US, has developed the so-called PIMA system; see "Evaluation of the PIMA Point-of-Care CD4 Analyzer in VCT Clinics in Zimbabwe" by Sekesai Mtapuri-Zinyowera et al. in J Acquir Immune Defic Syndr 2010;55:1-7. Therein the following is stated: For PIMA testing, each participant provided 1-2 drops of blood by lancet finger stick that were collected directly from the fingertip into the PIMA CD4 cartridge. A puncture depth of 1.8 mm with a blade-type lancet (Sarstedt) was used to achieve sufficient capillary blood flow. The PIMA cartridge collected the blood in a 25 μΙ_ receptacle. Of this initial volume, 5 μΙ_ of blood was drawn into the PIMA cartridge and further used for cytometric analysis. The cartridge was capped and inserted immediately into the PIMA analyzer to run the test. During the analysis process, the blood was automatically mixed with freeze-dried fluo- rescently labeled antibodies (anti-CD3 and anti-CD4) contained in the cartridge and transferred to a detection chamber where images were taken of the labeled cells to calculate the number of CD4 cells per μΙ_ of blood."
The PIMA system was a good progress for near-patient testing, but still the PIMA system is based on a sophisticated instrument comprising a complex cassette which is expensive in production.
Immunoassays are another particularly useful form of assay that exploit the specificity, strength and diversity of antibody-antigen reactions to analyze samples and detect specific components therein. A wide range of immunoassay techniques is available, such as those described in "The Immunoassay Handbook" Nature Publishing Group, 2001 . A wide range of methods for the detection of antibodies to specific antigens is also known. For example, the enzyme-linked immunosorbent assay (ELISA) or the radio-immunoassay (RIA) is routinely used in laboratories. Arrays and high-throughput screening methods are also employed. These methods generally require a high level of skill in laboratory techniques. A variety of methods has also been developed which require little skill and are rapid to perform, and which are therefore suitable for the detection of antibody to specific antigens, and/or the detection of specific antigens, at the point of care. In particular, lateral flow, dipstick and capillary tube kits have been developed to assay for a number of infections including viral infections. In one method of detecting CD4 cells, dynabeads coated with anti-CD4 antibodies are used to bind CD4+ T-lymphocytes. Monocytes, that express CD14 and CD4, are ex- eluded from fresh blood samples sample using beads coated with anti-CD14 antibodies. Reference is made to the publication "T regulatory-1 cells induce lgG4 production by B cells: role of IL-10" by Satoguina JS, Weyand E, Larbi J, Hoerauf A, in J Immunol (2005) 174:4718-4726. Thereafter, the isolated CD4 T-lymphocytes are lysed, stained with acridine orange and stained nuclei are enumerated by fluorescence microscopy.
A "TRAx CD4" test kit is described in Paxton et al., Clin. Diagn. Lab. Immunol., 2(1 ):104-1 14, 1995. This kit is an ELISA based method to measure total CD4 in whole blood samples. The antibodies used did not distinguish between cell-bound and soluble CD4 (see Lyamuya et al., J. Imm Methods, 195:103-1 12, 1996).
WO 2006/1 15866 describes an immunochromatographic device for measuring CD4 antigens. However, again there is no disclosure in this document of a capture reagent capable of distinguishing between cell-bound and soluble CD4 lacking a cytoplasmic domain in sample from a subject. Further, the device described in WO 2006/1 15866 depends upon the flow of sample over a series of numbered capture areas to capture CD4 by saturating consecutive capture areas on a test strip to subsequently provide a visual indication of the concentration of CD4 cells in the sample. In an illustrative embodiment, the method is used for evaluating in a blood sample from a subject the level of T-cell associated CD4 comprising a cytoplasmic (cytosolic) and an extracellular (ecto) domain or the level of CD4 T-cells, the method comprising:
(i) optionally contacting the sample with an agent capable of lysing or perme- abilizing CD4 T-cells;
(ii) contacting the sample with an antibody or antigen-binding fragment thereof that binds to the cytoplasmic domain of CD4; and
(iii) directly or indirectly evaluating the level or presence of bound CD4 in the sample. Anderson et al in US 8,409,818 describe a lateral flow method for evaluating from a subject the level of T-cell associated CD4 comprising a cytoplasmic (cytosolic) and an extracellular (ecto) domain or the level of CD4 T-cells. Said method comprises: a) applying the test sample to a sample portion of an immunochromatographic device wherein the sample portion is operably connected to a capture portion of the device and wherein components of the test sample flow from the sam- pie portion to and through the capture potion which comprises an antibody or antigen-binding fragment thereof that binds to the cytoplasmic domain of CD4 such that only CD4 comprising a cytoplasmic domain and not soluble CD4 that does not comprise a cytoplasmic domain binds to the antibody or frag- ment thereof to form a captured CD4;
b) contacting the capture portion with a second binding agent that binds to CD4 including the cytoplasmic or extracellular domain and which comprises a detection marker or which is capable of binding to a third or subsequent binding partner comprising a detection marker; and
c) optionally contacting the second binding agent with a third or subsequent binding agent comprising a detection marker; evaluating the presence of the detection marker.
This technology has been developed into the commercial product "VISITECT® CD4", produced by Omega Diagnostics, UK. The device which is a disposable, near-patient test device for the determination of CD4, comprising both the part of the CD4 receptor exposed on the surface and the intracellular part of the CD4 receptor of the cells. In this way, co-measurement of soluble parts of the CD4 receptor present in the blood plasma and not bound to the white blood cells is avoided. Furthermore, a magnetic separation of monocytes is an integral part of the test device. The test is easy-to-use and only requires a finger-prick blood sample to perform the test. It is a test device that is well suited for testing where sophisticated laboratory equipment is not available. However, it is a time consuming 40-minutes-test, which also requires a manual addition of a separate liquid reagent after 17 minutes, which opens for operator induced faults in the processing of the test. Furthermore, a complicated production with a built-in magnetic separation device and magnetic particles for processing the separation step is needed.
In lateral flow technology, the flow of reagents and sample is parallel to the surface of the device, typically a filtration device, with reagents - often in dry form - is connected to or placed or immobilized within the filter. Numerous such test devices have been made, both for qualitative, semi-quantitative and quantitative measurement of high number of analytes. In Anal Bioanal Chem (2009) 393:569-582, Geertruida A. Posthuma-Trumpie & Jakob Korf & Aart van Amerongen provide a review entitled "Lat- eral flow immunoassay: its strengths, weaknesses, opportunities and threats." In general, an alternative technology to lateral flow technology can be a vertical flow technology. Corresponding products have been made where sample and reagents are vertically passed through filtration devices, optionally with specific binders immobilized in the filter. Tests for antibodies against HIV viruses have been made e.g. by Medmira Inc, Canada, using immobilized antigen or antigen fragments in the filter device, as described by Owen et al in Journal of Clinical Microbiology, May 2008, p. 1588-1595 Vol. 46, No. 5 entitled "Alternative Algorithms for Human Immunodeficiency Virus Infection Diagnosis Using Tests That Are Licensed in the United States." The NycoCard CRP test is a 2-minute Point of Care test to indicate bacterial or viral cause of infection. NycoCard CRP measures C-reactive protein (CRP), an acute phase protein that increases rapidly after onset of infection, as described in "Evaluation of a near-patient test for C-reactive protein used in daily routine in primary healthcare by use of difference plots" by Dahler-Eriksen et al. in Clinical Chemistry November 1997 vol. 43 no. 1 1 2064-2075. The vertical flow assay is characterized by a sample volume of 5 μΙ_, an assay time of 2 minutes, sample material of whole blood, serum or plasma, measuring range: 8 - 200 mg/L for whole blood samples and 5 - 120 mg/L for serum and plasma samples. There is no sample crossover, it employs a disposable test device with immobilized antibodies to CRP in a nitrocellulose filtration membrane, and uses gold colloid particles conjugated to anti-CRP antibodies. There is low risk of sample contact, and a simple reflectometric reading of the color on the membrane surface is correlated to CRP concentration in the sample which is tested.
There is a need for a simple and fast, low cost method and device for the assessment of cell surface receptors specific for a subclass of blood cells of diagnostic or therapeutic interest, like for example T-cell associated CD4 receptors, in whole blood samples.
In earlier International patent application PCT/EP2016/067639 and earlier European patent application EP16180938.9, both filed July 25, 2016, by the present applicant, methods and devices for the assessment of cell surface receptors specific for a subclass of blood cells of diagnostic or therapeutic interest, like for example T-cell associated CD4 receptors, in whole blood sample, are described, the disclosure of which is herewith incorporated by reference.
Summary of the invention The above problem was surprisingly solved by the methods and devices as defined in the claims and described in more detail herein below. The present solution is based on a further improved development of the assay method described in Applicant's PCT/EP2016/067639.
The present invention very surprisingly made it possible to apply for the assessment of specific blood cells an even more simplified vertical flow principle, and, specifically, without any use of immobilized specific binder in the filter. The present invention em- ploys the use of colorimetric, or luminencene, i.p. fluorometrie or chemiluminescence, measurement of the color or luminescent, i.p.fluorimetric or chemiluminescent signals, developed on the surface of a filter, which is well known in vertical flow immunoassays, however antibody immobilization in the filter is not necessary. Said color or luminescence may either originate from a colored or luminenscent binding reagent (as for ex- ample comrising an antibody) or from a coloured or luminescent substrate of an enzyme marker attached to a per se non-colored or non-luminescent binding reagent.
The present invention relates in one embodiment to a method for the assessment of the amount of receptor molecules of a specific class of receptors bound to a "specific class" or "specific group" of cells (also designated as blood cells of interest (BCol)) in a sample of whole blood or a sample derived from whole blood. Typically, said class of receptor molecules is the class of CD4 receptors, and typically, the class (also designated herein as subclass, subset or subpopulation) of cells carrying said receptor molecules are T-lymphocytes.
The assay method of the present invention, in deviation from the teaching of PCT/EP2016/067639, is characterized by mixing in a "first step" the said sample or an aliquot of the said sample with a "first liquid" comprising antibodies against receptor molecules of said specific class of receptors bound to a "specific class" or "specific group" of cells (also designated as blood cells of interest (BCol)) and also comprising antibodies binding to other structures on the surface of "other cells" different from said above-mentioned specific group (or class) of cells but carrying said receptors (said "other cells" are also designated as disturbing blood cells (DBC)), forming particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in said specific group of cells (BCol). In one embodiment of the present invention, the said antibodies directed against said "other cells" in said "first liquid" have a specific affinity for CD14, which is very abundant on monocytes (and monocytes also carry CD4 receptors, and thus would otherwise disturb the assay).
In some embodiments the method of the present invention is further characterized by said "first liquid" carrying antibodies towards said "other cells" forming clusters of said "other cells" or the antibodies are polymerized or immobilized on particles or polymers or other large molecules facilitating the formation of particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in "said specific group" of cells.
In one embodiment of the present invention, the antibodies towards said "other cells" of said "first liquid" have a specific affinity for receptor CD14, thus enabling the formation of particles or aggregates or clusters of particles or cells comprising the monocytes of the said sample with a size significantly larger than the size of the cells in said "specific group" of cells. Preferentially, the said "first liquid" is selected to rapidly lyse the erythrocytes of the sample, and for this purpose may have a low ionic strength, and a high enough volume to maintain the said low ionic strength after being mixed with the sample to be analyzed. Hypotonic lysis of erythrocytes without lysis of leucocytes is described by Cunha et al in Anal. Methods, 2014,6, 1377-1383, entitled "Kinetics of hypotonic lysis of human erythrocytes". Lysis of the erythrocytes is very common in vertical flow immunoassays of whole blood samples. Other suitable solvent systems allowing lysis of erythrocytes without lysis of leucocytes are commercially available, as for example the whole blood lysis buffer ACK by Thermo Fisher Scientific Inc or the FACS Lysing Solution from BD.
The "second step" of the method of the present invention is a filtration step where the said particles or clusters or aggregates, with significantly larger size than the "specific group" of cells (i.e. BCol) to be analyzed which are either present in free form or are already reacted with said antibodies against receptor molecules on the surface of said specific group of cells, are filtered away with a first filter letting the "specific group" of cells (in free or bound form) to be analyzed through the filter.
In one embodiment of the invention, cells comprising CD14 receptors, including the monocytes of the sample, having formed clusters by reacting with antibodies with specific reactivity towards CD14 receptors - optionally with antibodies being conjugated to polymers or immobilized on particles - are in this way filtered away using a filter letting T cells (and T cells carrying CD4 receptors) (either in free form or already reacted with anti-CD4 antibody) through the filter. The said "first filter" must have a pore size which enables the passage of the "specific group" of cells to be analyzed. In an embodiment of the invention, the said "specific group" of cells is constituted by T-lymphocytes (either in free form or already reacted with anti-receptor antibody), hence - in the said embodiment - the said "first filter" must have a pore-size enabling said T-lymphocyte species to pass through the filter.
Filter materials having a low unspecific binding of cells and having a rather narrow distribution of pore size is preferred. A nylon web filter is a very good option, since it's pore-size is well defined, and it's unspecific binding is rather low. T-lymphocytes will easily pass through a 30 μηη filter, however aggregates of monocytes, especially when aggregated on rather large particles, e.g. 15 μηη to 50 μηη in diameter, particles carrying anti-CD14 receptor antibodies (and optionally also anti-CD4 receptor antibodies), will be withheld by such filters.
In a non-limiting sense, the said "first filter" may also consist of glass, glass fibre, poly- propylene, polyethylene, fluoropolymer, cellulose, nitrocellulose, polyamide and blends thereof. In general, a blocking treatment against unspecific binding of proteins and cells is preferred. When T-lymphocytes (either in free form or already reacted with anti- receptor antibody) is the said specific group of cells in a sample of whole blood or a sample derived from blood, a pore size of the filter of 10 to 50 μηη is suitable, however a preferred pore size is 12 to 40 μηι, and even more preferred is a pore size of 15- to 30 μπι.
In a next step ("third step"), the method of the present invention comprises passing the remaining mixture through a "second filter" retaining the said "specific group" of cells in said sample (either in free form or already reacted with anti-receptor antibody), however letting receptor molecules (either in free form or already reacted with anti-receptor antibody) in solution pass through the filter. The pore size of this "second filter" is, therefore, smaller than the pore size of the first filter.
In a non-limiting sense, the said "second filter" may consist of glass, glass fibre, poly- propylene, polyethylene, fluoropolymer, cellulose, nylon, nitrocellulose, polyamide and blends thereof. In general, a blocking treatment against unspecific binding of proteins and cells is preferred. If the said "specific group" of cells is T-lymphocytes, a suitable pore size of said membrane for capturing the T-cells are membranes with an average pore size 1 -15 μηη, preferentially 2-10 μηη, and even more preferred 3-8 μηη, allowing smaller particulate materials from the sample materials after the hypotonic solution to pass through the membrane. If the said "specific group" of cells is constituted by other cells, other pore sizes are preferred.
The said "second filter" may then optionally be washed by a washing buffer or a wash- ing solution.
In one embodiment of the invention, the said "specific group" of cells is constituted by the lymphocytes, including the T-lymphocytes, often called the CD4+ T-cells. In a next step ("fourth step") of the method of the present invention, the BCol retained on said "second filter" are assessed.
In labelled antibodies specifically reactive to said receptors (like CD4), said label may be selected from an enzyme or colored or fluorescent particle.
Brief Description of the Drawings
Figure 1 shows a vertical flow assay device which comprises an upper cover sheet
(101 ) provided with at least one circular liquid sample feed opening (102) and a lower absorbent layer (105) fixed to said upper cover sheet (101 ); a first circular filter (106), removably inserted into said at least one circular opening (102); a second filter (104) fixed between said upper cover sheet (101 ) and said lower absorbent layer (105), and separating said at least one feed opening (102) and the circular filter (106) inserted therein from the absorbent layer (105). Figure 2 shows a perspective view on another variant of a vertical flow assay device embodiment of the invention comprising an upper rotatable casing element (1 ) and a lower casing element (2) and a sample feed opening (3) and a reading opening (4).
Figure 3 shows the device of Figure 2 insertable into a corresponding opening
(10a) provided in a card (10).
Figure 4 shows a cross section of the assay device according to the Figure 2.
Figure 5 shows a top view of another embodiment of the assay device of the invention a sample provided with two pairs of feed openings and reading openings (3, 4 and 3', 4'). Figure 6 shows the results for monocyte removal in blood incubated in ACK lysing buffer with 2,5x105 anti CD14-pluribeads/ml and 0, 10, 20, or 30 g/ml ALP-anti-CD4.
Figure 7 shows the results for monocyte removal in blood incubated in ACK lysing buffer with 7,5x105 anti CD14-pluribeads/ml and 0, 10, 20, or 30 g/ml
ALP-anti-CD4.
Figure 8 shows the results for monocyte removal in blood incubated in ACK lysing buffer with 7,5x105 anti CD14-pluribeads/ml and 0 or 10 μς/ιτιΙ ALP-anti- CD4 filtered using different mesh size of nylon net filter.
Figure 9 shows a modification of a vertical flow assay device according to Figure 2.
A perspective exploded view from the top is shown. The assay device comprises an upper casing element (31 ) and a lower casing element (32). The upper element (31 ) has a sample feed opening (33), a washing opening (34), and a reading opening (35). The lower casing element (32) comprises a wedge (40), consisting of a flat piece of essentially non- transparent material and acting as an optical shield of filter which is rotat- ably mounted on the inner side of the lower casing element (32). In the reading position of the device a portion of the wedge (40) is positioned in between the lower absorbent layer (37) and the membrane element (36) so that background signals originating from the lower absorbent layer (37) is prevented from being transmitted through the membrane layer (36) and through the reading opening (35) to the detection device (not shown). Figure 10 shows a perspective exploded view of the device of Figure 9 from the bottom. shows a cross-section of the assay device of Figure 9 along an axis indi cated by the letter A (see also Section A indicated in Figure 9) immediate ly before assembly of upper (31 ) and lower (329 casing element:
Figure 12 shows a) the chemiluminescence image of five nitrocellulose membranes with increasing concentration of CD4 cells, b) An average intensity of the chemiluminescence signal for the five concentrations is shown. A blot im- aging system (Azure Biosystems c600) has been used.
Figure 13 shows a) the fluorescence image of five nitrocellulose membranes with increasing concentration of CD4 cells, b) An average intensity of the fluorescence signal for the five concentrations is shown. A blot imaging sys- tern (Azure Biosystems c600) has been used.
Detailed Description of the Invention
A. General Definitions
A "whole blood" sample as used in the assay method according to the invention is a sample derived from a mammal, in particular a human being. Any "whole blood sample" may be used. Said samples may be used "as is", i.e. without any pre-treatment, directly as taken from the blood donor, or may be pre-treated prior to the assay. Thus, for example whole blood in this context means a non-modified sample of whole blood or a sample where an anticoagulant has been added to the sample or a sample derived from whole blood, e.g. by adding a buffer or another liquid. Examples of suitable samples are native, untreated whole blood and pre-treated whole-blood, blood, like EDTA blood, citrate blood, heparin blood. The originally obtained samples may be further modified by dilution. Fractionation of whole blood to remove constituents which might disturb the assay is normally not required. Dilution may be performed by mixing the original sample with a suitable sample liquid, like a suitable buffer, in order to adjust the concentration of the constituents, as for example of the analyte. The sample may also be pre-treated by hemolysis, as for example selective hemolysis of erythrocytes. Such modified samples exemplify samples "derived from" the original whole blood sample collected or isolated from the body of the mammal.
An "analyte" to be assayed according to the invention is a cell marker, in particular a cell surface marker, more particularly CD4 or CD8. "CD4" (cluster of differentiation 4) is a glycoprotein found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells. It was discovered in the late 1970s and was originally known as leu-3 and T4 before being named CD4 in 1984. "CD4+ T-helper cells" are white blood cells that are an essential part of the human immune system. They are often referred to as CD4 cells, T-helper cells or T4 cells. They are called helper cells because one of their main roles is to send signals to other types of immune cells, including CD8 killer cells, which then destroy the infectious particle. If CD4 cells become depleted, for example in untreated HIV infection, or following im- mune suppression prior to a transplant, the body is left vulnerable to a wide range of infections that it would otherwise have been able to fight.
"CD8" (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co- receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocom- patibility complex (MHC) molecule, but is specific for the class I MHC protein. There are two isoforms of the protein, alpha and beta, each encoded by a different gene. The CD8 co-receptor is predominantly expressed on the surface of cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. "CD14" (cluster of differentiation 14), also known as CD14, is a human gene. The protein encoded by this gene is a component of the innate immune system. CD14 exists in two forms, one anchored to the membrane by a glycosylphosphatidylinositol tail (mCD14), the other a soluble form (sCD14). Soluble CD14 either appears after shedding of mCD14 (48 kDa) or is directly secreted from intracellular vesicles (56 kDa). CD14 is expressed mainly by macrophages and (at 10-times lesser extent) by neutrophils. It is also expressed by dendritic cells and monocytes. A "Blood cell of Interest" (BCol) as referred to herein belongs to a class or population or, more particular, to a sub-class or sub-population of cells typically present in a whole blood sample to be assessed according to the invention. Such (sub)- classes or (sub)- populations are distinguishable from each other in the test environment (whole blood sample) on the basis of a particular cell surface marker or a pattern of such markers which may be analyzed by means of corresponding antibody molecules specific for said marker or pattern of markers. A "sub-class", "sub-set" or "sub-population" of cells refers to a group of blood cells which are functionally and antigenically related. Examples thereof are (CD4+) T-Helper cells or (CD8+) cytotoxic T cells.
Examples of a "class" or "population" of blood cells are T-lymphocytes and B- lymphocytes.
"Free" describes cells (like CD4+ cells) or antigen (like non-cell surface bound forms of a cell surface receptor molecule, like for example CD4 receptor) as contained in the sample to be analyzed which are not or essentially not reacted with a reagent, like an antibody, as applied in an assay method of the invention.
"Bound" describes cells (like CD4+ cells) or antigen (like non-cell surface bound forms of a cell surface receptor molecule, like for example CD4) as contained in the sample to be analyzed which are quantitatively or at least essentially quantitatively reacted with a reagent, like an antibody, as applied in an assay method of the invention.
"Distinguishable" in the context of the present invention means that the particular marker is either "specific" for said particular BCol, i.e. is not detectable in any other body cell; or is "subclass-specific" and therefore not detectable in another cell popula- tion of the blood sample to be analyzed; or is "non-specific" as it is detectable on other blood cells which are present in the whole blood sample as well, however, which are either present in a very low proportion, and does not negatively affect or falsify the assay result, or are removed from the sample before the assessment of the BCol is performed. "Specific for" a class, population, sub-class or sub-population of cells in the context of the present invention, therefore, has to be understood broadly if not otherwise stated.
"Assessing" or "assessment" is intended to include both quantitative and qualitative determination in the sense of obtaining an absolute value for the amount or concentration of the analyte, present in the sample, and also obtaining an index, ratio, percentage, visual or other value indicative of the level of analyte in the sample. Assessment may be direct or indirect and the chemical species actually detected need not of course be the analyte itself but may for example be a derivative thereof.
The "accuracy" of an analytical method of the present invention, is the method's ability to accurately determine the concentration of the analyte in a sample, compared to the concentration as determined by an even more reliable reference method. The "precision" of an analytical method of the present invention, is the variation in the results when the concentration of the analyte in a sample is determined repeatedly.
A "robustness" of an assay according to the present invention is the method's ability to tolerate interfering substances and variations in assay conditions without influencing the resulting value of the analyte concentration determination.
An "inert protein" as used in the context of the invention is a protein of any origin (for example, human or non-human mammalian, microbial) which does not disturb the assay method of the invention; in particular, it should have substantially no or no detecta- ble affinity for the analyte to be analyzed and/or for the antibodies as used in the assay method of the invention.
The term "particle size" or "particle diameter" is if not otherwise stated herein defined as "mean particle size" or "mean particle diameter". Preferably the particles of the pre- sent invention, in particular the nanoparticles and immunoparticles derived therefrom by coupling of antibodies thereto, a characterized by a narrow, in particular an "essentially monomodal" or "monomodal" particle size distribution. Particle size determination may be performed in a manner known per se, as for example by applying particle size distribution measurements on a Malvern Mastersizer instrument. Typically, the meas- urement may be performed in 0.1 M NaOH. The mean particle size values stated herein are either D(0.5) or D(4.3) values which may slightly differ but which, nevertheless are in the indicated parameter range, D(0.5) represents the mean particle size in μηη at which 50% of the distribution is smaller and 50% of the size distribution is larger. D(4,3) represents the volume mean diameter. Mean particle sizes may also be determined microscopically, as for example by transmission electron microscopy (TEM).
"Antibody" relates to any class of "immunoglobulin molecule" (like IgA, D, E G, M, W, Y) and any isotype, including without limitation lgA1 , lgA2, lgG1 , lgG2, lgG3 and lgG4. Said term refers, in particular, to a functional (i.e. having the ability to bind to an antigen) monoclonal or polyclonal antibody (Ab) or fragment antibody (fAb) capable of binding to a particular antigen. Said Abs and fAbs are selected from chemically or en- zymatically produced molecules or may be produced non-recombinantly or recombi- nantly by prokaryotic or eukaryotic microorganism or cell lines, or may be produced by higher organisms, like mammalian, preferably non-human mammalian species, or non- mammalian species, preferably avian species, or plants. Said fAbs may be selected from the group consisting of: monovalent antibodies (consisting of one heavy and one light chain), Fab, F(ab')2 (or Fab2), Fab3, scFv, bis-scFv, minibody, diabody, triabody, tetrabody, tandab; and single antibody domains, like VH and VL domains, and fragments thereof; wherein polyvalent fragments thereof may bind to different or, preferably, the same antigenic determinant of the same antigen, like in particular CD4 or CD8.
"Luminescence" is herein understood as the emission of light by a substance not resulting from heat and is thus a form of cold-body radiation. It can, in particular, be caused by chemical or biochemical reactions or electrical energy. It encompasses in the context of the present invention "chemiluminescence", i.e. the emission of light as a result of a chemical reaction; and "photoluminescence" i.e. a result of the absorption of photons. Photoluminescence encompasses "fluorescence", i.e. a type of photoluminescence that is the result of singlet-singlet electronic relaxation (typical lifetime: nanoseconds); and "phosphorescence", i.e. a type of photoluminescence that is the result of triplet-singlet electronic relaxation (typical lifetime: milliseconds to hours), (see also definition of luminescence provided by Wikipedia).
Luminescent (like fluorescent or chemiluminescent) substrates are preferably used herein as substrates which, as a result of chemical or biochemical (i.p. enzymatic) reaction show luminescence (like fluorescence or chemiluminescence). The term "labelled antibody" as used herein, refers to an antibody molecule as defined above with a label incorporated that provides for the identification of the antibody (preferably after binding to the respective antigen). Particularly, the label is a "detectable marker", e.g., obtained by the incorporation of a radio-labelled amino acid or by the attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected in a manner known per se, as for example by optical or colorimetric methods or by luminescence, i.p. fluorescence or chemiluminescence, detection, depending on the type of substrate applied). Examples of labels for antibodies that may be applied in the present invention include, but are not limited to, the following:
- radioisotopes or radionuclides (e.g., 3H 14C, 35S, 90Y, "Tc, 111ln, 125l, 131 l, 177Lu, Ho, or Sm),
- fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors, Europium chelates);
- enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase) ;
- chemiluminescent markers;
- biotinyl groups;
- predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and
- polymer particles (e.g. colored, or luminescent, like fluorescent nanoparticles)
- metal particles (like gold nanoparticles)
- magnetic agents, such as gadolinium chelates and
- oligonucleotides.
In particular, luminescent particles, like fluorescent particles, when coupled to a binding molecule, like an antibody, allow a direct measurement or direct assessment of the analyte (i.e. measurement without requiring enzyme activity), which constitutes of the preferred methods of assessment of the present invention.
Depending on the type of substrate applied the enzymatic label may be used to generate a suitable colored or luminescent, i.p. fluorescent or chemiluminescent, signal. Alkaline phosphatase substrates suitable for chemiluminescence or fluorescence detection are commercially available from different suppliers (as for example CDP-Star™ Substrate or CSPD ® substrate from Thermofisher Scientific; or DuoLuX from Vector Laboratories). The term "epitope" or "antigenic determinant" includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to "specifically" bind an antigen when it at least preferentially or exclusively recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
"Present on the surface" of a cell means that said molecule (like cell surface marker) is either bound to the cell surface or is integral part of the cell membrane and extends beyond the cell membrane into the extra-cellular space and optionally also into the in- tra-cellular space (i.e. the cytoplasm).
"Specific for" in the context of a reaction comprising the binding of a binding agent (like an antibody) to a target (like in particular an antigen, like CD4 or CD8), defines the ability of the binding agent to specifically recognize and bind said particular intended target while showing no cross-reactivity with a different target (in particular antigen) which might also be present in the sample to be analyzed.
"Hemolysed" or "Hemolysis" defines, that the red blood cells (RBCs) as contained in a whole blood sample do undergo a hemolytic cell disruption during, and preferably prior to the analytical assessment according to the present invention. Unless otherwise stated it refers, to any suitable cell lysis system, like for example to a hypotonic lysing system, for the lysis of erythrocytes without lysis of leucocytes (as for example described by Cunha et al in Anal. Methods, 2014,6, 1377-1383, entitled "Kinetics of hypotonic lysis of human erythrocytes").
"Agglutination" and "aggregation" ("agglutinate" and "aggregate") are used as synonyms herein. These terms describe the clumping of particles e.g. when an antibody or other molecule binds multiple particles and joins them, creating a large complex.. Agglutination occurs if an antigen is mixed with its corresponding antibody (also called isoagglutinin). The term also describes the clumping of cells such as red blood cells or monocytes in the presence of an antibody or complement or other molecules like lectins.
A "non-aggregating" or "non-cross-linking" antibody does not aggregate the antigen, Either said antibody is mono-functional, i.e. contains one single antigen binding site. Alternatively the antibody may be bi- or polyfunctional, however, due to other processes, as for example steric hindrance, is unable to bind more than one antigen, Another cause may be that the antigen binding determinant on a larger antigen, like a cell, is singularly present, so that said antigen is bound by not more than one antibody mole- cule.
A "vertical flow assay" or "vertical flow immune assay" according to the present invention is characterized by the vertical flow of a fluid through the assay device. The assay device comprises a multiplicity (i.e. at least two or more particularly three) layers either of identical or, preferably, of different functionality, as for example with respect to selective permeability (size exclusion) or different absorption characteristics for liquids, stacked one upon the other. Such functional layers may be selected from grids, filter membranes and absorbent layers. An "absorbent layer" comprises a suitable natural or synthetic material which has the ability to physically absorb the liquid phase (including constituents dissolved or suspended therein) of the sample to be analyzed, the washing liquids added during the assay method as well as the liquid phase of the liquid reagent medium (solution or dispersion of required reagents in a liquid phase) added into the device as well as unre- acted constituents of said reagent medium. The size (volume) of said absorbent layer depends on the total volume of liquid to be absorbed and the absorption capacity of the absorbent material and should preferably exceed the volume of the liquid to be absorbed. Unless otherwise stated the term "upper" refers to the side of the device at which the sample to be analyzed (as for example an optionally pre-treated blood sample) is added and enters the device.
Unless otherwise stated the term "inner" refers to those parts of the device which are not or substantially not in direct contact with the surrounding environment. The "first configuration" of a device may also be designated as "sample addition configuration".
The "second configuration" of a device may also be designated as "reagent addition configuration" or "read-out configuration" or "reading configuration".
The "first opening" of a device may also be designated as "sample addition opening" or "sample feed opening". In said opening the optionally pre-treated blood sample is added and washed into the first filter layer, so that cell agglomerates optionally formed in said sample are retained by said filter.
The "second opening" of a device may also be designated as "reagent addition opening", "reading opening" or "read-out opening". A detectable signal formed upon addition of a reagent specific for the analyte (as for example cells or cell surface markers to be assessed) may be detected and read out from said opening.
"Multiplex" detection relates to the simultaneous detection of different analytes (like antigens) in the same sample, and preferably in the same assay device, at the same or different spots.
Multiplexing is easily achieved by spotting the same sample at one or more predetermined locations and/or patterns on the assay device. For easier visualization multiplexing can also be coupled with analytical probes (as for example antibodies) carrying distinguishable labels, as for example coupled to nanoparticles of different color. If different spots are applied for different analytes the presence of a particular antigen is easily detectable by the appearance of the corresponding label (like color or luminescence, like i.p. fluorescence and chemiluminescence) signal. If one single spot is applied a mixed label (like color or luminescence, like i.p., fluorescence and chemiluminescence) will appear if two or more different antigens are present in the sample, and the composition of the mixed label (like color or luminescence, like, i.p. fluorescence or chemiluminescence) will have to be analyzed in a suitable manner, as for example, spectroscopically.
Unless otherwise stated the term "essentially" designates values from about 80 to about 100%, in particular 80 to about 99,9 %, preferably about 95 to 99,5%, more preferably 98 to 99%. Herein disclosed are features, parameters and ranges thereof of different degree of preference (including general, not explicitly preferred features, parameters and ranges thereof). Unless otherwise stated, any combination of such two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description.
B. Preferred Embodiments
B1 . The present invention relates to the following particular embodiments
1 . An assay method for assessing in a liquid whole blood sample or a sample derived therefrom, one or more sub-classes of blood cells of interest (BCol), each sub-class carrying a first, preferably distinguishable, cell surface marker (or cell surface receptor molecule) (M1 ) for said sub-class of blood cells of interest, which means that the markers (M1 ) for different sub-classes of cells are different (i.e. antigenically different and therefore distinguishable) from each other,
wherein said sample may additionally comprise (or is suspected to comprise) disturbing blood cells (DBC), which carry at least one of said first cell surface markers (M1 ) as non-specific marker and would thus disturb the assessment of the said subclass of BCol also carrying at least one of said markers (M1 ), and/or wherein said sample may additionally comprise (or is suspected to comprise) at least one free (as for example dissolved), non-cell surface bound form, like a (for example soluble) extracellular fragment, of at least one, preferably of each of said first cell surface markers (M1 ), which method comprises
(1 ) exposing said sample to one or more reagents suitable for assessing one or more of said first cell surface markers (M1 )
(2) removing from said sample as obtained in step (1 ) any disturbing blood cells (DBC), which also carry at least one of said first cell surface markers (M1 );
(3) removing from said sample as obtained in step (2) any free, non-cell surface bound form of each of said first cell surface markers (M1 ); and
(4) assessing in the sample as obtained in step (3) each of said sub-classes of
BCol, carrying said first cell surface marker (M1 ). In said embodiment, steps (2) and (3) may occur simultaneously or sequentially. They also may start simultaneously but may be terminated sequentially, for example if an additional processing step, like a further washing step is required to complete step (3).
In particular, said whole blood sample is blood from a mammalian, preferably human, individual, like a blood donor, or a patient suffering from a disease or suspected to suffer from a disease affecting the cellular profile or composition of the population of whole blood cells, in particular of at least one of said BCol. It can be obtained e.g. from venous collection through a needle, or from capillary blood collected after a finger stick by a sharp object.
In a first particular alternative the present method comprises the assessment of one single sub-class of BCol, and steps (1 ) to (4) are performed once. Preferably, said one single sub-class comprises CD4+ cells, and the surface marker M1 is CD4. The DBC comprise CD14+ cells which also carry the M1 marker CD4, in particular said DBC comprise CD14+ monocytes. Said non-cell surface bound form of said first cell surface marker M1 is derived from CD4, i.e. comprises a soluble fragment thereof.
In a second particular alternative the present method comprises the multiplex assessment of two different sub-classes of BCol and steps (1 ) to (4) are performed separately for each subclass of cells.
In a variant of said second particular alternative the present method comprises the multiplex assessment of two different sub-classes of BCol (as for example CD4+ cells and CD8+cells) and steps (1 ) to (4) are performed for a first subclass of BCol (as for example CD4+ cells) and at least steps (2) to (4) are separately performed for the second sub-class of cells (as for example CD8+ cells) if no other blood cells would disturb the assessment of said second sub-class of cells.
Preferably, said two different sub-classes comprises CD4+ cells (the first subclass) and CD8+ cells (the second subclass) and the surface markers M1 to be assessed are CD4 (i.e. M1 a) and CD8 (i.e. M1 b). The DBC comprise CD14+ cells, in particular CD14+ monocytes, which also carry said CD4 marker (M1 a). Said non-cell surface bound form of said markers M1 a and M1 b is derived from CD4 and/or CD8, i.e. comprises a soluble, non-cell bound fragment of CD4 and/or CD8. In a third particular alternative the present method comprises the multiplex assessment of two different sub-classes of BCol and steps (1 ) to (4) are performed only once.
In a fourth particular alternative the present method comprises the multiplex as- sessment of two different subclasses of BCol and steps (1 ), (2) and (3) are performed only once while step (4) is performed for each of said subclasses separately.
Preferably in said above second, third and fourth alternatives, said two different sub-classes comprises CD4+ cells (the first sub-class) and CD8+ cells (the second subclass) and the surface markers M1 to be assessed are CD4 (i.e. M1 a) and CD8 (i.e. M1 b). The DBC comprise CD14+ cells, in particular CD14+ monocytes, which also carry said CD4 marker (M1 a). Said non-cell surface bound form of said markers M1 a and M1 b is derived from CD4 and/or CD8, i.e. comprises a soluble fragment of CD4 and/or CD8.
In the above embodiments the assessment step is, in a preferred aspect, based on the assessment of color or luminescence, more particularly fluorescence or chemiluminescence, like a change of color or change of luminescence, more par- ticularly fluorescence or chemiluminescence. The assay method of embodiment 1 , which is a vertical flow assay method, in particular a vertical flow immunoassay. The method of embodiment 1 or 2, wherein step (1 ) comprises
(a) contacting the sample with a reagent, allowing the removal of said DBCs by filtration; and
(b) contacting the sample with said one or more reagents suitable for assessing one or more of said first cell surface markers (M1 ),
wherein (a) and (b) are either performed simultaneously or sequentially in any order. Preferably (a) and (b) are performed simultaneously, and more preferably the corresponding reagents for (a) and (b) are contained in the same liquid reagent medium (like cell lysis buffer).
4. The assay method of one of the preceding embodiments, wherein in step (2) said DBCs are removed by filtration via a first filter (F1 ), in particular through a grid or net, as for example a Nylon net.
5. The assay method of embodiment 4, wherein said DBCs are aggregated, which aggregates are retained by the filter (F1 ) applied in step (2).
6. The method of embodiment 5, wherein said DBCs are aggregated by means of immunoglobulin molecules which do not bind said BCol.
7. The method of embodiment 6, wherein said DBCs are aggregated by means of immunoglobulin molecules, which bind to a second cell surface marker (M2) which is not present on the surface of said BCol (and thus may be identified as distinguishable marker), in particular wherein said second cell surface marker (M2) is distinguishable for, or may even be specific for said DBCs. 8. The method of embodiment 6 or 7, wherein said DBC binding immunoglobulins are selected from free antibodies, polymeric antibodies or antibodies, bound to the surface of solid particles, in particular polymer particles.
9. The method of one of the preceding embodiments, wherein in step (3) said non- cell surface bound form of said first cell surface marker (M1 ) is removed by filtration by applying a second filter (F2) which is permeable for said non-cell surface bound form of said first cell surface marker (M1 ) free or bound to said reagent for assessing M1 , but which retains said BCol. 10. The method of one of the preceding embodiments, wherein said assessment of step (4) is performed by means of immunoglobulin molecules, preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, (specifically) reactive with said first cell surface marker (M1 ), preferably an extracellular part of said marker.
1 1 . The method of embodiment 10, wherein said immunoglobulin molecules reactive with said first cell surface marker (M1 ) do not aggregate said BCols carrying said first cell surface marker (M1 ). 12. The method of embodiment 10 or 1 1 , wherein said immunoglobulin molecules reactive with said first cell surface marker (M1 ) are labelled. 13. The method of embodiment 12, wherein said label is selected from an enzyme, a luminescent, like i.p, chemiluminescent or fluorescent molecular marker, or colored molecular marker or a luminescent, like, i.p. fluorescent or chemiluminescent, or colored or metal colloid particle. 14. The method of one of the embodiments 4 to 13, wherein said first filter (F1 ) and said second filter (F2) differ in the size of their openings.
15. The method of embodiment 14, wherein the pore size of said first filter (F1 ) is such that DBC aggregates are retained by filter (F1 ), while
BCol carrying a first cell surface marker (M1 ),
BCol carrying a first cell surface marker (M1 ) and bound to said reagent for assessing said cell surface bound M1 ;
non-cell surface bound form of cell surface markers (M1 ); and
non-cell surface bound form of cell surface markers (M1 ); bound to said reagent for assessing said cell surface bound M1 .
are not retained.
16. The method of embodiment 15 wherein filter (F1 ) is a grid or sieve, having a grid or mesh size in the range of 10 to 50, preferably 12 to 40, in particular 15 to 30 μηι.
17. The method of one of the embodiments 9 to 16, wherein the pore size of said second filter (F2) is such that
BCol carrying a first cell surface marker (M1 ), and
BCol carrying a first cell surface marker (M1 ) and bound to said reagent for assessing said cell surface bound M1 ;
are retained; while
non-cell surface bound form of cell surface markers (M1 ); and
non-cell surface bound form of cell surface markers (M1 ); bound to said reagent for assessing said cell surface bound M1 .
are not retained. 18. The method of embodiment 17, wherein filter (F2) is a membrane filter, having a pore size in the range of 1 to 15, preferably 2 to10, in particular 3 to 8 μηη
19. The method of one of the preceding embodiments, wherein said BCol are selected from a sub-class of lymphocytes, in particular T-lymphocytes, and said DBCs are monocytes.
20. The method of one of the preceding embodiments, wherein said first cell surface marker (M1 ) is a T-lymphocyte marker (M1 a), in particular the CD4 cell surface receptor molecule.
21 . The method of one of the preceding embodiments, wherein said one or more sub-classes of blood cells of interest (BCol) to be assessed comprises CD4+ cells.
22. The method of one of the preceding embodiments, where said first cell surface marker (M1 a) is CD4 and said specific first sub-class of cells is T-helper cells. 23. The method of one of the preceding embodiments, where said method also comprises the assessment of a second sub-class of blood cells (BCol) carrying a second, preferably distinguishable, cell surface marker (M1 b) different from said first cell surface marker (M1 a). 24. The method of embodiment 23, wherein said cell surface marker (M1 b) is a T- lymphocyte marker different from (M1 a), in particular the surface marker CD8 and said specific second sub-class of cells comprises CD8+ cells.
25. The method of embodiment 24, where said surface marker (M1 b) is CD8 and said specific second sub-class of cells is cytotoxic T-cells.
26. The method of one of the embodiments 23 to 25, wherein the assessment of said second sub-class of BCol carrying said second cell surface marker (M1 b) is performed in step (4) together with the assessment of said first subclass of BCol, carrying a first cell surface marker (M1 a), in particular in the same sample.
27. The method of one of the embodiments 23 to 25, wherein the assessment of said second sub-class of BCol carrying said marker (M1 b) is performed separately. 28. The method of embodiment 27,
which method comprises
(5) optionally removing from said sample any disturbing macromolecular impurities which might disturb the assessment;
(6) removing from said sample (optionally as obtained in step (5)) any free, non-cell surface bound form of said first cell surface markers (M1 b); and
(7) assessing in the sample as obtained in step (6) said sub-class of BCol carrying said cell surface marker (M1 b). In said embodiment, steps (5) and (6) may occur simultaneously or sequentially.
They also may start simultaneously but may be terminated sequentially, for example if an additional processing step, like a further washing step is required to complete step (6). 29. The method of embodiment 28, wherein in step (6) said non-cell surface bound form of said second cell surface marker (M1 b) is removed by filtration by applying a filter which is permeable for said non-cell surface bound form of said cell surface marker (M1 b) free or bound to said reagent for assessing (M1 b) but which retains said sub-class of BCol carrying (M1 b) and BCol carrying (M1 b) and bound to said reagent for assessing (M1 b).
30. The method of embodiment 28, wherein said assessment of step (7) is performed by means of immunoglobulin molecules, preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, reactive with said cell surface marker (M1 b), preferably an extracellular part of said marker.
31 . The method of embodiment 30, wherein said immunoglobulin molecules are labelled. 32. The method of embodiment 31 , wherein said label is selected from an enzyme, a luminescent, like i.p. chemiluminescent or fluorescent, molecular marker or colored molecular marker or a luminescent, like i.p. chemiluminescent or fluorescent particle, or colored or metal colloid particle.
33. The method of one of the preceding embodiments, wherein said DBCs are CD14+ monocytes. The method one of the preceding embodiments, where the aggregation of DBCs in step (1 ) is performed by adding a first liquid comprising immunoglobulins, preferably monoclonal or polyclonal, non-human, like rodent or avian antibodies, said liquid being able to lyse erythrocytes contained in the sample. The method one of the preceding embodiments, comprising the steps of
(1 a) mixing the said sample or an aliquot of the said sample with a first liquid comprising
labelled antibodies specifically reactive to said CD4 receptors of said specific sub-group of cells, where said label is constituted by an enzyme or colored or luminescent, like i.p. chemiluminescent or fluorescent particle or metal colloid particle which, preferably, do not aggregate said sub-group of CD4 cells; and antibodies binding to other structures on the surface of other cells different from said specific sub-group of cells but carrying said CD4 receptors, forming particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in said specific sub-group of cells, and significantly larger than the size of the cells in said specific sub-group of cells bound to said labelled antibodies,
(1 b) filter away said formed particles or aggregates or cluster of particles or cells by means of a first filter that is constituted by a size exclusion filter, and
(2) passing the remaining mixture through a second filter retaining the said specific sub-group of cells in said sample but letting CD4 receptor molecules in solution (free or optionally bound to said labeled antibody) pass through the filter, optionally followed by a washing step,
(3a) optionally followed by adding a substrate to said enzyme generating a colored or luminescent, like i.p. fluorescent or a chemiluminescent substance, and (3b) measuring the intensity of the color or the luminescence, like i.p the fluorescence or the chemiluminescence on said second filter and correlating said intensity to the concentration of said class of CD4 receptors on the surface of the said specific sub-group of cells.
In said embodiment, steps (1 b) and (2) may occur simultaneously or sequentially. They also may start simultaneously but may be terminated sequentially, for example if an additional processing step, like a further washing step is required to complete step (2). The method of one of the preceding embodiments, wherein a preferably selective, preferably hypotonic, lysis of erythrocytes is performed to said blood sample prior to the assessment is performed. Preferably a hypotonic lysis of erythrocytes without lysis of leucocytes (as for example described by Cunha et al in Anal. Methods, 2014,6, 1377-1383, entitled "Kinetics of hypotonic lysis of human erythrocytes") is performed.
Lysis may be performed in any of the above described embodiments at any suitable period of time before the respective assessment step is performed. Particularly, lysis may be performed as an initial measure, i.e. even before steps (1 ) of embodiment 1 or (1 a) in embodiment 35 are performed. Lysis may also be performed together with step (1 ) of embodiment 1 , or together with step (1 a) and/or step (1 b) of embodiment 35. More particularly, lysis may be performed between step (1 ) and step (2) of embodiment 1 , or between step (1 a) and step (1 b) of embodiment 35. Lysis may also be performed for embodiment 3 simultaneously with part steps (a) and (b) or between part steps (a) and (b) or immediately after part steps (a) and (b) of embodiment 3 have been completed. Lysis may also be performed for embodiment 28 simultaneously with steps (5) and (6) or between steps (5) and (6) or immediately after steps (5) and 6) of embodiment 28 have been completed. Particular reference is made to the timing of the lysis step in the assay examples provided below.
The method of one of the preceding embodiments, wherein the cell count for the group of CD4+ cells is assessed (in number of cells/volume of sample).
The method of embodiment 37, wherein the cell count for the group of CD4+ cells, and at least for one further group of cells, different from CD4+ cells, in particular for the group of CD8+ cells, is assessed, in particular the CD4/CD8 ratio.
A method for assessing the quantity of CD4 receptors located on the surfaces of CD4+ cells and optionally for assessing the quantity of CD8 receptors located on the surfaces of CD8+ cells in a sample of whole blood or a sample derived from blood, which method comprises performing a method of one of the embodiments 1 to 38 and correlating the signal obtained for the assessment of the group of CD4+ cells with the quantity of cell-bound CD4+ receptor, and optionally correlat- ing the signal obtained for the assessment of the group of CD8+ cells with the quantity of cell-bound CD8+ receptor.
The method of one of the preceding embodiments, wherein said immunoglobulin molecules as applied in said method are antibodies, like monoclonal or polyclonal non-human, in particular non-rodent antibodies, like avian antibodies (in particular anti-CD4, anti-CD8 and anti-CD14 antibodies).
The method of one of the preceding embodiments, wherein the immunoglobulins applied for binding to (M1 a) and/or (M1 b), in particular to CD4+ or CD8+ cells, are covalently bound to colored or luminescent, like i.p. chemiluminescent or fluorescent latex or metal colloid particles having a mean particle diameter (before being coated with said immunoglobulins) in the range of 30 to 500 nm.
The method of anyone of the preceding embodiments, wherein said method further comprises before the assessment step (3), (3a) or (3b) a step of removing or blocking background signals.
The method of embodiment 42, which method comprises removing or blocking background signals caused by one or more constituents of the applied sample, which permeate filter (F2).
The method of embodiment 43, wherein said background signals are caused by constituents of the applied sample, that are retained by an absorbent layer provided to absorb sample material permeating filter (F2),
The method of embodiment 44, wherein said background signals are removed by applying on the surface of the absorbent layer a shield, shutter or filter impermeable to said background signal, or by removing the absorbent from the device before performing the assessment step, such that background signals are prevented from reaching the detection device
A vertical flow assay device for performing the method of any of the embodiments 1 to 41 , which device comprises an upper cover sheet (101 ) provided with at least one circular, preferably liquid, sample feed opening (102) and a lower absorbent layer (105) fixed to said upper cover sheet (1 );
a first circular filter (F1 )(106), removably inserted into said at least one circular opening (102);
a second filter (F2) (104) fixed between said upper cover sheet (101 ) and said lower absorbent layer (105), and separating said at least one feed opening (102) , and the circular filter (F1 ) (106) inserted therein from the absorbent layer (105). 47. The device of embodiment 46, wherein said first circular filter (F1 ) (106) is fixed via a carrier ring (108) to an adhesive tape (107), said ring (108) having an outer diameter slightly smaller than the diameter of the sample feed opening (102), and having an inner diameter chosen to define a free circular space sufficient for quantitatively taking up a predetermined sample volume.
48. The device of embodiment 47, wherein said tape (107) removably adheres to the upper side of said upper cover sheet (101 ).
49. The device of embodiment 46 to 48, wherein said first circular filter (106), remov- ably inserted intro said at least one circular opening (102), is removed from the device by removing the tape (107) from said upper cover sheet (101 ).
50. An assay device, comprising
an upper casing element (1 ) and a lower casing element (2),
the upper (1 ) and the lower casing element (2) being assembled in such a manner that a testing compartment is formed, which is suited to take up a stack of functional layers (5, 6, 7),
the testing compartment comprising an upper testing compartment inner surface (1 a) of the upper casing element (1 ) and a lower testing compartment in- ner surface (2a) of the lower casing element (2),
the upper casing element (1 ) being movable with respect to the lower casing element (2), thereby defining a first configuration and a second configuration of the assay device,
the upper casing element (1 ) having a first opening (3) and a second open- ing (4), which both provide access from the outside to the testing compartment, the first opening (3) and the second opening (4) being arranged in such a manner that the position of the first opening (3) with respect to the lower casing element (2) at the first configuration is essentially the same as the position of the second opening (4) with respect to the lower casing element (2) at the second configuration. 51 . The assay device according to embodiment 50, characterized in that the upper casing element (1 ) is rotatable with respect to the lower casing element (2).
Assay device according to one of the embodiments 50 and 51 , characterized in that the stack of functional layers comprises an upper membrane layer (6) and a lower absorbent layer (7), which are arranged on top of each other and extend essentially in parallel to the upper testing compartment surface (1 a) and the lower testing compartment surface (2a).
Assay device according to one of the preceding embodiments 50 to 52, characterized in that at least the upper membrane layer (6) is fixed to the lower casing element (2).
Assay device according to one of the embodiments 50 to 53, characterized in that a movement limiter (21 ) is formed in the upper casing element (1 ) and another movement limiter (22) is formed in the lower casing element (2), wherein the movement limiters (21 , 22) are provided in such a manner that the upper casing element (1 ) is movable with respect to the lower casing element (2) between a first extreme position corresponding to the first configuration and a second extreme position corresponding to the second configuration.
Assay device according to one of the embodiments 50 to 54, characterized in that the upper casing element (1 ) has several first openings (3, 3') and second openings (4, 4'), every one of the first openings (3, 3') being associated with one second opening (4, 4'), wherein the first openings (3, 3') and the second openings (4, 4') are arranged in such a manner that the positions of the first openings (3, 3') with respect to the lower casing element (2) at the first configuration are essentially the same as the position of the associated second openings (4, 4') with respect to the lower casing element (2) at the second configuration. 56. The device of one of the embodiments 46 to 55, wherein said filter (106, 5) has openings or pores retaining aggregated blood cells, in particular, aggregated CD14+ monocytes, and is permeable for non-aggregated blood cells, in particular CD4+ cells and optionally CD8+ cells. The device of embodiment 56, wherein said filter (106, 5) is a net filter having a grid size in the range of 10 to 50 μηη, preferably 12 to 40 μηη, more preferably 15 to 30 m. The device of one of the embodiments 46 to 57, wherein said second filter (104) or membrane element (6) has openings or pores retaining non-aggregated blood cells and is permeable to constituents soluble in said liquid sample. The device of embodiment 58, wherein said second filter (104) or membrane element (6) has a pore size in the range of 1 to 15 μηη, preferably 2 to 10 μηη, more preferably 3 to 8 μηι.
The device of any of the embodiments 46 to 59, wherein said absorbent layer 105, 7) has an absorbing capacity sufficiently high to absorb any liquid constituents of sample and reagents and washing solutions added to the sample feed opening (102, 3, 3') during the course of the vertical flow assay. Assay device, comprising
an upper casing element (31 ) and a lower casing element (32),
the upper (31 ) and the lower casing element (32) being assembled in such a manner that a testing compartment is formed, which is suited to take up a stack of functional layers (43, 36, 37),
the testing compartment comprising an upper testing compartment inner surface (31 a) of the upper casing element (31 ) and a lower testing compartment inner surface (32b) of the lower casing element (32),
the upper casing element (31 ) being rotatable with respect to the lower casing element (32), thereby defining a first, a second and a third configuration, of the assay device,
the upper casing element (31 ) having a first opening (33), a second opening (34), and a third opening (35) each of which provide access from the outside to the testing compartment,
the first opening (33) the second opening (34) and the third opening (35) being arranged in such a manner that the position of the first opening (33) with respect to the lower casing element (32) at the first configuration is essentially the same as the position of the second opening (34) with respect to the lower casing element (32) at the second configuration, and the position of the second opening (34) with respect to the lower casing element (32) at the second configuration is essentially the same as the position of the third opening (34) with re- spect to the lower casing element (32) at the third configuration,
wherein
the assay device is provided with said stack of functional layers (43, 36, 37), which is taken up by the testing compartment, said stack comprising an upper membrane layer (36) and a lower absorbent layer (37), which are arranged on top of each other and extend essentially in parallel to the upper testing compartment surface (31 a) and the lower testing compartment surface (32a),
and wherein at the third configuration of the assay device a wedge (40) is positioned between the membrane layer (36) and the absorbent layer (37). 62. The device of embodiment 61 , wherein at said third configuration the wedge (40) provides an optical shielding for said absorbent layer (37) versus said third opening (35).
63. The device of embodiment 62 or 63, wherein said testing compartment is provid- ed with a filter layer (43), which is arranged essentially in parallel to the upper membrane layer (36), wherein
the filter layer (43) is arranged in such a manner that it is positioned between the first opening (33) and the upper membrane layer (36) and
the filter layer (43) is attached to the upper testing compartment surface (31 a).
64. The use of a device as defined in anyone of the embodiments 46 to 63 for analytical or diagnostic purposes, in particular in medical diagnostics or analytics, and preferably for performing an assay as defined in anyone of the embodiments 1 to 45
In another embodiment of the invention the reagents applied in the assay methods as herein described may be provided in a liquid form. It may also be of advantage to provide individual or all reagents in solid form, as for example in freeze-dried, lyophilized form. Said solid reagents may be provided in separate form or as one mixture or more mixtures of two or more solid constituents. Prior to use said reagents may then be di- luted with a suitable conventional liquid solvent, like distilled water, for reconstitution. The provision of reagents in solid form may improve the storage stability of the reagents. For example, individual or, preferably all, reagents required for performing the initial method step (lysis buffer for erythrocyte lysis; reagent, required for removal of BDCs, like monocytes; reagent for the assessment of BCol, like CD4+ T-helper cells) may be included in said one or more, preferably one solid preparations.
In another embodiment the present invention provides an assay kit, comprising an assay device as described herein above, and the assay reagents, in solid or liquid form as described above, and optionally washing solutions, required for performing the assay method.
C. Further Embodiments C.1 CD4, CD8 or CD14 - binding immunoglobulins
If not otherwise stated herein, such immunoglobulins are preferably directed against an extracellular part (antigen binding domain) of one of said markers. If isoforms of one of said markers exist said immunoglobulins may be directed to individual or all isoforms to be found in /on the DBCs to be removed or BCols to be assessed according to the invention.
C.1.1 Polyclonal antibodies Polyclonal anti-human CD4, CD8 or CD14 antibodies can be prepared by methods well known in the art, such as those described for example by Chase, M. W., 1967, in "Methods of Immunology and Immunochemistry", ed. Williams, A. et al., M. W., pp. 197-209, Academic Press, New York. Briefly, animals of a suitable species (for example rabbits, goats, or sheep, or, preferably avian species, in particular poultry, like hens) are repetitively immunized with purified antigen in an appropriate adjuvant, for example Freund's complete or incomplete adjuvant. After immunization the animals are bled and the polyclonal antibodies are purified by methods such as for example ammonium sulfate or ammonium chloride precipitation, anionic exchange chromatography, immunoaffinity chromatography, and/or affinity chromatography. To achieve an optimum signal, antibodies of high avidity may be preferred. Since polyclonal antibodies comprise many different antibody molecules, an affinity constant cannot be calculated, however high avidity and affinity was obtained by conventional polyclonal antibody techniques. Rabbit antibodies obtained by conventional methods were used, however even better results were obtained with sheep antibodies. Even better results were obtained when avian antibodies were used. The avian antibodies may be according to the methods described in Larsson A, Baaloew R-M, Lindahl T, and Forsberg P-0 in Poultry Science 72:1807-1812, 1993. It is contemplated that the avians being genetically more distinct from humans are able to generate antibodies to- wards human CD4, CD8 or CD14 that have a higher avidity than polyclonal mammalian antibodies.
Polyclonal avian antibodies routinely are obtained from egg yolk (and are therefore designated IgYs). Egg yolk, however, contains large amounts of lipids making their further use problematic. IgY can be isolated from egg yolk by using stepwise ammonium sulphate (for example 25 to 40 %) and polyethylene glycol (PEG) precipitation. For initial purification also commercial IgY purification kits obtainable from Gallus Immuno- tech Inc, Cary, USA, or the Eggcellent Chicken IgY Purification Kit, obtainable from Pierce, Rockford, USA may also be employed considering the manufacturer's instruc- tions.
Furthermore, the avidity of polyclonal antibodies may be further increased by using antibodies that were purified by the use of antigen affinity purification methods, for example according to the teaching in "Affinity Purification of Proteins" downloaded from www.piercenet.com (April 2006) and incorporated by reference Affinity purification is described in more detail below.
"Increased avidity" was in particular observed when 20 % of the antibodies used had been antigen affinity purified, even more increase was observed when 50 % of the an- tibodies had been antigen affinity purified and even more when more than 75 %, like 75 to 100% of the antibodies had been obtained by antigen affinity purification methods.
For affinity purification of (for example avian) polyclonal anti-human CD4, CD8 or CD14 antibodies a suitable human CD4, CD8 or CD14 affinity column has to be prepared. Purified human CD4, CD8 or CD14 is fixed by a standard protocol to a suitable solid support as for example are Sepharose or Affi-Gel, activated to covalently bind the anti- gen to the support (suitable activated solid supports are for example available from Pierce, Rockford, USA). An affinity column is then prepared from said antigen-carrying resin. Successful affinity purification of antibody depends on effective presentation of the relevant epitopes on the antigen to binding sites of the antibody. If the antigen is small and immobilized directly to a solid support surface by multiple chemical bonds, important epitopes may be blocked or sterically hindered, prohibiting effective antibody binding. Therefore, it is best to immobilize antigens using a unique functional group (e.g., sulfhydryl on a single terminal cysteine in a peptide) and to use an activated support whose reactive groups occur on spacer arms that are several atoms long. For larger antigens, especially those with multiple sites of immobilization, the spacer arm length becomes less important since the antigen itself serves as an effective spacer between the support matrix and the epitope.
Little variation normally exists among typical binding and elution conditions for affinity purification of antibodies because at the core of each procedure is the affinity of an antibody for its respective antigen. Since antibodies are designed to recognize and bind antigens tightly under physiological conditions, most affinity purification proce- dures use binding conditions that mimic physiological pH and ionic strength. The most common binding buffers are phosphate buffered saline (PBS) and Tris buffered saline (TBS) at pH 7.2 and 150 mM NaCI (premixed buffer packs are for example available from Pierce, Rockford, USA). Once the antibody has been bound to an immobilized antigen, additional binding buffer is used to wash unbound material from the support. To minimize non-specific binding, the wash buffer may contain additional salt or detergent to disrupt any weak interactions.
Specific, purified antibodies are eluted from an affinity resin by altering the pH and/or ionic strength of the buffer (common elution buffers are for example available from Pierce, Rockford, USA). Antibodies in general are resilient proteins that tolerate a range of pH from 2.5 to 1 1 .5 with minimal loss of activity, and this is by far the most common elution strategy. In some cases, an antibody-antigen interaction is not efficiently disrupted by pH changes or is damaged by the pH, requiring that an alternate strategy has to be employed.
An example for an affinity purification protocol is given below: Step 1 : Wash the column (-1 ml resin bed) to remove residual protein before each use using 10 column volumes of the following sequence of buffers:
1 . 0.2 M glycine, pH 2.8 -10 ml
2. 0.1 M PBS, pH 7,2, 0.15 M NaCI -10 ml
3. Repeat the cycle with the above buffers twice. Then, equilibrate the column in the same PBS buffer with -5 ml
Step 2: Centrifuge 10 ml of crude antibody preparation to remove precipitates.
Step 3: Apply the crude antibody preparation to the column using a slow flow rate. Step 4: Wash the column extensively with 10 ml of 0.1 M PBS, pH 7,2, 0.15 M NaCI Step 5: Elute the antibody using 3 ml 0.15 M Ammonium Hydroxide, pH 10,5 ±0,2. Collect fractions into adequate tubes. Read the A2so of each fraction using an appropriate blank (i.e., 0.15 M Ammonium Hydroxide, pH 10,5 ± 0,2).
Step 6: Pool the appropriate fractions. Get an A2so of the pools and let the antibodies maturate at room temperature for a maximum of 2 weeks. If the antibodies are to be use immediately after maturation, follow the coating procedure. If contrary, the antibodies should be dialyzed against PBS containing a preservative, such as NaN3 or Proclin 950, and stored at 4°C.
Step 7: At the end, the column must be washed extensively with PBS containing a pre- servative, such as NaN3 or Proclin 950.
C.1.2 Monoclonal antibodies
Polyclonal antibodies are often more preferred than monoclonal antibodies in particle- enhanced assays. Polyclonal antibodies, contrary to monoclonals, are inherently reactive to many different epitopes on the antigens (or analytes), and therefore more easily create cross-bindings and networks between the antigens molecules per se, and between the antigens and the particles to which the antibodies are immobilized. In contrast, monoclonal antibodies generally bind to one type of epitopes only, which makes it more difficult to form cross-bindings and networks. The diagnostic industry often prefers, however, the use of monoclonal antibodies, because they are easier to standardized and to quality control to a predefined standard, especially over a product life-time of many years. Cocktails of different monoclonal antibodies, especially when they are composed of many different monoclonal antibodies with high affinity to CD4, CD8 or CD14, will result in good embodiments of the present invention. Monoclonal anti-human CD4, CD8 or CD14 antibodies also can be prepared by methods well known in the art, as for example those described by G. Kohler at al., 1975, Nature 256, 495, G. Galfre et al., 1981 , Meth. Enzymol. 73, 3-46, or R. Kennet, 1980, in: "Hybridomas: a new dimension in biological analysis", ed. R. Kennet et al., Plenum press, New York & London. Spleen cells or peripheral blood cells from immunized mice or rats are fused with a myeloma cell line, using for instance the polyethylene fusion method. After fusion the cells are grown under suitable conditions, for example on culture plates and a selection of correctly fused cells is performed using for example the hypoxanthine/ aminopterin/ thymidine (HAT) selection method. Antibody producing cell lines are identified by methods such as EIAs, RIAs or agglutination assays. After identification of the antibody producing cell line, the cells are repeatedly sub-cloned, as for example by the method of limited dilution, to guarantee that the new growing cell line derives from one single cell. C.1.3 Chimeric antibodies
Chimeric anti-human CD4, CD8 or CD14 antibodies can be obtained by methods well known in the art such as that described by G. L. Boulianne et al., 1984, Nature 312, 643-645. The procedure can be briefly described as follows. The DNA of the antigen- binding site from a monoclonal antibody of one species or parts thereof are transferred to the DNA of the antibody framework of another antibody of a different species. This new construct is cloned into an expression vector, which is transferred to the corresponding expression system to produce the antibody. C.1.4 Recombinant antibodies
Recombinant anti-human CD4, CD8 or CD14 antibodies can be obtained without using animal vehicles by methods known in the art, such as those described by G. Winter et al., 1991 , Nature, 349, 293 or J. S. Huston et al., 1988, Proc. Ntl. Acad. Sci. USA, 85, 5879. Those methods involve the following steps: introduction of DNA (cDNA or synthetic DNA) coding for an antibody or fragments thereof into a host cell, for example E. coli, fungi, yeast, plants or eukaryotic cells, selection of antibodies with the desired specificity and affinity and expressing the antibody or fragment thereof in the corresponding expression system.
C.1.5 Antibody fragments (fAbs) Fragments as described herein above, like Fab-, and F(ab')2 -fragments of polyclonal antibodies, monoclonal antibodies of any species (including chimeric antibodies and or recombinant antibodies) can be prepared by methods well known in the art, such as those described for example by A. Nissonoff et al., 1960, Arch Biochem Biophys, 89, 230, or R. P. Porter, 1959, Biochem J, 73, 1 19, or E. Harlow et al, 1988, in "Antibodies- -A Laboratory Manual", 626-631 , Cold Spring Harbour Press, New York, USA.
C1.6 Polymeric Antibodies
The preparation of polymeric multifunctional antibodies is well known in the art. A suitable method is for example described in EP-A-0 957 363.
C.2 Particles (latex particles) and their conjugates with antibody
Such particles are either applied in the step of agglutinating DBCs are applied for the detection of the cell surface markers M1.
The material for preparing the particles as used in the invention may be any natural or synthetic, inorganic, organic, non-polymer or polymer material suitable for generating and performing particle-enhanced light scattering assays. Such materials include for example selenium, carbon, gold; nitrides of carbon, silicon or germanium, for example Si3N4; oxides of iron, titanium or silicion, for example Ti02 or Si02 ; and polymeric materials such as for example, polystyrene, polyvinyl chloride), epoxy resins, poly(vinylidene chloride), poly(alpha-naphtyl methacrylate), poly(vinylnaphtalene), or copolymers thereof, in particular copolymers of styrene and a copolymerizable eth- ylenically unsaturated compound, for example styrene-(meth)acrylate co-polymers. Particles made of polymeric materials, as well as core-shell particles consisting of an inner core polymerized from styrene and an outer shell formed by copolymerization from styrene with a copolymerizable, ethylenically unsaturated compound, as described for example in U.S. Pat. No. 4,210,723, are also suitable.
Suitable polymeric particles for conjugation can be purchased from Bangs Particles Inc. or Interfacial Dynamics Inc, Merck SA, France, or other suitable sources. The particles can be activated for binding to antibodies according to numerous methods, a thorough teaching of such coupling chemistry can be found, e.g. in TechNote 205, Rev. 003, for example March, 2002, "Covalent Coupling" (incorporated by reference) which can be downloaded from Bangs Laboratories, Inc.'s web-site. For example, coupling may be achieved by means of particles carrying on their surface carboxyl-, amino-, hydroxyl-, hydrazide- or chloromethyl groups. The molecule to be coupled may either react direct- ly with such groups or by means of a suitable linker, as for example carbodiimides, glutaraldehyde, or cyanogen bromide.
For detection purposes of markers (M1 ) the particles (conjugated with a suitable anti M1 -antibody may be further modified by the attachment of a detectable marker, like a fluorophore or a chromophore. Corresponding particles are commercially available or may be produces by suitable preparative methods well known in the art (see for example: Site-specific labelling of proteins using cyanine dye reporters described in CA 2493309 A1 ; Protein specific fluorescent microspheres for labelling a protein described in US 4326008 A; or Protein specific fluorescent microspheres for labelling a protein described in US 4326008 A).
C.3 Performing the method of the invention and equipment therefore C.3.1 Devices
Suitable devices are also disclosed in co-pending EP application, application number EP16180938.9 by Gentian AS, which document is herewith incorporated by reference.
A non-limiting example of a simple device for performing a vertical flow assay of the present invention is shown in Figure 1 . Figure 1 is a vertical section of such a device illustrating in particular the sequence of different layers of filter and adsorbent materials required for performing the assay.
In an upper square disc layer 101 a central circular aperture 102 is provided. Under- neath the said square disc on its lower surface, a thin layer 103 of glue is provided in order to fix a circular piece of a filter 104 with a suitable pore size, to the lower side of said disc layer 101 , with its center in the middle of the central aperture 102 of said disc. The glue layer 103 also fixes to the lower side of the said disc 101 a square absorbent pad 105 of about the same size as that of the disc 101 . In the central hole 102 of the disc 101 , on top of the underlying filter 104, a disc of a suitable net filter 106, attached to a ring 108 is inserted into the central aperture and is removably fastened to the up- per side of disc 101 by means of an adhesive tape 107 fixed to the upper side of the ring 108. In tape 107 a central aperture is formed which allows adding the sample to be analyzed, and washing reagents on top of the net filter 106. Filter 106 may be removed from the device after sample addition and washing is completed by pulling off the tape 107. Washing buffer and further reagents may then be added to the remaining "opened" device through aperture 102 directly onto filter 104. The test result (as for example a color reaction, may be visually inspected and further analyzed through said aperture 102. Figures 2, 3 and 4 illustrate another non-limiting embodiment of a vertical flow assay device.
As can be taken from Figure 2, the assay device comprises an upper casing element 1 and a lower casing element 2. The upper casing element 1 has a first opening 3, in the depicted case a sample feed opening, and a second opening 4, in the depicted case a reading opening 4. The upper 1 and the lower casing element 2 are assembled on top of each other. The assembly comprising the upper 1 and lower casing element 2 has the shape of a flat round disc, i.e. the radius of the resulting assembly is larger than the thickness of the disc.
In an optional variant of this embodiment, a card 10 is provided with a hole 10a, which is suited to take up the assembled assay device. Particularly, the shape of the hole 10a of the card 10 is formed in such a way that it is suited to interlock with at least one portion of the lower casing element 2. The hole 10a may also comprise a notch, which is suited to hold the lower casing element 2 in place and to prevent it from a rotation with respect to the card 10.
In another variant of this embodiment, an explanatory imprint may be provided on the card 10, e.g., instructions for the use of the assay device or information to facilitate the quantification of measurements using the assay device, as for example reference colored spots as explained above.
With reference to Figure 4, a cross-section of the assay device according to Figure 2 is described. The upper 1 and lower casing element 2 comprise an upper 1 a and a lower testing compartment inner surface 2a, which are facing each other and extend essentially in parallel to each other. The upper 1 and lower casing element 2 are furthermore formed in such a way that a testing compartment is formed between them. The upper 1 a and lower testing compartment inner surface 2a form the top and bottom surfaces of a cylindrical testing compartment.
The testing compartment is provided with an upper membrane layer 6 and a lower absorbent layer 7, which are arranged on top of each other and extend essentially in par- allel to the upper 1 a and the lower testing compartment inner surface 2a. In this embodiment, the testing compartment is essentially filled out by the upper membrane layer 6 and the lower absorbent layer 7, i.e. said layers as inserted in form-locking manner. In further embodiments, the upper membrane layer 6 is spaced apart from the upper testing compartment inner surface 1 a while still being inserted in the lower testing compartment in form-locking manner.
If appropriate, in line with the description of the particular embodiment of Figures 9 to 1 1 , below, a movable wedge (or shutter) comprising a flat piece of essentially non- transparent material acting as an (preferably) optical shield or filter may be provided, which is pushed in between membrane layer 6 and absorbent layer 7 when the upper part 1 of the casing is rotated from its first (sample feed) into its second (reading) configuration. In the latter position said wedge is then positioned in the area under the second opening 4, thereby preventing background signals originating from material washed into the absorbent layer 7 to be detected through opening 4.
The second, lower testing chamber inner surface 2a is provided with a protrusion 2b that is suited to hold the lower absorbent layer 7 in place by restricting its mobility, in particular by inhibiting any mobility during the rotational movement of the assay device during the assay procedure, particularly by completely avoiding rotational movement inside the testing compartment. In other embodiments of the invention, the protrusion 2b further extends into the testing compartment and is suited to also hold the upper membrane layer 6 in place. In other embodiments, the lower absorbent layer and/or the upper membrane layer are kept in place alternatively or additionally by other attachment means, e.g., by glue. The assembly further comprises a filter layer 5, which in the depicted embodiment is arranged inside a recession 5a of the upper testing compartment inner surface 1 a right below the first opening 3. The filter layer 5 is attached to the upper casing element 1 , particularly to restrict its motion with respect to the upper casing element 1 . In the de- picted case, the filter 5 is glued to the upper casing element 1 such that the first opening 3 is covered on the side facing the testing compartment.
In this embodiment, the second opening 4 of the upper casing element 1 serves primarily as a reading opening 4, wherein the second opening offers direct optical access from outside through the upper casing element 1 to the testing compartment and an unobstructed view of the upper membrane layer 6. The second opening is also used for the optional addition of reagent solutions and the addition of washing solutions on top of the membrane layer carrying the analyte (like particular blood cells) retained on the surface of said membrane layer 6.
In this embodiment, the lower absorbent layer 7 comprises an absorbent material for taking up lower molecular substances and liquid which are not retained by the upper membrane 6. The upper membrane layer 6 comprises a semi-permeable membrane retaining the analyte, in particular blood cells suspected to carrying the analyte in said cells, or preferably, on the cell surface, and retaining the analyte, in particular blood cells carrying the analyte on the cell surface, and bound to the detection reagent, like a labelled detection antibody. Furthermore, the filter layer 5 comprises a semi-permeable membrane or preferably grid, permeable for non-agglutinated blood cells and smaller constituents of the sample, while retaining larger agglomerates of blood cell which have to be removed before the analytical detection reaction on the surface of the upper membrane is finally performed.
The assembly of the upper 1 and lower casing element 2 comprises an interlocking mechanism in which the upper casing element 1 takes up a portion of the lower casing element 2. Due to the round shape of the interlocking portions of the upper 1 and lower casing element 2, the upper 1 and lower casing element 2 may be rotated with respect to each other, wherein a rotational angle defines a position of the two casing elements 1 , 2 to each other. Latches 12 are provided on the interlocking portion of the lower casing element 2, which are suited to hold the assembly of the upper 1 and lower casing element 2 firmly in place and leave essentially only a rotational degree of freedom for motion of the casing elements 1 , 2 relative to each other. Furthermore, latches 13 are provided on a portion of the lower casing element 2 interlocking with the hole 10a in the card 10 as shown in Figure 3. The latches 12, 13 may be formed in different ways, as a person skilled in the art will appreciate. Furthermore, corresponding grooves are formed in the upper casing element 1 corresponding to the latches 12 of the lower casing element. Similar structures may be formed in the card 10 in order to facilitate the interlocking action with the lower casing element 2.
In Figure 5 a top view of another non-limiting embodiment of an assay device is depicted. The configuration of the assay device is analogous to the structures described above with reference to Figures 2 to 4. For simplification purposes, the lower casing element 2 is not depicted in Figure 5 except for the rotation stop 22. The upper casing element 1 is provided with two rotation stops 21 , which engage with the rotation stop 22 of the lower casing element 2 in the first and second configuration, respectively. Herein, the first configuration of the assay device is shown and the second configuration can be reached by rotating the upper casing element 1 anticlockwise with respect to the lower casing element 2 towards the second extreme rotation angle that is defined by the rotation stops 21 , 22.
A first pair 3, 4 and a second pair 3', 4' of first 3, 3' and second openings 4, 4' are formed in the upper casing element 1 , wherein the first openings 3, 3' are provided with ridges 3a, 3a' around their respective circumference. Also, the filter layers 5, 5' that extend across the first openings 3, 3' on the bottom side of the upper casing element 1 are shown by a hatching inside the first openings 3, 3'. The pairs of openings 3, 4, 3', 4' are arranged in such a manner that, in the second configuration (after rotation), the positions of the second openings 4, 4' relative to the lower casing element, which is represented by its rotation stop 22, will be essentially the same as the positions of the first openings 3, 3' in the first configuration.
Furthermore, the label 1 1 is arranged on the top surface of the upper casing element 1 and the explanation imprint 1 1 b is visible to a user of the assay device. Also, circum- ferential imprints 4a, 4a' are provided in the label 1 1 around the second openings 4, 4'. Different hatching of the circumferential imprints 4a, 4a' illustrate the differences in col- oring that, e.g., help the user to easily differentiate the individual second openings 4, 4' from each other or give a reference color for the interpretation of a colorimetric read-out of the assay. Another embodiment of the assay device according to the invention is shown in Figures 9 to 1 1. Figures 9 and 10 show the assay device in a perspective exploded view from the top and from the bottom, respectively. Figure 1 1 shows a cross-section of the assay device along an axis indicated by the letter "A" in Figures 9 and 1 1 . The terms "upper" and "lower" relate to the directions in the drawings.
The assay device comprises an upper casing element 31 and a lower casing element 32. The upper element 31 has a first opening 33, in the depicted case a sample feed opening 33, a second opening 34, in the depicted case a washing (or color or signal development) opening 34, and a third opening 35, in the depicted case a measurement (or reading) opening 35. The sample feed opening 33 is provided with a ridge 33a, similar to the one 3a described in the previous embodiment (Figure 5). Also, a filter layer 43 is arranged below the sample feed opening 33 on the inner surface 31 a of the upper casing element 31 , covering the sample feed opening 33 such that any sample passing through the opening 33 will have to pass the filter layer. The filter layer 43 is not covering the second 34 or third opening 35 as to allow free access to parts of the device located below and/or unhindered sight from the top of the device through the second 34 and third opening 35.
Furthermore, the upper casing element 31 is provided with an upper groove 39 extend- ing in a circular arch around the center R of the casing element 31 and an upper guiding pin 38a on its inner surface 31 a. Complementary to the upper groove 39 and the upper guiding pin 38a, the upper surface 32b of the lower casing element 32 is provided with a lower groove 38 extending in a circular arch around the center of the casing element 32 and a lower guiding pin 39a, which are configured such that they may en- gage with the upper guiding pin 38a and the upper groove 39, respectively. The center of the casing elements 31 and 32 define the rotation axis of the assembled device during the rotational movement of the upper 31 and lower casing element 32 relatively to each other. When the upper 31 and lower casing element 32 of the assay device are assembled, the upper guiding pin 38a engages with the lower groove 38 and may slide along it upon rotation of the upper 31 and lower casing element 32 relatively to each other. In an analog fashion, the lower guiding pin 39a engages with and may slide along the upper groove 39 upon rotation. Thus, the rotational movement of the upper 31 and lower casing element 32 relatively to each other is restricted and guided by the movement of the upper 38a and lower guiding pin 39a in the lower 38 and upper groove 39, respectively. Different mechanisms to ensure a secure rotational movement may be utilized in further embodiments. In particular, features of the previously described embodiments may be used to secure the assembly of the upper 31 and lower casing element 32, and/or stoppers may be provided to define certain rotational angles.
The lower casing element 32 comprises a recess, which is in the present case cuboid, suitable to act as a testing compartment similar to the one formed by the upper 1 and lower casing elements 2 of the previously described embodiment. The recess has a lower testing compartment inner surface 32a, which in the present case is quadratic. The compartment formed by the recess takes up the lower absorbent layer 37. Above the lower absorbent layer 37, a membrane element 36 is arranged. In the present em- bodiment, the membrane element 36 is formed as a stripe, which is fixed to the upper surface 32b of the lower casing element 32, spanning the whole width of the recess. In particular, the membrane element 36 is configured such that it is held in a fixed position relatively to the lower casing element 32, even when the upper 31 and lower casing elements 32 are rotated relatively to each other.
In analogy to the embodiments of Figures 2, 4 and 5 described previously, the openings 33, 34, 35 are arranged within the upper casing element 31 such that a rotation relatively to the lower casing element 32 will lead to different configurations at defined rotation angles, here for example 90°, where one of the openings 33, 34, 35 will take the same position relatively to the lower casing element 32 as another opening 33, 34, 35 will take in another configuration. However, in contrast to the previous embodiment, one more (third) opening 35 is provided in addition to the first 33 and second opening 34. Thus, three different configurations of the device are defined by a certain reference position relatively to the lower casing element 32, as for example a specific section of the membrane element 36, which may be taken by the first 33, second 34 and third opening 35, respectively. Herein after, the "sample feed configuration" will be referred to as a first configuration (opening 33 is positioned immediately above the reference position on the membrane element 36) ; the "washing configuration" will be referred to as the second configuration (opening 34 is positioned immediately above the reference position on the membrane element 36); and "measurement configuration" will be re- ferred to as the third configuration (opening 35 is positioned immediately above the reference position on the membrane element 36).
In the first, sample feed configuration, the sample feed opening 33 is positioned above the membrane element 36 and the lower absorbent layer 37. As described above, a filter layer 43 is fixed to the upper casing element 31 , spanning the area of the sample feed opening 33. Thus, sample constituents of a sample which are smaller than the mesh size of the filter 43, added through the sample feed opening 33 will pass through the filter layer 43 and then reach the membrane element 36. Sample constituents for which the membrane element 36 is permeable, will be absorbed by the lower absorbent layer 37. Constituents of bigger size are retained on top of the membrane element 36.
To reach the second, washing configuration, the upper casing element 31 is rotated, here by an angle of 90° with respect to the lower casing element 32 such that the washing opening 34 is positioned above the same position of the lower casing element 32 as the sample feed opening 33 had taken in the sample feed configuration. Thus, the membrane element 36 is accessible through the washing opening 34, for example for adding a washing buffer, which may penetrate through the membrane element 36 by capillary suction towards the lower absorbent layer 37.
Finally, to reach the third, measurement configuration, the rotation is continued, here again for an angle of 90°, until the measurement opening 35 is positioned above the same spot of the membrane element 36 as the washing opening 34 had taken in the washing configuration. To reduce the intensity of background signal caused by material that has penetrated membrane 36 and that is embedded in the absorbent layer 37, the present embodiment provides a "shutter" 40 that is shielding the signal from the lower absorbent layer 37 from the measurement opening 35. To this end, the lower casing element 32 further comprises a wedge (which may also be designated as shutter) 40, comprising a flat piece of essentially non-transparent material. The wedge 40 is rotatably mounted on the lower guiding pin 39a, which thereby also serves as a rotational axis 39a for the wedge 40. A portion of the wedge 40 is arranged such that it is partially covering the lower groove 38 of the lower casing element 32. Thus, when the upper 31 and lower casing elements 32 of the assembled assay device are rotated with respect to each other, the upper guiding pin 38a slides along the lower groove 38 and will, at certain rotation angles, reach the wedge 40 and pushes it out of the area of the lower groove 38. Thus, the wedge 40 may be rotated around its rotational axis 39a. Essentially, two states are defined for the arrangement of the wedge 40: In the first (sample feed) and second (washing) configuration of the assay device, the wedge 40 is arranged in such a way that only a small portion of the wedge 40 is arranged between the membrane element 36 and the lower absorbent layer 37. In particular, the wedge 40 is not positioned in the area under the first 33 and second opening 34, respectively. At the same time, a portion of the wedge 40 is covering the lower groove 38.
To reach the measurement configuration of the assay device, the upper 31 and lower casing elements 32 of the assembled assay device are further rotated with respect to each other. Thus, the upper guiding pin 38a will slide along the lower groove 38 and, after a certain rotational angle has been reached, pushes the wedge 40 out of its way. The rotation of the wedge 40 is configured such that a portion of the wedge 40 is pushed in between the lower absorbent layer 37 and the membrane element 36. When the measurement configuration is reached, the wedge 40 is spanning the whole area that is covered by the measurement opening 35 with respect to the membrane element 36. This measurement configuration is shown in the cross-section of Figure 1 1. Thus, any signal originating from the lower absorbent layer 37 is prevented from being transmitted through the membrane layer 36 and from being detected during the measurement through the measurement opening 35. The wedge 40 may be made of different materials, for example polymer materials or plastics. Whatever material is used, it is in a preferred embodiment, essentially non- transparent for background signals that are to be expected from the lower absorbent layer 37, which might disturb the assessment of the analyte to be determined. It another embodiment the wedge 40 may also be reflective for the desired signal, stemming from the membrane element. For example, the wedge 40 may be white or at least reflective for a range of wavelengths that are emitted by a marker compound that is used in the assay. Therefore, the noise from washed-out markers in the lower absorbent layer 37 is reflected away from the measurement opening 35, while the desired signal from marker material on the membrane element 36 is reflected towards the measurement opening 35. The wedge 40 may therefore both prevent background noise and amplify the signal to be measured and also the signal to noise ratio.
The upper casing element 31 further comprises corner elements 42a, 42b, 42c, 42d that are arranged at its upper surface. The upper 31 and the lower casing element 32 may be assembled on top of each other and the assembled assay device is suitable to be inserted into a reading device (not shown). A suitable commercially available reading devices is for example the Optricon Cube reflectance reader manufactured by opTricon, Berlin, Germany. Herein, the corner elements 42a, 42b, 42c, 42d are suita- ble to guide the assembly upon insertion of the cube onto the device of the invention and ensure a predefined insertion position of the assembly. To this end, at least one of the corner elements 42a, 42b, 42c, 42d may be formed asymmetrically with regard to the others, such that the insertion direction is unambiguously defined. In further embodiments of the invention, the assay device of the invention may comprise other structural features acting as equivalent guiding elements, as an alternative to or acting in combination with the corner elements 42a, 42b, 42c, 42d of the present embodiment. C.3.2 Performing the assay method of the invention C.3.2.1 CD4 assessment
This embodiment refers to the assessment of CD4 receptors on CD4 + lymphocytes, in particular T helper cells.
In said embodiment of the invention, the labelled antibody is reactive to the CD4 receptor, and said label is constituted by an enzyme or colored or fluorescent particle. If an enzyme is used as label, a preferred embodiment is characterized by the subsequent exposure to the said filter of a substrate forming a colored substance, preferentially a precipitating colored substance, or a fluorescent or a chemiluminescent substance.
The correlation between color or fluorescence or chemiluminescence generated in the method of the present invention and the concentration of said class of receptor mole- cules, can be performed as follows: There is a direct relationship between the amount of the said specific receptor molecules and the color to be measured, since the amount of colored particles bound relates to the amount of said specific receptor molecules present in the sample to be tested. This color is then detectable either visually with comparison to pre-evaluated, pre-calibrated and/or predetermined coloristic diagrams or by measurement of the amount of color by electronic color detectors either freely available on the market or the one developed for the present invention. Corresponding fluorescent or chemiluminescent methods can be used.
Measurement instruments used are easily calibrated and adjusted to colored substances or immunoparticles used, their color scheme and detection range needed. In calibration for detection instruments a known amount of analyte is used, giving a good ratio of background vs. signal, and will allow users to be provided with exact calculated readouts.
If an enzyme - including but not limited to peroxidase enzymes or alkaline phosphatase - is used in the place of colored or fluorescent substances, a color generating a chemiluminescence generating or a fluorescent generating substrate for said enzymes are used. Measurements of two components with different color deposited on a filter by measurement of reflectance at two and more wavelengths is well known to the skilled man of the art. It was already described in Clinical Chemistry 43:12 2390 -2396 (1997) in the article "Glycohemoglobin filter assay for doctors' offices based on boronic acid affinity principle" by Frank Frantzen et al., in US 5,702,952 by Erling Sundrehagen and Frank Frantzen, and in US 5,506,144 by Sundrehagen and Frantzen. Frantzen et al used a specialized reflectometer measuring reflectance (%R) at 620 and 470 nm. Measurements at these wavelengths were used to quantitate the blue-colored boronic acid conjugate and red hemoglobin (Hb), respectively. The instrument automatically performed Kubelka-Munk transformations (Kubelka P. New contributions to the optics of intensely light scattering materials. J Opt Soc Am 1948;38:448-57) to linearize the recorded reflectance data. A "Portable rapid diagnostic test reader" is described in EP 2 812 675 and a "Spectroscopic sensor on mobile phone" is described in US 2006/0279732. Today the camera function on the mobile phone is commonly used for reflectometric measurements of filter based test devices in diagnostic medicine.
Such systems are also described in EP 0 953 149 (B1 ) by Sundrehagen and Bremnes. Many companies today deliver reflectometric scanning instruments or digital camera imaging software for measuring intensity and wavelength of reflected light from test spots on diagnostic devices, comprising software for calibration for computing the con- centration on samples from intensity and wavelength of reflected light. The Scansmart system from Skannex AS, Oslo, Norway, is an example of an automated and dedicated system for this application, which has been sold for reflectometry spot intensity measurement for vertical flow tests to customers in Norway. Also standard "smartphones" with digital cameras can be used to obtain digital images of the color signal obtained. Typically, the digital images are then uploaded into Adobe Photoshop electronic program. This method allows graphic presentation of the results. This method also allows the determination of when the signal is strongest versus when the background is lowest. Standardization and calibration of the signals can be obtained by using reference spots with known intensity and concentration of the analyte to be measured.
If enzymatic color system generation is used, for example based on color development or luminescence, like i.p., fluorescence or chemiluminescence, then kinetic measurements can be employed, and the measurement can be performed using a "video" mode.
The software Adobe Photoshop Elements 13© and the program "Eyedropper tool" to determine HSL and Red, Green and Blue and other color schemes to determine color of uploaded images. The HSL (hue, saturation and lightness) scheme provides a de- vice-independent way to describe color. Especially instructive is http://www.handprint.com/LS/CVS/color.html on the internet. (July 2015).
In a special embodiment of the present invention, reference colored spots are placed or fastened in close proximity to the membrane with immobilized antibodies or bother binding molecules or fragments thereof, preferentially on the holder of the assay membrane. As a part of the measurements of the assay of the present inventions, these reference spots are measured as well. The measurement of said reference spot can - by the software of the measurement instrument, be used to compensate for instrument- to-instrument and other hardware variations, to increase the overall accuracy of the assay.
These reference spots may define color scale for each color in the analytical measurement. The instrument, e.g. the camera on a mobile telephone take a picture or a series of pictures of the surface to be measured, and also the reference spots on the device. Different software programs can convert the pixels measured into numeric values and define color rooms in different numeric system. Very common is the RGB (Red Green Blue) color room. The RGB color model is an additive color model in which red, green, and blue light are added together in various ways to reproduce a broad array of colors. The name of the model comes from the initials of the three additive primary colors, red, green, and blue. (Wikipedia 16 July 2016)
HSL and HSV are the two most common cylindrical-coordinate representations of points in an RGB color model. The two representations rearrange the geometry of RGB in an attempt to be more intuitive and perceptually relevant than the cartesian (cube) representation. Developed in the 1970s for computer graphics applications, HSL and HSV are used today in color pickers, in image editing software, and less commonly in image analysis and computer vision.
A very modern and free software package in use today to measure and analyse color spots and give them numerical values in a color room, is GIMP. GIMP /gimp/ (GNU Image Manipulation Program) is a free and open-source raster graphics editor used for image retouching and editing, free-form drawing, resizing, cropping, photo-montages, converting between different image formats, and more specialized tasks. See www.gimp.org, where all aspects are explained. The result is reported in the number of CD4-lymphocyt.es and CD8-lymphocyt.es per volume unit and/or as a ratio between the two numbers.
The CD4 assessment may be performed with a simple device as depicted in Figure 1 , as follows:
1 . A whole blood sample was mixed with dilution buffer adapted to cell lysis, as for example, but not limited to hypotonic lysis, of erythrocytes as contained in the sample while not lysing the leucocytes. The dilution buffer also contains anti CD14 antibody in a form suitable to agglomerate CD14 monocytes, and it con- tains anti-human CD4 receptor antibodies with detectable marker (like enzyme, colored particles) for example, but not necessarily limited to, in a form which do not or essentially not agglomerate CD4 cells.
2. After a short incubation time an aliquot of said mixture was transferred to the aperture 102 of the device of Figure 1 , and is immediately sucked into the coarse (nylon mesh) filter 106 inserted into said aperture and retaining agglutinated CD14 cells while letting pass through CD4 + T-helper cells (n free or antibody bound but non-agglutinated form).
3. Thereafter, a wash solution was transferred to the aperture 102 of the filtration device and was sucked into the nylon mesh filter 106 and the filter 104 positioned below 106.
4. Thereafter, the nylon mesh filter 106 was removed from the device.
5. Thereafter, if an enzyme is used as marker, the corresponding substrate was transferred to the hole 102 of the filtration device and was sucked into the filter 104.
6. A defined time (as for example 5 minutes) thereafter, the color developed was measured, as for example reflectometrically using a SkanSmart CE reader with software delivered by Skannex AS, Norway.
7. The reading was compared to a calibration curve stored in the software generated by calibration samples with known content of CD4+ lymphocytes, analyzed in identical experiments, and the content of CD4+ lymphocytes was calculated.
Corresponding methods using luminescent, like i.p. fluorescent and chemiluminescent marker molecules can be used.
The CD4 assessment may be performed with a more advanced device as depicted in Figures 2, 3 and 4 as follows:
1 . A whole blood sample was mixed with dilution buffer adapted to hypotonic lysis of erythrocytes as contained in the sample while not lysing the leucocytes. The dilution buffer also contains anti CD14 antibody in a form suitable to agglomerate CD14 monocytes, and it contains anti-human CD4 receptor antibodies with detectable marker (like enzyme, colored particles) for example, but not necessarily limited to, in a form which do not or essentially not agglomerate CD4 cells.
2. After a short incubation time an aliquot of said mixture was transferred to the aperture 3 of the device of Figure 4, and is immediately sucked into the coarse (ny- Ion mesh) filter 5 positioned underneath said aperture 3 and retaining agglutinated CD14 cells while letting pass through CD4 + T helper cells (in free or antibody bound, but non-agglutinated form) which will be retained on the surface of the next filter layer 6.
3. Thereafter, a wash solution was transferred to the aperture 3 of the filtration de- vice and was sucked into the nylon mesh filter 5, and the filter 6. 4. Thereafter, the nylon mesh filter 5 was removed by performing twisting the upper casing element 1 of the device (angle of rotation about 180 °) so that opening 4 is now exactly in the previous position of opening 3 relative to the filter 6, i.e. the section of the filter where said CD4+ helper cells are adsorbed on the filter.
5. Thereafter, if an enzyme is used as marker, the corresponding substrate was transferred to the hole 4 of the filtration device and was sucked into the filter 6.
6. A defined time (as for example 5 minutes) thereafter, the color developed was measured, as for example reflectometrically using a SkanSmart CE reader with software delivered by Skannex AS, Norway.
7. The reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated. If the detectable marker is for example a colored particle, then step 5 and the color development according to step 6 is not of course not necessary.
The CD4 assessment as described above for the more advanced device as depicted in Figures 2, 3 and 4, may in analogy also be performed with a device depicted in Figure 5 where two blood samples may be assessed simultaneously. The angle of rotation of the uppercasing element 1 is in this case about 90 °.
C.3.2.2 CD4 and CD8 assessment The assessment may be performed in analogy to the assessment of CD4, as described above by applying a device of Figure 1 or a device as depicted in Figures 2 to 4 comprised the following steps:
Steps 4 and 6 may be performed in identical manner.
In Step 1 a suspension anti-CD4 antibodies and anti-CD8 antibodies conjugated each conjugated to different, distinguishable markers, as for example latex particles of different color (like to red carboxylated latex and blue carboxylated latex) was provided. Immediately thereafter, the color on the filter 104 or 6 was measured reflectometrically using a standard Apple i-Phone telephone and its inbuilt flash-light. Simultaneously two for example red (a weak and a strong) and two for example blue color dots (a weak and a strong) (depending on the color of the latex particles) for example, are also placed on top of the device was depictured. For all five spots, the BGR file obtained (see above) was used (by converting the files to gray scale) the place and the limits of the dots were decided. By the GIMP program (see above), all the pixels were transformed to HSV color values. The maximum and the minimum responses with respect to the two blue color dots defined the blue color room, and the maximum and the minimum responses with respect to the two red color dots defined the red color room. The HSV value from the test spot (with both red and blue articles) was then interpolated into the red and the blue HSV color rooms, and HSV values for all pixels were calculated and normalized.
The obtained normalized values were then compared to the values obtained with the calibrating samples of known CD4 and CD8 positive lymphocytes (who had also been analyzed with for example a conventional Becton Dickinson Excalibur Flow Cytometry system), which had been stored in the calibrating file of the computer in the i-Phone system, and the results were reported on the display and in the electronic output. The result is reported in numbers per volume unit CD4-lymphocyt.es, CD8-lymphocyt.es per volume unit and as a ratio between the two.
The CD4 and CD8 assessment as described above for the more advanced device as depicted in Figures 2, 3 and 4, may in analogy also be performed with a device depicted in Figure 5 where two blood samples, one for CD4 and the other for CD8, may be assessed simultaneously in different pairs of openings (3,4 and 3', 4'). The angle of rotation of the uppercasing element 1 is in this case about 90 °.
1 . Two identical aliquots of whole blood sample was mixed with dilution buffer adapted to hypotonic lysis of erythrocytes as contained in the sample while not lysing the leucocytes. The dilution buffer also contains anti CD14 antibody in a form suitable to agglomerate CD14 monocytes. One aliquot of the sample also contained anti-human CD4 receptor antibodies with detectable marker (like im- munoparticles of a first color or enzyme). The other aliquot of the sample also contained anti-human CD8 receptor antibodies with detectable marker (like im- munoparticles of a second color or enzyme) 2. After a short incubation time an aliquot of each of said two mixtures was transferred to the respective aperture 3 and 3' of the device of Figure 5, and is immediately sucked into the coarse (nylon mesh) filter 5 positioned underneath said aperture 3, 3' and retaining agglutinated CD14 cells while letting pass through CD4 + and CD8 + cells which will be retained on the surface of the next filter layer 6. (as for the CD8 assessment CD14 cell would not disturb the assay the filter 5 might also be omitted for the CD8 sample opening).
3. Thereafter, a wash solution was transferred to the apertures 3, 3' of the filtration device and was sucked into the nylon mesh filter 5, and the filter 6.
4. Thereafter, the nylon mesh filter 5 was removed by performing twisting the upper casing element 1 of the device (angle of rotation about 90 °) so that openings 4, 4' are now exactly in the previous position of openings 3, 3' relative to the filter 6, i.e. the section of the filter where said CD4+ helper cells are adsorbed on the filter.
The further steps (color development in the case of an enzyme as marker) and measurement of colored spots may be performed as described above.
The following non-limiting examples further illustrate the present invention. Based on said teaching a person of ordinary skill in the art will be able to provide, without the need of undue experimentation or inventive effort, further embodiments of the invention.
Experimental Part
Unless otherwise stated all reagents and chemical compounds as used herein are of analytical grade.
Preparation Example 1 : Preparation of Nitrocellulose Filters with a mean pore size 3, 5 and 8 Mm
Whatman nitrocellulose filter (catalogue no 7193-002 for 3 μηη pore size, cat no. 7195- 004 for 5 μηη pore size, and 104001 12 for 8 μηη pore size.)were soaked for 30 minutes in SuperBlock T20 (TBS) Blocking Buffer (Thermo Scientific, prod. No. 37536).. This blocking procedure was performed to avoid unspecific binding of protein and cells to the filters when later used in the vertical filtration devices. Preferably, a 20 μηι nylon net filter (Millipore prod. No. NY3002500) can be supported in the periphery by a ring of polystyrene or another stiffer material, since the nylon net filter is a fluffy material. The said stiffer material should be glued, e.g. by Clearsol Cas- co glue, or melted to the nylon net filter to hinder liquids from leaking in between the ring and the nylon net filter (see filter (106) and ring (108) in Fig.1 as described in Preparation Example 7, below).
Preparation Example 2: Preparation of polyclonal antibodies anti-human CD14 receptor antibodies from chicken eggs
Human CD14 receptor, tailor-made from Novoprotein Inc, US, with the following amino acid sequence (SEQ ID NO:1 ) MNHKV HMELDDEDFR CVCNFSEPQP DWSEAFQCVS AVEVEIHAGG LNLEPFLKRV 61
DADADPRQYA DTVKALRVRR LTVGAAQVPA QLLVGALRVL AYSRLKELTL EDLKITGTMP 121
PLPLEATGLA LSSLRLRNVS WATGRSWLAE LQQWLKPGLK VLSIAQAHSP AFSCEQVRAF 181
PALTSLDLSD NPGLGERGLM AALCPHKFPA IQNLALRNTG METPTGVCAA LAAAGVQPHS 241
LDLSHNSLRA TVNPSAPRCM WSSALNSLNL SFAGLEQVPK GLPAKLRVLD LSCNRLNRAP 301
QPDELPEVDN LTLDGNPFLV PG was suspended in Freund's complete adjuvants (FCA.
Polyclonal anti human CD14 antibodies can be prepared by methods well known in the art, such as those described for example by Chase, M. W., 1967,. in "Methods of Immunology and Immunochemistry", ed. Williams, A. et al., M. W., pp. 197-209, Academic Press, New York. Briefly, animals of a suitable species (for example rabbits, goats, or sheep, or, preferably avian species, in particular poultry, like hens) are repetitively immunized with purified antigen in an appropriate adjuvant, for example Freund's com- plete or incomplete adjuvant. After immunization the animals are bled and the polyclonal antibodies are purified by methods such as for example ammonium sulfate or ammonium chloride precipitation, anionic exchange chromatography, immunoaffinity chromatography, and/or affinity chromatography. Polyclonal avian antibodies routinely are obtained from egg yolk (and are therefore designated IgYs). Egg yolk, however, contains large amounts of lipids making their further use problematic. IgY can be isolated from egg yolk by using stepwise ammoni- um sulphate (for example 25 to 40 %) and polyethylene glycol (PEG) precipitation. For initial purification also commercial IgY purification kits obtainable from Gallus Immu- notch Inc, Cary, USA, or the Eggcellent Chicken IgY Purification Kit, obtainable from Pierce, Rockford, USA may also be employed considering the manufacturer's instruc- tions.
Furthermore, the avidity of polyclonal antibodies may be further increased by using antibodies that ware purified by the use of antigen affinity purification methods, for example according to the teaching in "Affinity Purification of Proteins" downloaded from www.piercenet.com (April 2006) and incorporated by reference Affinity purification is described in more detail below.
Two to four hens were used for each immunization experiment. 0,1 mg peptide dissolved in 1 ml water was emulsified with equal volume of Freund's complete adjuvant and injected into the breast muscle of hens. The injection was repeated every 4 weeks. 10 weeks after the start of the injections, eggs were collected. The egg yolk was isolated from the eggs, and the IgY fraction from the egg yolk was isolated by ammonium chloride precipitation, in a conventional manner according to prior art methods of egg antibody isolation (see for example Larsson A, Baaloew R-M, Lindahl T, and Forsberg P-0 in Poultry Science 72:1807-1812, 1993). Further immunizations were performed every four weeks. After 10 weeks, eggs were collected and the egg yolk was isolated manually from the egg white. The total antibody fraction (the IgY fraction) from the egg yolk was isolated by ammonium chloride precipitation, in a conventional manner according to prior art methods of egg antibody isolation (see for example Larsson A, Baaloew R-M, Lindahl T, and Forsberg P-0 in Poultry Science 72:1807-1812, 1993).
10 mg of highly pure human CD14 receptor was then immobilized on a HITRAP NHS- Active HP column from Amersham Pharmacia Biotech, following the prescription in the package insert of the column. The IgY fraction isolated from egg yolk was diluted to 2 mg/ml in phosphate buffered saline. 200 ml of this IgY solution was passed through the column, followed by 50 ml phosphate buffered saline with no IgY. The antibodies with specific affinity for the immobilized CD14 receptor was eluted with 35 ml of 0.1 M citrate buffer pH = 3.0. The eluted specific anti- CD14 antibodies were dialyzed against phosphate buffered saline and concentrated to 3 mg/ml using an Amicon Centricon centrifugation filtration device with molecular weight cut-off of 30.000 Dalton. Preparation Example 3: Conjugation of anti-human CD14 antibodies to carboxylated polystyrene particles 15 μηι carboxylated polystyrene particles (2500 μη-iol/g COOH) were purchased from Microparticles GmbG, Germany. 31 mg of chicken anti-human CD14 antibodies, prepared according to Preparation Example 2 above, were dialyzed to 20 ml buffer (pH = 9.5, 5 mM borate, 7.5 mM sodium chloride). 4500 mg of said carboxylated polystyrene particles were washed by centrifugation and suspended in 20 ml water. 12,5 mg EDC (1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (Sigma, US) was dissolved in the particle suspension. The antibody solution was added to the latex suspension, and stirred for 5 hours. The particles in the suspension were then washed 4 times with 1 mM NaCI, 0,5 mM sodium borate, 0,025 % Tween 20, 0,5 mM glycine, pH = 9,5. This stock solution was then diluted 1 :3 in 30mM borate buffer (pH 9,1 -9,3 with 150mM sodium chlo- ride, 0,1 % Tween 20, 0,5 mg/ml PSA (porcine serum albumin) and 0,1 % ProClin™ 950 biocide).
Preparation Example 4: Polymerization of anti-CD14 antibodies 10 mg of anti CD14 IgY antibodies, prepared as described in Preparation Example 2 above, was added dropwise 1 ml of a PBS solution of 4 mg of dithiobis (sulfosuccin- imidyl propionate) (manufactured by Pierce Corp., hereinafter referred to as DTSSP) with stirring at room temperature. After stirring the mixed solution at 35°C for 30 minutes, the mixed solution was filtered through a Sepharose gel (manufactured by Pharmacia Fine Chemical Inc., Sephadex G25M column). This gave approximately 6 ml of the PBS solution containing IgY polymer (hereinafter referred to as IgYagg. This procedure was in analogy to the polymerization procedure described in EP-A-0 957 363. Preparation Example 5: Sample dilution buffer containing anti-CD14 conjugated beads and Alkaline phosphate enzyme conjugated monoclonal mouse anti- human CD4 receptor antibodies
ACK lysing buffer (155 mM ammonium chloride 10 mM photassium bicarbonate, 1 mM EDTA, pH >=7.0 to <=7.6) have been used for lysis of erythrocytes without lysis of leucocytes. Commercially available anti-CD14-coated beads, 32 μηη CD14 S-pluriBeads® anti- human (PluriSelect, prod. No. 19-01400-10), was added to said buffer solution at an amount (7,5 <105 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the present invention. If, for example, 20 μΙ whole blood will be analyzed, and a volume of 400 μΙ of sample dilution buffer shall be used, an amount of anti-CD14 antibodies which will bind all monocytes of 20 μΙ blood must be present in 400 μΙ of the sample dilution buffer. A titration of the amount necessary can be done by setting up a dilution series of said antibodies in 400 μΙ samples of dilution buffer, and test by applying the method described in reference example 1 . In the same buffer, Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti-CD4) (EDU- 2 clone of monoclonal anti-human CD4 receptor, Diatec AS, Oslo, Norway) was added. The amount ALP-anti-CD4 will be adjusted to result in sufficient binding to CD4+ lym- phocytes in the presence of free CD4 receptors and CD4 bound to monocytes. Typically the concentration is between 5 and 30 μg ml.
Preparation Example 6: Washing buffer A solution of 0,14 M sodium chloride, 1 g/l of Tween 20 (Sigma), 0,01 M of 2-amino-2- hydroxymethyl-propane-1 ,3-diol (Sigma), 1 gram bovine serum albumin (Sigma) per liter and 1 g/l of Proclin 300, pH adjusted to 7.4 was prepared.
Preparation Example 7: A vertical flow assay device similar to that according to Figure 1
A vertical filtration device was formed around a 0,20 mm thick square polystyrene disc measuring 22 x 22 mm (101 ). In the center of the polystyrene disc a 5 mm circular hole (102) is punched out with a standard punching instrument.
Underneath the circular hole of the said square polystyrene disc, a circular piece of nylon net filter, 15, 20 or 30 μη"ΐ,(106) is attached by melting. Underneath the nylon net filter a piece of a nitrocellulose (104) with a mean pore size of 3, 5 or 8 μηι, prepared according to Preparation Example 1 above, having a diameter of 10 mm, is placed, covering the circular hole of the polystyrene disc with the nylon net filter in between.
Thereafter, underneath the nitrocellulose filter(104), the said polystyrene disc was cov- ered by a CF7 absorbent pad with an area of about 360 mm2 (105) (100% cotton linter material) from GE Health Care Life Sciences.
In an alternative embodiment in the hole of the polystyrene disc (102), on top of the underlying nitrocellulose filter, a 5 mm diameter disc of a nylon net filter (106), may be fastened to the polystyrene disc by the use of Clearseal Casco glue and a ring of polystyrene (108) and a 22 x 10 mm piece of adhesive tape (107), with a central aperture of 3 mm in the adhesive tape (107).
Reference Example 1 : Blood Cell analysis by means of automated cell sorting
1. Materials and Equipment
• Sample dilution buffer prepared according to Preparation Example 5
• ACK lysing buffer
• Anti-CD14-conjugated beads such as CD14 pluribeads or anti-CD14-polymer · Dynabeads CD14 (product no. 1 1 149D, Life Technologies)
• Blood
• Rocking table
• Vortex
• Centrifuge
· Nylon net filter 15 μηη (pluriStrainer 15μηι (Cell Strainer) product no. 43-50015-
03, Pluriselect)
• Nylon net filter 20 μηι (pluriStrainer 20μηι (Cell Strainer) product no. 43-50020- 03, Pluriselect)
• Nylon net filter 30 μηη mounted in a filtration device (product no. NY3002500, Millipore)
• ACK Lysing Buffer (155 mM Ammonium Chloride, 10 mM Potassium Bicarbonate and 1 mM EDTA, pH >=7.0 to <=7.6)
• PBS (Phosphate buffered saline, Gibco® PBS tablets pH 7.4, product number 18912-014, Thermo Fisher Scientific or equivalent) • Alexa Fluor® 647 Mouse Anti-Human CD14, product number 562690, BD Biosciences
• DAPI-staining reagent (NucBlue™ Live Cell Stain ReadyProbes™ Reagent, product number R37605, Thermo Fisher Scientific)
· Countess® II FL automated cell counter, product number AMQAF1000, Thermo
Fisher Scientific
o EVOS® Light Cube, CY 5®, product number AMEP4656, Thermo Fisher Scientific
o EVOS® Light Cube, DAPI, product number AMEP4650, Thermo Fisher Scientific
• Countess™ cell counting chamber slides, product number C10283, Thermo Fisher Scientific
• 1 .5-2 ml microcentrifuge tubes
• Pipettes and tips
2. Sample preparation and measurements
The blood is incubated in sample preparation buffer prepared according to Preparation Example 5. Control samples are prepared in ACK lysing buffer, and in ACK lysing buff- er with pluribeads only. The buffers can also be prepared with other anti-CD14- conjugated beads or anti-CD14-polymer. Another control sample were prepared in ACK lysing buffer and incubated with CD14-Dynabeads in an amount sufficient to bind most monocytes after removal by magnetic separation. The samples were mixed on a rocking table during incubation. After incubation, the samples were filtrated through 15 μηη, 20 μηη or 30 μηη nylon net filters to remove particle bound monocytes. The monocytes bound to dynabeads in the positive control are removed by magnetic separation. After monocyte elimination, 700 μΙ is removed from each sample and prepared for Countess® II FL measurements according to the following protocol:
• Centrifuge the 700 μΙ samples at 100xg for 10 minutes.
· Withdraw 550 μΙ of the supernatant, carefully not to touch the pellet. 150 μΙ sample now remains in each tube.
• Add 1 ml ACK lysis buffer to each sample to lyse remaining whole red blood cells and wash the samples. Mix all samples for a few seconds using a vortex and let stand for 3 minutes for lysing to occur.
· Centrifuge the samples at 100xg for 10 minutes. • Withdraw 1 ml of the supernatant, carefully not to touch the pellet and mix all samples by vortexing for 30 seconds.
• Add 10 μΙ Alexa Fluor® 647 Mouse Anti-Human CD14 to each sample and mix by pipetting.
· Incubate the samples covered with aluminium foil or another opaque material for 30 minutes with slight agitation to allow for antibody binding.
• Wash the samples by adding 1 ml PBS and mix by inverting the tubes.
• Centrifuge the samples at 10Oxg for 10 minutes.
• Withdraw 1010 μΙ of the supernatant. 150 μΙ sample now remains in each tube. · Mix all samples by vortexing for 30 seconds.
• Add 50 μΙ DAPI-staining reagent to each sample and mix by pipetting. All samples now contain 200 μΙ.
• Incubate the samples covered with aluminium foil or another opaque material for 15 minutes with slight agitation to allow for binding.
· Keep the samples covered and under slight agitation while performing the Countess® II FL measurements.
• To measure a sample in the Countess® II FL load the Countess™ cell counting chamber slide with 10 μΙ sample by first removing one sample tube from under the covering, inverting it a few times, withdraw 10 μΙ sample and load the cell counting chamber. Make sure the whole chamber is covered with sample prior to analysis.
o When analysing the dynabeads positive control sample hold a magnet against the tube for 30 seconds right after mixing to remove remaining dynabeads. Thereafter withdraw 10 μΙ sample and load the cell counting chamber.
The samples were analysed for number of monocytes (Alexa Fluor® 647 Mouse Anti- Human CD14 stained cells) and total number of leucocytes (DAPI stained cells, DAPI stains all cells with a nucleus). All samples were analysed in triplicate using Countess® II FL automated cell counter. The settings were adjusted to minimize the background for each light source (bright field, CY 5 and DAPI). After capturing a picture the intensity settings were adjusted to encompass all visible cells. It was also determined that all cells measured as CY 5-positive (Alexa Fluor® 647 Mouse Anti-Human CD14 stained monocytes) had a DAPI-stained nucleus and was measured as DAPI-cells.
Assay Example 1 : A vertical flow CD4 assay using Alkaline Phosphatase (ALP)- antiCD4 conjugates The purpose of the experiments described in this example is to evaluate different parameters in the CDcard (as depicted in Figure 3 equipped with an assay device as depicted in Figure 5) for a simplified test setup of the present invention. The main purpose is to verify that all steps of the simplified test method works. Examples of suitable rang- es for mesh size of nylon filter, pore size of nitrocellulose filter and concentration of ALP-antiCD4 in lysis buffer will be determined. These parameters will be varied. Anti- CD14 bead concentration, incubation time, blocking of nitrocellulose, absorbent size will be fixed parameters but may be further varied if necessary. The results for monocyte removal will be evaluated using Countess II FL (see Reference Example 1 above)and the results for CD4-cell staining in the CD-card prototype.
1. Materials and Equipment
• ACK lysing buffer (155 mM Ammonium Chloride, 10 mM Potassium Bicar- bonate and 1 mM EDTA, pH >=7.0 to <=7.6)
• Venous blood
• Commercially available anti-CD14-coated beads, 32 μηη (2,5x106 beads/ml) CD14 S-pluriBeads® anti-human (PluriSelect, prod. No. 19-01400-10)
• Alkaline phosphatase enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti-CD4) (0,5 mg/ml in TBS with 50% glycerol, EDU-
2 clone of monoclonal anti-human CD4 receptor, Diatec AS, Oslo, Norway)
• Nylon net filter 15 μηη (pluriStrainer 15μηι (Cell Strainer) product no. 43-50015- 03, Pluriselect)
• Nylon net filter 20 μηι (pluriStrainer 20μηι (Cell Strainer) product no. 43-50020- 03, Pluriselect)
• Nylon net filter 30 μηη mounted in a filtration device (product no. NY3002500, Millipore)
• Nylon net filter 20 μηη mounted in the CDcard test device (product no NY2000010, Millipore)
· CDcard test devices
• Whatman nitrocellulose filter 3 μηη, 5 μηη, and 8 μηη (catalogue no 7193-002 for
3 μηη pore size, cat no. 7195-004 for 5 μηη pore size, and 104001 12 for 8 μηη pore size. cat. No ),
• SuperBlock T20 (TBS) Blocking Buffer (Thermo Scientific, prod. No. 37536). • Washing buffer: 0,14 M sodium chloride, 1 g/l of Tween 20 (Sigma), 0,01 M of 2- amino-2-hydroxymethyl-propane-1 ,3-diol (TRIS)(Sigma), 1 g/l bovine serum albumin (Sigma), 1 g/l of Proclin 300, pH adjusted to 7.4
• Centrifuge
· Absorbent pad cut for CDcard device (360 mm2/sample)
• Seramun Purple S-008-NBT liquid enzyme substrate Seramun GmbH
• General lab glassware and consumables
• 0,1 M HCI for stopping the color development (optional)
• AP StabilPLUS for diluting ALP-antiCD4 and applying after sample (no incuba- tion)
• Countess II FL Automated Cell Counter (product no AMQAF1000 Thermo Fisher Scientific (Life technologies) and other equipment and chemicals for cell counting (appendix 1 for details) 2. Removal of monocytes
2.1 Removal of monocytes in ACK lysing solution with anti-CD14 pluribeads and ALP-anti-CD4 at different concentrations
In the following sections all samples were prepared in ACK lysing buffer.
2.2 Experimental: Pluribead concentration of 2,5*105beads/ml
The sample preparation buffers used in the experiment is described in Table 1 .
Table 1: Sample preparation buffers for 2,5x105 beads/ml
Figure imgf000069_0001
Before adding ALP-anti-CD4, the anti-CD14 pluribeads were washed once with ACK lysing buffer. All other components (Table 1 ) were added to the samples and incubation was performed on a rocking table for 30 minutes. The samples were filtered through a 20 μηη nylon mesh filter (pluristrainer) and further prepared for analysis using Countess II FL (see Reference Example 1 ). 2.3. Results: Pluribead concentration of 2,5*105beads/ml
The results for samples prepared according to Table 1 evaluated for monocyte removal using Countess II FL are shown in Fig.6. The result show that 0 μg ml ALP-anti-CD4 efficiently remove monocytes (less than 20 % remains), 10 μg ml ALP-anti-CD4 in incubation decrease the efficiency and slightly above 40 % remain, For 20 and 30 μg ml ALP-anti-CD4 most monocytes remain in solution after filtration. The ALP-anti-CD4 obviously interferes with the pluribeads binding but this could be compensated by increasing the bead concentration,
2.4 Experimental: Pluribead concentration of 7,5*105beads/ml
In order to increase the efficiency of removal the pluribeads concentration was increased to 7,5x105beads/ml. The other sample details were identical as in Table 1 . The sample preparation buffers used in the experiment is described in Table 2. Table 2: Sample preparation buffers for Pluribeads concentration of 7,5*105beads/ml
Figure imgf000070_0001
Before adding ALP-anti-CD4, the anti-CD14 pluribeads were washed once with ACK lysing buffer. All other components (Table 2) were added to the samples and incubation was performed on a rocking table for 30 minutes.
The samples were filtered through a 20 μηη nylon mesh filter (pluristrainer) and further prepared for analysis using Countess II FL (see Reference Example 1 )
2.5 Results: Pluribead concentration of 7,5*105beads/ml
The results for samples prepared according to Table 2 evaluated for monocyte removal using Countess II FL are shown in Figure 7. The result show that an efficient removal is achieved for all samples with 7,5x105 pluribeads/ml and that the removal is comparable with the positive control prepared with anti-CD14-dynabeads and magnetic separation. All ALP-anti-CD4 concentrations result in similar efficiency of removal as for the sample with no added ALP-antiCD4. The same samples 2, 3, and 4 were analysed for CD4 staining in the CD card after 60 minutes incubation (section 3.2 below).
2.6 Experimental: Pluribead concentration of 7,5x105 beads/ml and different mesh size for nylon net filter The bead concentration of 7,5x105beads/ml was selected as sufficient to remove most monocytes in the samples. In the next step different mesh sizes of the nylon net filter were evaluated. All samples were prepared using the ALP-anti-CD4 concentration of 10 μg ml. The sample preparation buffers used in the experiment is described in Table
Table 3: sample preparation for Pluribead concentration of 7,5x105beads/ml and different mesh size for nylon net filter
Figure imgf000071_0001
Before adding ALP-anti-CD4, the anti-CD14 pluribeads were washed once with ACK lysing buffer. All other components (Table 3) were added to the samples and incubation was performed on a rocking table for 30 minutes.
The samples were filtered through the nylon filter specified in Table 3 and further pre- pared for analysis using Countess II FL (see Reference Example 1 )
2.7 Results: Pluribead concentration of 7,5x105 beads/ml and different mesh size for nylon net filter The results for samples prepared according to Table 3 evaluated for monocyte removal using Countess II FL are shown in Figure 8. The result show that all filter mesh sizes (15, 20 and 30 μηη) result in a comparable removal of monocytes. The same samples 2, 3, and 4 were tested for CD-4 staining in CDcard and sample 3 was tested for CD4- staining in CDcard on different Nitrocellulose filter pore sizes (section 3.4 and 3.6 below)
3. Evaluation of CD4-cell staining
In all tests the nitrocellulose filter was blocked with superblock T20 (TBS) for 30 minutes and dried in air at RT.
3.1 Experimental: Evaluation of CD4-cell staining samples prepared with 7,5x105beads/ml and different ALP-anti-CD4 concentrations
The blood was mixed with sample buffer including anti-CD14 pluribeads at a concentration of 7,5x105beads/ml and ALP-anti-CD4 at concentration of 10, 20 or 30 μς/ιτιΙ I ACK lysing buffer. The samples were incubated for 60 minutes before CD card analy- sis. The control plasma samples were prepared with the same concentration of ALP- anti-CD4 concentration but no pluribeads. The plasma samples were not incubated in this experiment but prepared just before use. The blood samples that were used are the same as in table 2 above but the samples were tested in CDcard after 60 minutes. The samples are described in Table 4. All samples are filtered in the test described in 0 and 2.5 and filtered again through the 20 μηη nylon filter melted on the CDcard. For all samples 5 μηη nitrocellulose filters were used.
Table 4: samples tested for CD4 staining using 7,5x105beads/ml and different ALP- anti-CD4 concentrations in incubation buffer.
Figure imgf000073_0001
The test was performed as follows:
The prototype for CDcard with two separate sample application sites was used. In this prototype a 20 μηη nylon net filter is attached by melting underneath the sample appli- cation well. After sample application and washing, the top lid is rotated 90° to remove the nylon filter and allow the color development to be performed directly on the nitrocellulose filter. The blood samples were always applied to the left and plasma control to the right. The sample information was written on the card and the sample no. indicated on the reading site.
Test:
1 . Apply 100 μΙ sample prepared in sample buffer and incubated for the desired time
2. Wash with 200 μΙ washing buffer
3. Rotate the top part of the CDcard 90° to remove the nylon net filter
4. Apply 20 μΙ Seramun Purple S-008-NBT liquid enzyme substrate or one of the substrates creating fluorescent or chemiluminescent molecules, e.g.
CSPD™ Substrate (0.25 mM Ready-To-Use) with Nitro-Block™ Enhancer 5. Read the signal for 10 minutes. Optionally, stop the reaction after the desired time by addition of e.g. 0, 1 M HCI.
3.2 Results: Evaluation of CD4-cell staining samples prepared with 7,5x105beads/ml and different ALP-anti-CD4 concentrations
The staining of blood incubated with ALP-anti-CD4 at concentrations of 10, 20 or 30 μg result in similar CD4-staining in CDcard. The staining after 5 minutes color development and optionally stopping the reaction with 0,1 M HCI was evaluated (Results not shown)
There is a good contrast between blood and plasma for all samples and for all samples it was shown that the monocytes were removed (Figure 7). The staining for plasma is slightly higher compared to the tests described below in section 3.3, 3.4 and 3.5, 3.6 below. Incubation of plasma with anti-CD14 pluribeads and ALP-antiCD4 result in less background compared to a sample that is mixed right before use. Also noteworthy, is that the staining after 60 minutes incubation with ALP-anti-CD4, result in significantly stronger staining when compared to after 30 minutes incubation as in section 3.3, 3.4 and 3.5, 3.6, below.
3.3 Experimental: Evaluation of CD4-cell staining samples prepared with 7,5x105beads/ml, 10 Mg/ml ALP-anti-CD4 varying the mesh size of the nylon net filter. The blood was mixed with sample buffer including anti-CD14 pluribeads at a concentration of 7,5x105beads/ml and ALP-anti-CD4 at concentration of 10 μg ml in ACK lys- ing buffer. The samples were incubated for 30 minutes and filtered through nylon net filters of different mesh size before CD card analysis. The control plasma samples were prepared with the same concentration of ALP-anti-CD4 and pluribeads concentration as the blood samples but was filtered through 20 μηη nylon net filter only. The samples are described in Table 5. The samples were only filtered once and there was no filter in the CDcard test device but filtration was done before applying the sample to the test card. The test was performed according to section 3.1 with the difference that the 20 μηη nylon net filter was removed. For all samples 5 μηη nitrocellulose filters were used. Table 5: Samples tested in CD card using 7,5*105beads/ml and, 10 ng/ml ALP-anti- CD4 in the incubation buffer but varying the mesh size of the nylon net filter.
Figure imgf000075_0001
3.4 Results: Evaluation of CD4-cell staining samples prepared with 7,5*105beads/ml, 10 Mg/ml ALP-anti-CD4 varying the mesh size of the nylon net filter.
The staining of blood and plasma incubated with 7,5x105 CD14 pluribeads/ml and 10 μg/ml ALP-anti-CD4 for 30 minutes followed by filtration using nylon net filters of different mesh size was investigated.
The staining after 5 minutes color development and stopping the reaction with 0,1 M HCI was evaluated (Results not shown). There is a good contrast between blood and plasma for all samples and for all samples it was also shown that the monocytes were removed (Figure 8). The CD4-staining for the sample filtered through 30 μηη nylon net filter is slightly stronger compared to 20 μηη and 15 μηι. 3.5 Experimental: Evaluation of CD4-cell staining samples prepared with 7,5x105beads/ml, 10 Mg/ml ALP-anti-CD4, filtered through 20 Mm nylon net filter and varying the pore size of the nitrocellulose in the CDcard. The blood was mixed with sample buffer including anti-CD14 pluribeads at a concentration of 7,5x105 beads/ml and ALP-anti-CD4 at concentration of 10 μg ml in ACK lys- ing buffer. The samples were incubated for 30 minutes and filtered through a 20 μηη nylon net filter. The samples were applied to the CDcard test device with a nylon net filter (20 μηη) and with varying pore sizes for the nitrocellulose filter (3, 5 or 8 μηη). The main purpose was to investigate the level of staining of CD4 cells as well as staining for plasma sample. A higher staining for plasma samples with decreasing pore size would be a sign of antibody mediated aggregation of free CD4 that is stuck to the nitrocellulose filter and contributes to the staining. The control plasma samples were prepared in an identical manner as the corresponding blood samples. The samples are described in Table 6. The samples were filtered through the nylon filter twice, before cell counting and in the CDcard test device.
Table 6: Samples tested in CD card using 7,5*105beads/ml and, 10 ng/ml ALP-anti- CD4 in the incubation buffer, 20 μηι nylon net filter and varying the pore size of the nitrocellulose (NC) in the CDcard.
Sample Blood/ Pluribeads ALP-anti- Incubation Nylon net NC filter Blood/plasma no plasma cone. CD4 cone. time filter mesh pore + lysis buffer size volume (pi)
1.1 blood 7,5x10° 10 pg/ml 30 min 2x20 pm 3 pm 50 pi + 1000 beads/ml pi buffer
1.2 plasma 7,5x10° 10 pg/ml 30 min 2x20 pm 3 pm 50 pi + 1000 beads/ml pi buffer
2.1 blood 7,5x10° 10 pg/ml 30 min 2x20 pm 5 pm 50 pi + 1000 beads/ml pi buffer
2.2 plasma 7,5x10° 10 pg/ml 30 min 2x20 pm 5 pm 50 pi + 1000 beads/ml pi buffer
3.1 blood 7,5x10° 10 pg/ml 30 min 2x20 pm 8 pm 50 pi + 1000 beads/ml pi buffer
3.2 plasma 7,5x10° 10 pg/ml 30 min 2x20 pm 8 pm 50 pi + 1000 beads/ml pi buffer 3.6 Results: Evaluation of CD4-cell staining samples prepared with 7,5x105beads/ml, 10 Mg/ml ALP-anti-CD4, filtered through 20 μιτι nylon net filter and varying the pore size of the nitrocellulose in the CDcard. The staining of blood and plasma incubated with 7,5x105 CD14 pluribeads/ml and 10 μg ml ALP-anti-CD4 for 30 minutes followed by filtration using a 20 μηη nylon net filter and application to the CDcard test device including a 20 μηη nylon net filter and varying pore sizes for the nitrocellulose filters was investigated. The staining after 5 minutes color development (Serum Purple Prec) and stopping the reaction with 0,1 M HCI was evaluated (Results not shown).
There is a good contrast between blood and plasma for all samples and there is almost no staining for the plasma controls. Application of the sample on 3, 5 or 8 μηη nitrocellu- lose filters result in similar staining. There is no increase in staining for the plasma controls with decreasing pore size and therefore no indication of formation of large aggregates of ALP-anti-CD4 and free CD4 that disturbs the measurement.
Assay Example 2: A vertical flow CD4 assay using enzyme immunoconjugates
The assay performed with a device of Figure 1 comprised the following steps:
1 . A whole blood sample of 25 μΙ was mixed with 500 μΙ dilution buffer according to
Preparation Example 5 above.
2. The sample was incubated for 30 minutes allowing lysis of erythrocytes, binding of anti-CD14 beads to monocytes and of ALP-anti-CD4 to CD4+ cells and free
CD4 receptor. 30 minutes thereafter, 100 μΙ of said mixture was applied on top of the nylon mesh filter in the filtration device according to Preparation Example 7 above, and is immediately sucked into the device.
3. Thereafter, 200 μΙ of wash solution according to Preparation Example 6 above was transferred to nylon mesh of the filtration device according to Preparation
Example 7 above, and was sucked into the device.
4. Thereafter, the nylon mesh filter (106) was removed.
5. Thereafter, 20 μΙ of Seramun Purple S-008-NBT liquid enzyme substrate Sera- mun GmbH was transferred to the hole of the polystyrene disc (102) of the filtra- tion device according to Preparation Example 7 above, and was sucked into the nitrocellulose filter (104) and color developed for 5 minutes.
6. Five minutes thereafter, the color development was stopped by washing with 100 μΙ 0,1 M HCI and the color development was evaluated reflectometrically using a SkanSmart CE reader with software delivered by Skannex AS, Norway.
7. The reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated.
Alternative procedure:
In order to avoid the use of time-consuming enzymatic signal generation, the said anti- human CD4 antibody can be coupled to a colored substance or a luminescent, i.p. fluo- rescent or chemiluminescent substance which can be read immediately after step 4, above. Colored immunoparticles comprise antibodies or immunoreactive fragments thereof and particulate materials exhibiting a color or luminescence, like i.p. fluorescence. The particulate material can be coupled to the said antibodies or fragments by physical absorption or covalent coupling, often with a spacer or bridging molecules. The colored material may be constituted - but is not limited - to pigments in or on latex particles or polymer particles, which can be made from many different materials, or metal colloids like gold colloids or ferric colloids or carbon particles. Fluorescent particles like Bangs Particles product code FCEU001/FC02F can also be used. Such colored and fluorescent particles are described in the prior art and are well known by the skilled man of the art. They are typically purchased from suppliers like Merck France, Life technologies (US) and/or Bangs Laboratories (US). Polymer particles are supplied in all sizes and colors, also as fluorescent particles. The size and color intensity of the particles must be adjusted to the sensitivity and the capacity needed for the assay methods, as well as to the pore size of the membranes used in the product of the in- vention.
As mentioned above, the present invention employs a membrane for capturing a specific group of cells, and the measurement of receptors associated with said groups of cells. Depending on the size of the cells or the specific group of cells, the said mem- brane has a pore size adapted to capture said group of cells, and allowing other smaller particles to pass through the said membrane. In one embodiment of the present in- vention, the specific group of cells are T lymphocytes, and a suitable pore size of said membrane for capturing the T-cells are membranes with an average pore size 1 -10 μηη, preferentially 3-9 μηη, and even more preferred 3-5 μηη, allowing smaller particulate materials contained in the sample material to pass through the membrane. In preferred embodiments of the present invention, the present invention employs colored immunoparticles in a size generating a good signal from the colored immunoparticles, but small enough to pass through the said membrane which captures the said specific group of cells. Typically, the present invention employs immunoparticles sized from 60 to 400 nm, more preferred from 80 to 300 nm, even more preferred 95 to 200 nm in diameter.
Preparation Example 8: Anti-CD4 antibodies conjugated to blue carboxylated latex In one embodiment of the present invention, blue carboxylated latex particles from Mil- lipore, Europe (Prod. No. PSI 90-91 ), with a mean diameter of 1 17 nm, were employed. 5 mg EDU-2 clone of monoclonal anti-human CD4 receptor antibodies, from Diatec AS, Norway, were dialyzed to 5 ml buffer (pH = 9.5 in 5 mM borate, 7.5 mM sodium chloride). 23,4 mg of said carboxylated blue latex particles were washed by centrifugation and are suspended in 2 ml water. 0,8 mg EDC (Sigma, US) was dissolved into the particle suspension and the antibody solution was mixed with the latex suspension, and stirred for 5 hours. The particles in the suspension were then washed 4 times with 1 mM NaCI, 0,5 mM sodium borate, 0,025 % Tween 20, 0,5 mM glycine, pH = 9,5. This stock solution was then diluted 1 :3 in 30mM borate buffer (pH 9,1 -9,3 with 150mM so- dium chloride, 0,1 % Tween 20, 0,5 mg/ml PSA and 0,1 % ProClin 950).
Assay Example 3: A vertical flow CD4 assay using blue latex immunoparticles
The assay performed with a device of Figure 1 comprised the following steps:
1 . A whole blood sample of 25 μΙ was mixed with 500 μΙ dilution buffer according to Preparation Example 5 above but prepared with anti-CD4-conjugated colored beads instead of ALP-anti-CD4 according to Preparation example 8 above. The sample was incubated for 30 minutes. 2. 30 minutes thereafter, 150 μΙ of said mixture was transferred to the nylon mesh filter of the filtration device according to Preparation Example 7 above, and is immediately sucked into the device (106).
3. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above was transferred to the nylon mesh filter and was sucked into the device.
4. Thereafter, the nylon mesh filter (106) was removed.
5. Immediately thereafter, the color on the nitrocellulose filter was measured reflec- tometrically using a SkanSmart CE reader with software delivered by Skannex AS, Norway.
6. The reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated. Preparation Example 9: Anti-CD8 antibodies conjugated to red carboxylated latex
Red carboxylated latex particles from Merck Estapor (Prod. No. 784 K1 -010) with a mean diameter of 190 nm, were employed. 5 mg of UCHT-4 clone monoclonal anti- human CD8 receptor antibodies, from Diatec AS, Norway, were dialyzed to 5 ml buffer (pH = 9.5, 5 mM borate, 7.5 mM sodium chloride). 35 mg of said carboxylated red latex particles were washed by centrifugation and suspended in 2 ml water. 0,8 mg EDC (Sigma, US) was dissolved into the particle suspension and the antibody solution was mixed with the latex suspension, and stirred for 5 hours. The particles in the suspension were then washed 4 times with 1 mM NaCI, 0,5 mM sodium borate, 0,025 % Tween 20, 0,5 mM glycine, pH = 9,5. This stock solution was then diluted 1 :3 in 30mM borate buffer pH 9,1 -9,3 with 150mM sodium chloride, 0,1 % Tween 20, 0,5 mg/ml BSA and 0,1 % ProClin 950.
Assay Example 4: A vertical flow CD4 and CD8 assay using blue and red latex immunoparticles
The assay performed with a device of Figure 1 comprised the following steps:
4. A whole blood sample of 20 μΙ was mixed with 400 μΙ dilution buffer according to Preparation Example 5 above including both anti-CD4 conjugated blue beads and anti-CD8 conjugated red beads as described in Preparation Example 9 above. The sample is incubated for 30 minutes.
5. 30 minutes thereafter, 150 μΙ of said mixture was transferred to the nylon mesh filter of the filtration device according to Preparation Example 7 above, and is immediately sucked into the device (106).
6. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above was transferred to the nylon mesh filter of the filtration device according to Preparation Example 7 above, and was sucked into the device.
7. Thereafter the nylon mesh filter (106) was removed.
8. Immediately thereafter, the color on the nitrocellulose filter was measured reflec- tometrically using a standard Apple i-Phone telephone and its inbuilt flash-light. Simultaneously two red (a weak and a strong) and two blue color dots (a weak and a strong) placed on the device was depictured. For all five spots, the BGR file obtained (see above) was used (by converting the files to gray scale) the place and the limits of the dots were decided. By the GIMP program (see above), all the pixels were transformed to HSV color values. The maximum and the minimum responses with respect to the two blue color dots defined the blue color room, and the maximum and the minimum responses with respect to the two red color dots defined the red color room.
9. The HSV value from the test spot (with both red and blue articles) was then interpolated into the red and the blue HSV color rooms, and HSV values for all pixels were calculated and normalized.
10. The obtained normalized values were then compared to the values obtained with the calibrating samples of known CD4 and CD8 positive lymphocytes (who had also been analyzed with the Becton Dickinson Excalibur Flow Cytometry system), which had been stored in the calibrating file of the computer in the i-Phone system, and the results were reported on the display and in the electronic output. The result is reported in numbers of CD4-lymphocyt.es per volume unit, number of CD8-lymphocyt.es per volume unit and as a ratio between the two.
Preparation Example 10: A vertical flow assay device similar to that according to Figure 2. A vertical filtration device according to Figures 2 and 4 was provided in shape from a 5 mm thick circular polystyrene capsule measuring 50 mm in diameter. In the top part (2) of the polystyrene capsule there are two 3 mm circular holes, one deeper (3) for sample loading and one shallow hole (4) for reading the signal. Underneath the deeper circular hole (3), a circular piece of nylon net filter (5), 15, 20 or 30 μηη mesh size, is attached by melting. Underneath the nylon net filter (5) a piece of a nitrocellulose membrane (6) with a mean pore size of 3, 5 or 8 μηι, prepared according to Preparation Example 1 above, having a diameter of about 10 mm, is placed, covering the circular hole (3) with the nylon net filter (5) in between.
Thereafter, underneath the nitrocellulose filter (6), the bottom part of the said polysty- rene capsule, was covered by a CF7 absorbent pad (7) with an area of about 360 mm2 (100% cotton linter material) from GE Health Care Life Sciences.
In an alternative embodiment (not shown in the Figures) in the hole (3) of the polystyrene capsule, on top of the underlying nitrocellulose filter (6), a 5 mm diameter disc of a nylon net filter (5), may be fastened to the inner surface of the top part of (2) of the polystyrene disc by the use of Clearseal Casco glue and a ring of polystyrene and a 22 x 10 mm piece of adhesive tape, with a central aperture of 3 mm in the adhesive tape ( corresponding to the aperture of the circular hole (3). The nylon net filter (5) attached to the top part (2) of the polystyrene capsule can be removed from the nitrocellulose membrane (6) by turning the top part (2) of the capsule in an angle of 180°.
Preparation Example 11 : A vertical flow assay device similar to that according to Figure 9 to 11.
A vertical filtration device according to Figures 9 to 1 1 was provided in the shape of a 5 mm thick circular polystyrene capsule measuring 50 mm in diameter. In the top part (31 ) of the polystyrene capsule there are three 3 mm circular holes (33), (34) and (35), one deeper for sample loading (33) and two shallower for color development (34, position 2 ) and reading the signal (35), distributed at equal distances to and at equal angels of 90° around the central rotational axis of the device. Underneath the deeper circular hole (33), a circular piece of nylon net filter (43), 15, 20 or 30 μηη mesh size, is attached by melting to the inner surface of the (31 a) of the top part (31 ). Underneath the nylon net filter (42) a piece of a nitrocellulose with a mean pore size of 3, 5 or 8 μηη (36), prepared according to Preparation Example 1 above, is placed, covering the circular hole (33) with the nylon net filter (43) in between.
Thereafter, underneath the nitrocellulose filter (36), the bottom part (32) of the said polystyrene capsule, was covered by a CF7 absorbent pad with an area of about 360 mm2 (37) (100% cotton linter material) from GE Health Care Life Sciences.
Between the nitrocellulose (36) and the absorbent (37) a thin piece or sheet of a non- transparent material (e.g. plastic or metal) (40) is included. With the polystyrene cap- sule in the position for sample application (configuration 1 ) and after rotating for a first angle of 90° the first time (to reach configuration 2) the non-transparent material (40) is placed between nitrocellulose (36) and the absorbent (37) but not underneath the sample loading well (33), or the color development well (34). After rotating the top part for an additional angle of 90° to reach the reading position (configuration 3), the non- transparent material (40) is moved into a position between the nitrocellulose (36) and the absorbent (37) and at a position underneath the reading hole (35). This movement is performed by a tap or pin (38a) located on the inner surface (31 a) of the top part (31 ) of the capsule forcing the non-transparent material (40) in place. The non-transparent material is fixed on a pivot tap or pin (39a) located on the inner surface (32a) of the bottom part (32) of the capsule. The non-transparent material (40) is now covering the absorbent underneath the reading hole (35) and blocking background signal originating from the absorbent (37).
In an alternative embodiment (not shown in the Figures) in the deeper sample loading well (33) of the polystyrene capsule, on top of the underlying nitrocellulose filter (36), a 5 mm diameter disc of a nylon net filter (43), may be fastened to the inner surface (31 a) of the top part (31 ) of the polystyrene disc by the use of Clearseal Casco glue and a ring of polystyrene and a 22 x 10 mm piece of adhesive tape, with a central aperture of 3 mm in the adhesive tape corresponding to the aperture of the circular hole (33)..
Preparation example 12: Sample incubation buffer containing anti-CD14 conjugated beads and Alkaline Phosphate enzyme conjugated monoclonal mouse an- tihuman CD4 receptor antibodies Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti- CD4) can be used as sample incubation buffer. Commercially available anti-CD14-coated beads, 32 μηη CD14 S-pluriBeads® antihu- man (PluriSelect, prod. No. 19-01400-10), were added to said buffer solution at an amount (8,3 <105 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the present invention. If, for example, 25 μΙ of whole blood will be analyzed, and a volume of 75 μΙ incubation buffer shall be used, an amount of anti-CD14 antibodies which will bind all monocytes of 25 μΙ blood must be present in 75 μΙ of the sample dilution buffer (more than 6,3x 104 beads). In the same buffer, Alkaline phosphate enzyme conjugated monoclonal mouse anti-human CD4 receptor antibodies (ALP-anti-CD4) (EDU- 2 clone of monoclonal anti-human CD4 receptor, Diatec AS, Oslo, Norway) was added. The amount ALP-anti-CD4 will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes. Typically the concentration is between 0,3 and 3,0 μg ml.
Preparation example 13: Sample incubation buffer containing anti-CD14 conjugated beads and anti-CD4 conjugated 80 nm gold particles
Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and anti-CD4-cojugated gold nanoparticles can be used as sample incubation buffer.
Commercially available anti-CD14-coated beads, 32 μηη CD14 S-pluriBeads® antihu- man (PluriSelect, prod. No. 19-01400-10), were added to said buffer solution at an amount (8,3*105 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the pre- sent invention. If, for example, 25 μΙ of whole blood will be analyzed, and a volume of 75 μΙ incubation buffer shall be used, an amount of anti-CD14 antibodies which will bind all monocytes of 25 μΙ blood must be present in 75 μΙ of the sample dilution buffer (more than 6,3x104 beads). In the same buffer, anti-CD4 conjugated gold nanoparticles according to Preparation Example 15 was added. The amount of anti-CD4 conjugated gold nanoparticles will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes. Typically, the concentration is between 1 x 1010 and 1 x 1012 particles/ml.
Preparation example 14: Sample incubation buffer containing anti-CD14 conju- gated beads and anti-CD4 conjugated 100 nm Europium particles
Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and anti-CD4-cojugated Europium nanoparticles can be used as sample incubation buffer. Commercially available anti-CD14-coated beads, 32 μηη CD14 S-pluriBeads® antihu- man (PluriSelect, prod. No. 19-01400-10), were added to said buffer solution at an amount (8,3x 105 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the present invention. If, for example, 25 μΙ of whole blood will be analyzed, and a volume of 75 μΙ incubation buffer shall be used, an amount of anti-CD14 antibodies which will bind all monocytes of 25 μΙ blood must be present in 75 μΙ of the sample dilution buffer (more than 6,3x 104 beads). In the same buffer, anti-CD4 conjugated Europium nanoparticles according to Preparation Example 16 were added. The amount of anti-CD4 conjugated Europium nanoparticles will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes. Typically, the concentration is between 1 x1010 and 1 x1012 particles/ml.
Preparation Example 15: Anti-CD4 antibodies conjugated to gold nanoparticles In one embodiment of the present invention, 80 nm gold nanoparticles from Innova Biosciences (Prod. No. SKU: 231 -0005 ), were conjugated with anti-human-CD4.
The manufacturer's instruction was followed for conjugation to EDU-2 clone of monoclonal anti-human CD4 receptor antibodies, from Diatec AS, Norway. Basically, the antibody was diluted to 0,05 mg/ml in antibody diluent (provided in the conjugation kit). 12 μΙ antibody solution was mixed with 42 μΙ reaction buffer (provided with the kit). 45 μΙ of the said solution was used to reconstitute the freeze-dried gold particles and the conjugation reaction was left for 15 minutes. After 15 minutes, 5 μΙ quencher (provided with the kit) was added to stop the conjugation reaction. Finally, the particles were washed, and unconjugated antibody removed, by adding 500 μΙ quenching solution diluted 1 :10 in ultrapure water. The particles were centrifuged for 10 minutes at 9000 g and 500 μΙ of the supernatant was withdrawn. The particles were resuspended in the buffer before use.
Preparation Example 16: Anti-CD4 antibodies conjugated to 100 nm carboxylated Europium particles.
In one embodiment of the present invention, 100 nm Europium nanoparticles from Bangs laboratories Inc. (Prod. No. FCEU001 ) were conjugated with anti-human-CD4. The manufacturer's instruction was followed for conjugation to EDU-2 clone of monoclonal anti-human CD4 receptor antibodies, from Diatec AS, Norway. The particles (1 ml_, 10 mg/mL) were washed 2 times in 10ml_ of 0,1 M MES Buffer; pH 5.7 and then resuspended in 10ml_ of the same buffer. 100mg of EDC were added to the particles and was allowed to react with the particles for 15 minutes at room temperature (18- 25°C), with continuous mixing. The activated particles were washed 2 times in 10 ml coupling buffer (0,2 M sodium borate buffer pH 8,5 i.e. 0,2 M boric acid adjusted to pH 8,5 using NaOH). After washing the particles were resuspended in 5ml_ of coupling buffer. The antibody was diluted to 5 ml using the same coupling buffer. 0.15 mg antibody result in maximum coverage on 10 mg of particles. The antibody was used in 10 times excess, i.e. 1 ,5 mg. The antibody solution and particle solution were combined, and the coupling reaction was performed for 2,5 h with constant mixing. The coupling buffer was removed by filtration and 10 ml quenching solution (Tris buffer saline, Etha- nolamine 40 mM and Ovalbumin 10 g/L, pH 7,1 ) was added. The solution was stirred overnight.
The next day, the particles were washed by filtration (1000 kD MWCO filter) and the buffer changed by washing 3 times with 10 ml storage buffer (0,01 M Tris buffer saline, pH 7,4 with 0,1 % BSA and 0,05 % tween). Finally, the particles were resuspended in 10 ml storage buffer and sonicated to remove aggregates before use. Preparation example 17: Sample incubation buffer containing anti-CD14 conjugated beads and fluorescent Alexa Fluor® 647 Mouse Anti-Human CD4
Tris buffer saline pH 7,4 (TBS) with anti-CD14-coated beads and fluorescent Alexa Fluor®647 Mouse Anti-Human CD4 (BD Biosciences Catalog No. 557707) can be used as sample incubation buffer. Commercially available anti-CD14-coated beads, 32 μηη CD14 S-pluriBeads® antihu- man (PluriSelect, prod. No. 19-01400-10), were added to said buffer solution at an amount (8,3 <105 beads/ml) sufficient to bind and aggregate all monocytes in a whole blood volume which shall be analyzed for CD4 receptors using the method of the pre- sent invention. If, for example, 25 μΙ whole blood will be analyzed, and a volume of 75 μΙ incubation buffer shall be used, an amount of anti-CD14 antibodies which will bind all monocytes of 25 μΙ blood must be present in 75 μΙ of the sample dilution buffer (more than 6,3*104 beads). In the same buffer, fluorescent Alexa Fluor®647 Mouse Anti- Human CD4 (BD Biosciences Catalog No. 557707) was added. The amount of fluores- cent Alexa Fluor®647 Mouse Anti-Human will be adjusted to result in sufficient binding to CD4+ lymphocytes in the presence of free CD4 receptors and CD4 bound to monocytes. Typically the added volume is between 5 and 50 μΙ to 75 μΙ incubation buffer.
Assay Example 5: A vertical flow CD4 assay using enzyme immunoconjugates and colorimetric substrate
The assay performed with a device of Figures 9 to 1 1 comprised the following steps:
1 . A whole blood sample of 25 μΙ was mixed with 750 μΙ sample incubation buffer according to Preparation Example 12 above.
2. The sample was incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and of ALP-anti-CD4 to CD4+ cells and free CD4 receptor.
3. The sample was mixed with FACS™ lysing solution (BD Biosciences, catalogue no. 349202) to a final volume of 500 μΙ, and erythrocytes were lysed for 5 minutes
4. After 5 minutes, 100 μΙ of said mixture was applied on top of the nylon mesh filter in the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50- 150 μΙ.
5. Thereafter, 200 μΙ of wash solution according to Preparation Example 6 above was transferred to nylon mesh of the filtration device according to Preparation Example 1 1 above, and was sucked into the device.
6. Thereafter, the nylon mesh filter was removed by turning the top part 90° 7. Thereafter, 50 μΙ of Seramun Purple S-008-NBT liquid enzyme substrate Seramun GmbH was transferred to the color development hole of the polystyrene disc of the filtration device according to Preparation Example 1 1 above, and was sucked into the nitrocellulose filter. Typically, the substrate volume can be 10-100 μΙ.
8. Immediately thereafter, the top part was turned another 90° to the reading well and the non-transparent material was moved into position between the nitrocellulose and the absorbent. The color developed for 5 minutes.
9. Five minutes thereafter, the color development was stopped by washing with 25 μΙ 0,1 M HCI and the color development was evaluated colorimetrically using the Cube reader from opTricon Gmbh.
10. The reading was compared to a calibration curve stored in the software generated by calibration samples with known content of T-cell associated CD4 receptor molecules, analyzed in identical experiments, and the content of T-cell associated CD4 receptor molecules was calculated.
Assay Example 6: A vertical flow CD4 assay using colloidal gold immunoparti- cles The assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
1 . A blood sample containing 100, 500, 1000 and 1500 CD4 cells/μΙ of 25 μΙ is mixed with 75 μΙ of a solution of gold particles conjugated with anti-CD4 according to Preparation Example 13. 2. The solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and Au-anti-CD4 to CD4+ cells and free CD4 receptors.
3. The solution is then mixed with BD FACS™ Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 μΙ and erythrocytes are lysed for 5 minutes. 4. Thereafter, 100 μΙ of said mixture is transferred to the nylon mesh filter of the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50-150 μΙ. 5. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
6. Thereafter, the nylon mesh filter was removed by turning the top part 90° and then the top part was turned another 90° to the reading well and the non- transparent material was moved into position between the nitrocellulose and the absorbent.
7. Immediately thereafter, the color on the nitrocellulose filter is measured.
Assay Example 7: A vertical flow CD4 assay using fluorescent immunoparticles (e.g. Europium chelates)
The assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
1 . A blood sample containing 100, 500, 1000 CD4 cells/μΙ of 25 μΙ is mixed with 75 μΙ of a solution of fluorescent particles conjugated with anti-CD4 according to Preparation Example 14.
2. The solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and Au-anti-CD4 to CD4+ cells and free CD4 receptors.
3. The solution is then mixed with BD FACS™ Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 μΙ and erythrocytes are lysed for 5 minutes.
4. Thereafter, 100 μΙ of said mixture is transferred to the nylon mesh filter of the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50-150 μΙ.
5. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
6. Thereafter, the nylon mesh filter was removed by turning the top part 90° and then the top part was turned another 90° to the reading well and the non- transparent material was moved into position between the nitrocellulose and the absorbent.
7. Immediately thereafter, the fluorescence on the nitrocellulose filter is measured. Assay Example 8: A vertical flow CD4 assay using enzyme immunoconjugates and fluorescent substrate (e.g. DuoLux™ from Vector Laboratories)
The assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
1 . A blood sample containing 100, 500, 1000 and 1500 CD4 cells/μΙ of 25 μΙ is mixed with 75 μΙ of a solution of anti-CD4 according to Preparation Example 12.
2. The solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and anti-CD4 to CD4+ cells and free CD4 receptors.
3. The solution is then mixed with BD FACS™ Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 μΙ and erythrocytes are lysed for 5 minutes.
4. Thereafter, 100 μΙ of said mixture is transferred to the nylon mesh filter of the fil- tration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50-150 μΙ.
5. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
6. Thereafter, the nylon mesh filter was removed by turning the top part 90°. 7. Thereafter, 25 μΙ of the fluorescent substrate is added to the color development hole of the polystyrene disc of the filtration device according to Preparation Example 1 1 above, and was sucked into the nitrocellulose filter. Typically, the substrate volume can be 10-100 μΙ.
8. Immediately thereafter, the top part was turned another 90° to the reading well and the non-transparent material was moved into position between the nitrocellulose and the absorbent. The fluorescent substance is developed for 5 minutes.
9. Immediately thereafter, the fluorescence on the nitrocellulose filter is measured.
Assay Example 9: A vertical flow CD4 assay using enzyme immunoconjugates luminescent substrate (e.g. CDP-Star™ from ThermoFisher Scientific) The assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
1 . A blood sample containing 100, 500, 1000 and 1500 CD4 cells/μΙ of 25 μΙ is mixed with 75 μΙ of a solution of anti-CD4 according to Preparation Example
12.
2. The solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and anti-CD4 to CD4+ cells and free CD4 receptors.
3. The solution is then mixed with BD FACS™ Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 μΙ and erythrocytes are lysed for 5 minutes.
4. Thereafter, 100 μΙ of said mixture is transferred to the nylon mesh filter of the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50-150 μΙ. 5. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device.
6. Thereafter, the nylon mesh filter was removed by turning the top part 90°
7. Thereafter, 25 μΙ of the luminescent substrate (e.g. CDP-StarTM from Ther- moFisher Scientific) is added to the color development hole of the polystyrene disc of the filtration device according to Preparation Example 1 1 above, and was sucked into the nitrocellulose filter. Typically, the substrate volume can be 10-100 μΙ.
8. Immediately thereafter, the top part was turned another 90° to the reading well and the non-transparent material was moved into position between the nitrocellulose and the absorbent. The luminescence was developed for 5 minutes.
9. Immediately thereafter, the luminescence on the nitrocellulose filter is measured using a gel doc system (e.g. from Azure Biosystems).The results are shown in Figure 12 a) and b). Assay Example 10: A vertical flow CD4 assay using fluorescent Alexa Fluor®647 Mouse Anti-Human CD4
The assay to be performed with a device of Figures 9 to 1 1 comprises the following steps:
1 . A blood sample containing 100, 500, 1000 and 1500 CD4 cells/μΙ of 25 μΙ is mixed with 75 μΙ of a solution of fluorescent Alexa Fluor®647 Mouse Anti- Human CD4 according to Preparation Example 17.
2. The solution is incubated for 10 minutes allowing binding of anti-CD14 beads to monocytes and Alexa Fluor®647 Mouse Anti-Human CD4 to CD4+ cells and free CD4 receptors.
3. The solution is then mixed with BD FACS™ Lysing Solution (BD Biosciences Catalogue No. 349202) to a final volume of 500 μΙ and erythrocytes are lysed for 5 minutes. 4. Thereafter, 150 μΙ of said mixture is transferred to the nylon mesh filter of the filtration device according to Preparation Example 1 1 above, and is immediately sucked into the device. Typically, the sample volume can be 50-150 μΙ.
5. Thereafter, 200 μΙ of washing solution according to Preparation Example 6 above is transferred to the nylon mesh filter and is sucked into the device. 6. Thereafter, the nylon mesh filter was removed by turning the top part 90° and then the top part was turned another 90° to the reading well and the non- transparent material was moved into position between the nitrocellulose and the absorbent.
7. Immediately thereafter, the fluorescence on the nitrocellulose filter is measured using a gel doc system (e.g. from Azure Biosystems). The results are shown in Figure 13 a) and b).
The disclosure of prior art documents as cited herein is incorporated by reference.

Claims

CLAIMS An assay method for assessing in a liquid whole blood sample or a sample derived therefrom one or more subclasses of blood cells of interest (BCol), each of which carrying a first cell surface marker (M1 ) for said sub-class of blood cells of interest , wherein said sample may additionally comprise disturbing blood cells (DBC), which carry at least one of said first cell surface markers (M1 ) as non-specific marker, and/or at least one free non-cell surface bound form of any of said first cell surface markers (M1 ) which method comprises
(1 ) exposing said sample to one or more reagents suitable for assessing one or more of said first cell surface markers (M1 )
(2) removing from said sample as obtained in step (1 ) any disturbing blood cells (DBC),
(3) removing from said sample as obtained in step (2) any free, non-cell surface bound form of each of said first cell surface markers (M1 ); and
(4) assessing in the sample as obtained in step (3) each of said sub-classes of BCol, carrying said first cell surface marker (M1 ).
The method of claim 1 , which is a vertical flow assay method.
The method of claim 1 or 2, wherein step (1 ) comprises
(a) contacting the sample with a reagent, allowing the removal of said DBCs by filtration; and
(b) contacting the sample with said one or more reagents suitable for assessing one or more of said first cell surface markers (M1 ),
wherein (a) and (b) are either performed simultaneously or sequentially in any order.
4. The method of one of the preceding claims, wherein in step (2) said DBCs are removed by filtration via a first filter (F1 ).
5. The method of claim 4, wherein said DBCs are aggregated, which aggregates are retained by the first filter (F1 ) applied in step (2).
6. The method of claim 5, wherein said DBCs are aggregated by means of immunoglobulin molecules which do not bind said BCol.
7. The method of claim 6, wherein said DBCs are aggregated by means of immunoglobulin molecules, which bind to a second cell surface marker (M2) which is not present on the surface of said BCol.
8. The method of claim 6 or 7, wherein said DBC binding immunoglobulins are selected from free antibodies, polymeric antibodies or antibodies bound to the surface of solid particles, in particular polymer particles.
9. The method of one of the preceding claims, wherein in step (3) said non-cell surface bound form of said first cell surface marker (M1 ) is removed by filtration by applying a second filter (F2) which is permeable for said non-cell surface bound form of said first cell surface marker (M1 ) free or bound to said reagent for as- sessing M1 , but which retains said BCol.
10. The method of one of the preceding claims, wherein said assessment of step (4) is performed by means of immunoglobulin molecules reactive with said first cell surface marker (M1 ).
1 1 . The method of claim 10, wherein said immunoglobulin molecules reactive with said first cell surface marker (M1 ) do not aggregate said BCols carrying said first cell surface marker (M1 ).
12. The method of claim 10 or 1 1 , wherein said immunoglobulin molecules reactive with said first cell surface marker (M1 ) are labelled.
13. The method of claim 12, wherein said label is selected from an enzyme, a luminescent, in particular chemiluminescent or fluorescent molecular marker, or a colored molecular marker, or a luminescent, in particular chemiluminescent or fluorescent particle, or colored particle, or metal colloid particle.
14. The method of one of the claims 4 to 13, wherein said first filter (F1 ) and said second filter (F2) differ in the size of their openings.
15. The method of claim 14, wherein the pore size of said first filter (F1 ) is such that DBC aggregates are retained by filter (F1 ), while BCol carrying a first cell surface marker (M1 ),
BCol carrying a first cell surface marker (M1 ) and bound to said reagent for assessing said cell surface bound M1 ;
non-cell surface bound form of cell surface markers (M1 ); and
non-cell surface bound form of cell surface markers (M1 ); bound to said reagent for assessing said cell surface bound M1.
are not retained.
16. The method of claim 15 wherein filter (F1 ) is a grid or sieve, having a grid or mesh size in the range of 10 to 50, preferably 12 to 40, in particular 15 to 30 μηη.
17. The method of one of the claims 9 to 16, wherein the pore size of said second filter (F2) is such that
BCol carrying a first cell surface marker (M1 ), and
BCol carrying a first cell surface marker (M1 ) and bound to said reagent for as- sessing said cell surface bound M1 ;
are retained; while
non-cell surface bound form of cell surface markers (M1 ); and
non-cell surface bound form of cell surface markers (M1 ); bound to said reagent for assessing said cell surface bound M1.
are not retained.
18. The method of claim 17, wherein filter (F2) is a membrane filter, having a pore size in the range of 1 to 15, preferably 2 to10, in particular 3 to 8 μηη.
19. The method of one of the preceding claims, wherein said BCol are selected from a sub-class of lymphocytes, in particular T-lymphocytes, and said DBCs are monocytes.
20. The method of one of the preceding claims, wherein said first cell surface marker (M1 ) is a T-lymphocyte marker (M1 a), in particular the CD4 cell surface receptor molecule.
21 . The method of one of the preceding claims, wherein said one or more subclasses of blood cells of interest (BCol) to be assessed comprises CD4+ cells.
22. The method of one of the preceding claims, where said first cell surface marker (M1 a) is CD4 and said first sub-class of cells is T-helper cells.
23. The method of one of the preceding claims, where said method also comprises the assessment of a second sub-class of BCol carrying a cell surface marker (M1 b) different from said first cell surface marker (M1 a).
24. The method of claim 23, wherein said cell surface marker (M1 b) is a T- lymphocyte marker different from M1 a, in particular the surface markerCD8 and said second sub-class of BCol comprises CD8+ cells.
25. The method of claim 24, where said surface marker (M1 b) is CD8 and said second sub-class of cells is cytotoxic T-cells.
26. The method of one of the claims 23 to 25, wherein the assessment of said second sub-class of BCol carrying said second cell surface marker (M1 b) is per- formed in step (4) together with the assessment of said first subclass of BCol, carrying a first cell surface marker (M1 a), in particular in the same sample.
27. The method of one of the claims 23 to 25, wherein the assessment of said second sub-class of BCol carrying said marker (M1 b) is performed separately.
28. The method of claim 27,
which method comprises
(5) optionally removing from said sample any disturbing macromolecular impurities which might disturb the assessment
(6) removing from said sample, optionally as obtained in step (5), any free, non-cell surface bound form of said second cell surface markers (M1 b); and (7) assessing in the sample as obtained in step (6) said sub-class of BCol carrying said cell surface marker (M1 b).
29. The method of claim 28, wherein in step (6) said non-cell surface bound form of said first cell surface marker (M1 b) is removed by filtration by applying a filter which is permeable for said non-cell surface bound form of said cell surface marker (M1 b) free or bound to said reagent for assessing (M1 b) but which retains said sub-class of BCol carrying (M1 b) and BCol carrying (M1 b) and bound to said reagent for assessing (M1 b).
30. The method of claim 28, wherein said assessment of step (7) is performed by means of immunoglobulin molecules reactive with said cell surface marker (M1 b).
31 . The method of claim30, wherein said immunoglobulin molecules are labelled.
32. The method of claim 31 , wherein said label is selected from an enzyme, a lumi- nescent, in particular chemiluminescent or fluorescent molecular marker, or colored molecular marker or a luminescent, in particular chemiluminescent or fluorescent particle, or colored particle or metal colloid particle.
33. The method of one of the preceding claims, wherein said DBCs are CD14+ mon- ocytes.
34. The method one of the preceding claims, where the aggregation of DBCs in step (1 ) is performed by adding a first liquid comprising immunoglobulins, said liquid being able to lyse erythrocytes contained in the sample.
35. The method one of the preceding claims, comprising the steps of
(1 a) mixing the said sample or an aliquot of the said sample with a first liquid comprising
labeled antibodies specifically reactive to said CD4 receptors of said specific sub- group of cells, where said label is constituted by an enzyme or colored or luminescent, in particular chemiluminescent or fluorescent particle or metal colloid particle which, preferably, do not aggregate said subgroup of CD4 cells; and antibodies binding to other structures on the surface of other cells different from said specific sub-group of cells but carrying said CD4 receptors, forming particles or aggregates or clusters of particles or cells with a size significantly larger than the size of the cells in said specific sub-group of cells, and significantly larger than the size of the cells in said specific sub-group of cells bound to said labeled antibodies,
(1 b) filter away said formed particles or aggregates or cluster of particles or cells by means of a first filter that is constituted by a size exclusion filter, and (2) passing the remaining mixture through a second filter retaining the said specific sub-group of cells in said sample but letting CD4 receptor molecules in solution pass through the filter, optionally followed by a washing step,
(3a) optionally followed by adding a substrate to said enzyme generating a col- ored substance, or luminescent, in particular fluorescent or chemiluminescent substance
(3b) measuring the intensity of the color or the luminescence, in particular fluorescence or chemiluminescence, on said second filter and correlating said intensity to the concentration of said class of CD4 receptors on the surface of the said specific sub-group of cells
36. The method of one of the preceding claims, wherein a lysis of erythrocytes is performed to said blood sample prior to the assessment is performed.
37. The method of one of the preceding claims, wherein the cell count for the group of CD4+ cells is assessed.
38. The method of claim 37, wherein the cell count for the group of CD4+ cells, and at least for one further group of cells, different from CD4+ cells, in particular for the group of CD8+, cells is assessed, in particular the CD4/CD8 ratio.
39. A method for assessing the quantity of CD4 receptors located on the surfaces of CD4+ cells and optionally for assessing the quantity of CD8 receptors located on the surfaces of CD8+ cells in a sample of whole blood or a sample derived from blood, which method comprises performing a method of one of the claims 1 to 38 and correlating the signal obtained for the assessment of the group of CD4+ cells with the quantity of cell-bound CD4+ receptor, and optionally correlating the signal obtained for the assessment of the group of CD8+ cells with the quantity of cell- bound CD8+ receptor.
40. The method of one of the preceding claims, wherein said immunoglobulin molecules as applied in said method are antibodies, like monoclonal or polyclonal non-human, in particular non-rodent antibodies, like avian antibodies.
41 . The method of one of the preceding claims, wherein the immunoglobulins applied for binding to (M1 a) and / or (M1 b) are covalently bound to particles, selected from coloured or luminescent, in particular chemiluminescent or fluorescent, latex particles or metal colloid particles, the particles having a mean particle diameter in the range of 30 to 500 nm.
42. The method of anyone of the preceding claims, wherein said method further comprises before the assessment step (3), (3a) or (3b) a step of removing or blocking background signals.
43. The method of claim 42, which method comprises removing or blocking back- ground signals caused by one or more constituents of the applied sample, which permeate filter (F2).
44. The method of claim 43, wherein said background signals are caused by constituents of the applied sample, that are retained by an absorbent layer provided to absorb sample material permeating filter (F2),
45. The method of claim 44, wherein said background signals are removed by applying on the surface of the absorbent layer a shield or filter impermeable to said background signal, or by removing the absorbent from the device before perform- ing the assessment step, such that background signals are prevented to reach the detection device
46. A vertical flow assay device for performing the method of any of the claims 1 to 41 , which device comprises
an upper cover sheet (101 ) provided with at least one circular (liquid) sample feed opening (102) and a lower absorbent layer (105) fixed to said upper cover sheet (101 );
a first circular filter (F1 ) (106), removably inserted into said at least one circular opening (102);
a second filter (F2) (104) fixed between said upper cover sheet (101 ) and said lower absorbent layer (105), and separating said at least one feed opening (102) and the circular filter (F1 )(106) inserted therein from the absorbent layer (105).
The device of claim 46, wherein said first circular filter (F1 ) (106) has openings or pores retaining aggregated blood cells, and is permeable for non-aggregated blood cells or blood cells bound to a reagent suitable for assessing said cells, in particular CD4+ cells and optionally CD8+ cells.
48. The device of claim 47, wherein said first circular filter (F1 ) (106) is a net filter having a grid size 10 to 50, preferably 12 to 40 in particular 15 to 30 μηη 49. The device of one of the claims 46 to 48, wherein said second filter (F2) (104) has openings or pores retaining non-aggregated blood cells or blood cells bound to a reagent suitable for assessing said cells, and is permeable to constituents soluble in said liquid sample. 50. The device of claim 49, wherein said second filter (F2) (104) has a pore size in the range of 1 to 15, preferably 2 to10, in particular 3 to 8 μηη.
The device of one of the claims 46 to 50, wherein said first circular filter (F1 ) (106) is fixed via a carrier ring (108) to an adhesive tape (107), said ring (108) having an outer diameter slightly smaller than the diameter of the sample feed opening (102), and having an inner diameter chosen to define a free circular space sufficient for quantitatively taking up a predetermined sample volume.
The device of claim 51 , wherein said tape (107) removably adheres to the upper side of said an upper cover sheet (101 ).
The device of one of the claims 46 to 52, wherein said a first circular filter (F1 ) (106), removably inserted intro said at least one circular opening (102), is removed from the device by removing the tape (107) from said upper cover sheet (101 ).
The device of any of the claims 46 to 53, wherein said absorbent layer has an absorbing capacity sufficiently high to absorb any liquid constituents of sample and reagents and washing solutions added to the sample feed opening (102) during the course of the vertical flow assay.
Assay device, comprising
an upper casing element (31 ) and a lower casing element (32), the upper (31 ) and the lower casing element (32) being assembled in such a manner that a testing compartment is formed, which is suited to take up a stack of functional layers (43, 36, 37), the testing compartment comprising an upper testing compartment inner surface (31 a) of the upper casing element (31 ) and a lower testing compartment inner surface (32b) of the lower casing element (32),
the upper casing element (31 ) being rotatable with respect to the lower cas- ing element (32), thereby defining a first, a second and a third configuration, of the assay device,
the upper casing element (31 ) having a first opening (33), a second opening (34), and a third opening (35) each of which provide access from the outside to the testing compartment,
the first opening (33) the second opening (34) and the third opening (35) being arranged in such a manner that the position of the first opening (33) with respect to the lower casing element (32) at the first configuration is essentially the same as the position of the second opening (34) with respect to the lower casing element (32) at the second configuration, and the position of the second opening (34) with respect to the lower casing element (32) at the second configuration is essentially the same as the position of the third opening (34) with respect to the lower casing element (32) at the third configuration,
wherein
the assay device is provided with said stack of functional layers (43, 36, 37), which is taken up by the testing compartment, said stack comprising an upper membrane layer (36) and a lower absorbent layer (37), which are arranged on top of each other and extend essentially in parallel to the upper testing compartment surface (31 a) and the lower testing compartment surface (32a),
and wherein at the third configuration of the assay device a wedge (40) is positioned between the membrane layer (36) and the absorbent layer (37).
The device of claim 55, wherein at said third configuration the wedge (40) provides an optical shielding for said absorbent layer (37) versus said third opening (35).
The device of claim 55 or 56, wherein said testing compartment is provided with a filter layer (43), which is arranged essentially in parallel to the upper membrane layer (36), wherein
the filter layer (43) is arranged in such a manner that it is positioned between the first opening (33) and the upper membrane layer (36) and the filter layer (43) is attached to the upper testing compartment surface
(31 a).
The use of a device as defined in anyone of the claims 46 to 57 for performing assay method as defined in anyone of the claims 1 to 45.
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