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WO1999005528A1 - ANALYSE ET DISPOSITIF PERMETTANT D'IDENTIFIER UNE INFECTION A $i(TRYPANOSOMA CRUZI) - Google Patents

ANALYSE ET DISPOSITIF PERMETTANT D'IDENTIFIER UNE INFECTION A $i(TRYPANOSOMA CRUZI) Download PDF

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Publication number
WO1999005528A1
WO1999005528A1 PCT/US1998/015888 US9815888W WO9905528A1 WO 1999005528 A1 WO1999005528 A1 WO 1999005528A1 US 9815888 W US9815888 W US 9815888W WO 9905528 A1 WO9905528 A1 WO 9905528A1
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WO
WIPO (PCT)
Prior art keywords
cruzi
support
sample
assay
antigen
Prior art date
Application number
PCT/US1998/015888
Other languages
English (en)
Inventor
Andrew E. Levin
Original Assignee
Immunetics
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Immunetics filed Critical Immunetics
Priority to AU86042/98A priority Critical patent/AU8604298A/en
Publication of WO1999005528A1 publication Critical patent/WO1999005528A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa

Definitions

  • Chagas' disease or American trypanosomiasis is caused by Trypanosoma cruzi (T. cruzi) infection and is transmitted by triatomine insect vectors or blood transfusion. It is endemic in Central and South America and as such is recognized as an important public health problem. In a global perspective, the World Health Organization ranks the control of Chagas disease in third place after malaria and schistosomiasis control.
  • Chagas disease develop in one-third of those infected, some 10-20 years after the initial acute phase, and include: chronic cardiopathy (in 27% of those infected); chronic digestive lesions (in 6%), and neurological disorders (in 3%). Patients with severe chronic disease become progressively sick and ultimately die, usually as a result of heart failure. It is estimated that 2,740,000 Disability- Adjusted Life Years (DALYS) are lost.
  • DALYS Disability- Adjusted Life Years
  • Chagas disease diagnoses are complicated by the complex life cycle of the parasite (e.g., T. cruzi is in an epi- or pro- mastigote stage in the insect vector, a trypomastigote stage in blood and amastigote stage intracellularly (e.g. intramacrophage)).
  • T. cruzi parasite hosts exhibit diverse immune responses, which involve both humoral and cell-mediated responses to the array of parasite antigens. Further, there have been persistent problems with false positive reactions in sera from patients with leishmaniasis.
  • the invention features novel assays for detecting the presence of T. cruzi or Leishmania sp. antibodies in a sample.
  • the assays involve contacting the sample with a support containing resolved antigens from T. cruzi of an appropriate stage for the sample being detected; and determining the relative location of the antibody-antigen complexes on the support and comparing the location to the location of complexes known to indicate the presence of T. cruzi or Leishmania sp. antibodies in the sample. Prior to determining the relative location, a detection step may be necessary to detect labeled antibody-antigen complexes.
  • the sample is blood or serum and the T. cruzi is in the trypomastigote stage and the antibody-antigen complex forms at an antigen having a molecular weight of about 4, 7, 10, 17, 21, 24, 26, 31, 35, 39 and 58 k Da.
  • the invention features novel supports containing resolved T. cruzi antigens.
  • the support is selected from the group consisting of: a membrane, bead, particle, tube or well.
  • the membrane is made of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride, glass fiber, cellulose, cellulose acetate, polycarbonate, polysulfone, nylon and nitrocellulose.
  • the T. cruzi antigens have been resolved and the antigens on the support comprise bands of increasing molecular weight.
  • the solid support additionally comprises at least one isolated T. cruzi and/or Leishmania sp.
  • the support is comprised of a membrane that has been cut into strips, each strip containing antigens from T. cruzi and/or L. chagas.
  • the invention features a removable cassette for use in a rapid flow-through binding assay device for specifically detecting T. cruzi antibodies or Leishmania sp. antibodies in a sample.
  • the removable cassette comprises a multi- channeled plate attached to a support as described above.
  • the removable cassette comprises a support disposed between two multi-channeled plates.
  • the removable cassette comprises a multi-channeled top plate, a support, support plate and base.
  • kits comprising a support of the present invention and reagents for carrying out the specific binding assay.
  • the kit may further comprise instructions for use as well as appropriate packaging.
  • the instant disclosed assays and devices accurately detect T. cruzi or Leishmania sp. antibodies in a sample. Therefore the assays and devices can be used to detect infected blood samples or to diagnose a subject with Chagas disease or Leishmaniasis. Preferred assays can be readily performed in less than about 30 minutes' time.
  • Figure 1 is a perspective view of an assay device having a removable cassette
  • Figure 2 is an exploded view of a first removable cassette
  • Figure 3 is an exploded view of a second removable cassette
  • Figure 4 is an exploded cross-sectional view of the assay device taken along lines 4-4 of FIG. 1;
  • Figure 5 is an exploded cross-sectional view of the assay device taken along lines 5-5 of FIG. 1;
  • Figure 6 is a top view of the assay device base plate
  • Figure 7 is an exploded view of a third removable cassette.
  • Figure 8 is an illustration of a removable cassette encased in a sealed plastic packaging.
  • the invention is based on the identification of T. cruzi antigens to which sera from Chagas patients, but not Leishmaniasis patients react, and cross- reactive antigens to which sera from both Chagas and Leishmaniasis patients react. Based on these findings, the invention features, assays, kits, kit components and devices for specifically detecting T. cruzi and/ 'or Leishmania sp. antibodies in a sample and therefore identifying a blood supply as being contaminated by T. cruzi or Leishmania sp. ; or diagnosing a subject as having Chagas disease or Leishmaniasis.
  • a “biological sample” shall refer to any material obtained from a subject (e.g. human or animal).
  • the biological material can be in any form including a solid material (e.g. tissue, cell pellet, stool sample, biopsy), or biological fluid (e.g. blood, serum, plasma, saliva, cerebrospinal fluid, urine, amniotic fluid).
  • Chronic cardiopathy chronic digestive lesions and neurological disorders.
  • Leishmania sp refers to any Leishmania species that can be detected and distinguished from T. cruzi according to the method of the invention, including but not limited to: L. chagasi, L. donovani, L. i ⁇ fantum, L. major, L. amazonensis, L. braziliensis, L. panamensis, L. tropica, L. uyanensis and L. rangeli.
  • an "isolated Leishmania antigen” as used herein refers to an antigen that is characteristic of Leishmania sp. in general or a particular Leishmania sp., whether recombinant, purified or synthetic.
  • Preferred Leishmania antigens generate antibodies in a subject that can be detected as an indication of Leishmaniasis.
  • preferred antigens occur predominantly on the amastigote form of the parasite (i.e., the form that replicates and is responsible for pathology) and not the promastigote form (i.e., the form transmitted by sand flies).
  • Leishmania antigens for diagnosing visceral Leishmaniasis in a subject include: the Leishmania chagasi rK39 antigen (U.S. Patent No. 5,411,865 by Reed, Badaro, R. et al., (1996) J. Infect. Dis. 173:758-61, Singh, S. et al., (1995) J. Parasitol 81: 1000-1003, and Burns, J.M. et al., (1993) Proc. Natl. Acad. Sci., USA 90: 775-779, available from Corixa Corp., Seattle, WA); antigens having molecular weights of 20,000; 40,000; 70,000; 113,000 or 140,000 (U.S.
  • Patent No. 4,992,273 by Monjour et al. an epitope of the L. chagasi ribosomal antigen LcPO (WO 96/33414 by Reed); certain antigens encoded by Leishmania sp. genes that are diSerentially expressed at higher levels in the amastigote, but not the promastigote stage of the life cycle (WO 95/06729)); an antigenic portion of dp72 or g ⁇ 70-2 (EP 0 293 827 by Jaffe et al.); an antigenic portion of the Leishmania major protein described in WO 95/30006 by Glaichenhaus et al., and the Leishmania-speciRc antigens described in WO 89/01045 by Berneman, et al.; and the Ldp23 antigen (WO 97/11180 by Reed et al.).
  • Leishmaniasis refers to any of a variety of symptoms, ranging from self- healing skin lesions to severe visceral involvement of the spleen, liver and lymph nodes, which result from infection by a protozoan parasite of the genus Leishmania.
  • Visceral Leishmaniasis (VL) a widely distributed disease with high morbidity and mortality, is caused by a member of the Leishmania donovani complex (e.g. L. chagasi, L. donovani and L. infantum).
  • a “support” shall refer to any material to which an antigen can be attached or immobilized.
  • preferred supports include: membranes (e.g. polyethylene, polypropylene, polyamide, polyvinylidenedifluoride, glass fiber or cellulose, cellulose acetate, polycarbonate, polysulfone, nylon, nitrocellulose), beads, particles, tubes (e.g. capillaries, straws or pipettes) or wells made of a variety of materials including (e.g. glass, plastic and metal).
  • T. cruzi refers to any strain of T. cruzi, which is currently known or to be identified. The origin, isolation and maintenance of various T. cruzi strains is published in O'Daly J.A. et. al., (1987) Exp. Parasitol. 64:1%- 87. T. cruzi is in an epi- or pro- mastigote stage in the insect vector, a trypomastigote stage in blood and amastigote stage intracellularly (e.g. in a macrophage).
  • An "isolated T. cruzi antigen” as used herein refers to an antigen that is characteristic of T. cruzi, whether recombinant, purified or synthetic.
  • T. cruzi antigens are trypomastigote or amastigote stage antigens.
  • Particularly preferred T. cruzi antigens are: the 2 D E antigen (available from Corixa Corp. Seattle, WA) Gp90, Gp 60/50 or LPPG antigen (U. S. Patent No. 5,550,027), the Ptcl 00 antigen (WO 96/05312 by
  • Paranhosbaccala et al. an antigen of a 90,000 kDa glycoprotein (U.S. Patent No. 4,615,973 by Lizardi et al.)
  • Leishmania sp. antibodies in a sample are carried out by contacting the sample with a support containing resolved antigens from T. cruzi of an appropriate stage for the sample being detected, thereby allowing antibody-antigen complexes to form on the support; and determining the location of antibody-antigen complexes on the support and comparing the location of the complex(es) to the location of complexes known to indicate the presence of T. cruzi or Leishmania sp. antibodies in the sample.
  • a detection reaction may be required to detect labeled antibody-antigen complexes prior to determining the location on a support.
  • the assays can be accomplished using any of a number of competitive or non-competitive assay procedures.
  • competitive immunoassays are performed by adding labeled antibody to be detected to the biological sample, so that exogenously supplied labeled antibody and endogenous unlabeled antibody compete for a limited number of antigen binding sites resulting in the formation of unlabeled antibody-antigen and labeled antibody-antigen complexes.
  • concentration of labeled antibody and antigen By maintaining the concentration of labeled antibody and antigen constant, the amount of labeled antibody-antigen complex formed is inversely proportional to the amount of antibody present in the sample. A quantitative determination of the antibody can therefore be made based on the labeled complex.
  • competitive assays can be homogeneous (i.e., not requiring separation) or heterogeneous (i.e., requiring separation).
  • non-competitive assays involve incubating a biological sample with an immobilized antigen for a period of time sufficient to result in a detectable level of antibodies bound to antigen.
  • the antigen can be directly or indirectly labeled.
  • indirect labeling can be carried out after a wash step to remove unbound antibody by contacting the immobilized antigen-antibody complexes with a second, labeled antibody that is specific for the antibody-antigen complex. Following a second wash step to remove unbound second antibody, the amount of bound second antibody can be detected and measured as an indication of bound antibody.
  • Exemplary competitive and non-competitive immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).
  • FPIA fluorescence polarization immunoassay
  • FIA fluorescence immunoassay
  • EIA enzyme immunoassay
  • NIA nephelometric inhibition immunoassay
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Labeling can be performed in any manner that facilitates detection of antibody- antigen complex.
  • Preferred labels include enzymes (e.g. horseradish peroxidase, alkaline phosphatase, urease, and ⁇ -galactosidase), enzyme co-factors, radioisotopes (e.g. 3 H, 14 C, 125 1, 32 P,
  • fluorescent compounds e.g. fluorescein, rhodamine, allophycocyanin, phycoeiythin, erythrosin, europian, luminol, luciferin and coumarin
  • colored or uncolored beads or particles e.g. silica gel, controlled pore glass, magnetic, Sephadex/Sepharose, cellulose, metal (e.g. gold) or latex.
  • Detection can be carried out as appropriate for the label using procedures, which are well-known in the art.
  • the assay comprises a Western Blot in which supports are individually processed in reagent solutions (e.g. in narrow trays, each typically holding
  • the support is incubated with a blocking solution containing a non-specific protein, (e.g., non-fat dry milk, bovine serum albumin, newborn calf serum or gelatin).
  • a blocking solution typically a physiological saline buffer containing a low percentage of detergent
  • the support is then incubated with the biological sample. Unbound antibody is then washed off with buffer, and the support is incubated in the detection reagent.
  • the detection reagent could be goat-anti-human IgG-alkaline phosphatase conjugate.
  • Unbound detection reagent is washed off with buffer, and finally the substrate (for alkaline phosphatase, a common substrate is 5-bromo-4-chloro-3-indolyl phosphate plus nitroblue tetrazolium) for the detection enzyme is added.
  • the conversion of the substrate to a visually detectable product is allowed to proceed until optimal visualization of bands, and then substrate is washed away. Bands on the support indicating antibody reactivity can be compared with control supports to determine the specificity of the immunoreaction.
  • a positive T. cruzi test result is defined as the appearance of at least one band selected from the group consisting of 4, 7, 10, 17, 21, 24, 26, 31, 35, 39 and 58 k Da.
  • a combination of at least two of the above-identified bands is detected as an indication of a positive T. cruzi test.
  • Further confirmation can be provided by antibody-antigen complex formation with an isolated T. cru ⁇ antigen.
  • the isolated T. cruzi antigen is on the same support as the resolved T. cruzi antigens.
  • a positive Leishmania sp. test is defined as the appearance of at lest one band selected from the group consisting of: 14, 15, 16, 64, 82 or 103 k Da.
  • a combination of at least two of the above-identified bands is detected as an indication of a positive Leishmania sp. result.
  • Further confirmation can be provided by antibody-antigen complex formation with an isolated Leishmania sp. antigen.
  • the isolated Leishmania sp. antigen is on the same support as the resolved T. cruzi antigens.
  • T. cruzi antigens refer to distinct T. cruzi antigens (e.g., surface antigens) from a whole T. cruzi organism.
  • the antigens have been resolved by molecular weight (e.g. via electrophoresis or chromatography) and the resolved antigens are arranged on the support according to increasing molecular weight. If the antigens have been resolved by electrophoresis, the resolved antigens can be blotted onto a support using standard procedures, so that antigens on the support are ordered top to bottom by decreasing molecular weight.
  • isolated (e.g., native or recombinant) antigens like the resolved T. cruzi antigens can be incorporated onto the support using any of a number of procedures, which are well-known to one of skill in the art.
  • isolated Leishmania sp. or T. cruzi antigens are directly applied, for example, using a pen, spray device, ink jet device or a flow-through device such as the MinislotTM (Immunetics, Cambridge, MA).
  • the isolated antigens can be applied to the solid support before, during or after application of the resolved antigens, preferably the isolated antigens are applied after the resolved antigens.
  • the support can also be in any shape that facilitates the reading and interpretation of a result.
  • the support can be in the form of strips, each bearing the identical pattern of antigen bands.
  • the applied antigens i.e., resolved and/or isolated
  • the bands on a support are configured to indicate a "+" (i.e., infection) if the appropriate antibody-antigen complexes form and "-" if not.
  • the support can be packaged alone or as a component of a removable cassette for use in performing a rapid flow-through assay.
  • the removable cassette can comprise a multi- channeled plate attached to a membrane.
  • the removable cassette can comprise the above-described membrane disposed between two multi-channeled plates.
  • the removable cassette can comprise a multi- channeled top plate, the above-described membrane, support plate and base.
  • Supports comprising the above described antigens alone or as a removable cassette can be packaged as a kit, which optionally includes appropriate processing reagents, as well as instructions for use.
  • suitable reagents for performing a Western blot can include: 1) blocking solution (e.g., proteinaceous solution), 2) primary antibody (diluted human serum), 3) wash solution (e.g. buffer containing phosphate- buffered saline and Tween-20 detergent), 4) secondary antibody (e.g., diluted anti-human lgG-alkaline phosphatase conjugate), and 5) enzyme substrate (e.g., 5-bromo-4 ⁇ chloro-3- indolyl phosphate and nitroblue tetrazolium solution).
  • blocking solution e.g., proteinaceous solution
  • primary antibody diluted human serum
  • wash solution e.g. buffer containing phosphate- buffered saline and Tween-20 detergent
  • secondary antibody e.g., diluted anti-human lgG-alkaline phosphatase conjugate
  • enzyme substrate e.g., 5-bromo-4 ⁇ chloro-3- indolyl phosphate and
  • FIG. 1 shows a perspective view of an assembled assay device 20 of a preferred embodiment of the present invention.
  • the assay device 20 generally has a base plate 22, a cover plate 24, and a removable cassette 26 for receiving assay samples.
  • the cover plate 24 and base plate 22 are generally rectangular in shape, although their size and shape can vary provided that the removable cassette 26 is suitably accommodated.
  • the base plate 22 and cover plate 24 are preferably formed from a solution-resistant material such as, but not limited to, plastic, aluminum, stainless steel or other synthetic material.
  • the removable cassette 26 may be disposed between the base plate 22 and cover plate 24, with the cover plate 24 having an aperture so as to expose the sample introduction channels 25 of the removable cassette 26.
  • the base plate 22 has a central pocket (not shown) and a vacuum/aspiration port 36.
  • the central pocket of the base plate 22 serves as a collector for liquid aspirated through a membrane component of the cassette 26.
  • the vacuum/aspiration port 36 is connected via a tubing adapter 38 to a length of tubing (not shown) leading to a trap (not shown) and ultimately to a vacuum source (not shown).
  • test samples and assay reagents are applied to the membrane through the channels of the cassette 26 under vacuum.
  • the cover plate is not essential for the proper operation of the assay device 20.
  • the cassette 26 may be held in the base plate with the application of pressure. In view of this, small actuated clips or cam fasteners on the base plate may be used to apply the pressure needed. Furthermore, the presence of the vacuum that is applied to the central pocket of the base plate 22 may be sufficient pressure in and of itself.
  • FIG. 2 presents an exploded view of a removable cassette 26 of a first preferred embodiment of the present invention.
  • the removable cassette 28 has a top plate 30, membrane 34, and an optional wicking member 36.
  • the top plate 30 preferably made by machine processing or injection molding of, for example, acrylic, styrene plastic or polymethylpentene, has a channel portion 29 that is skirted by a flange portion 31.
  • the top plate 30 also has an array of eight parallel channels 32 projecting perpendicularly from the top surface of the channel portion 29. It will be appreciated, however, that the plate may have one or more channels.
  • the channels 32 project completely through the top plate 30 and are preferably 10 centimeters long by 0.35 centimeters wide. With the eight channel configuration, up to eight samples may be received for assay. However, it should be readily understood that the number of channels may be increased or decreased to provide the ability to receive a greater or lesser number of specimens, and the channel dimensions may also vary depending upon the assays for which the particular cassette is designed.
  • the channels 32 are relatively long and narrow with very little structure over the channels obstructing the ability to apply solutions to the entire channel length (i.e., the channels 32 are open at the top surface 33 of the channel portion 29). Furthermore, even if the channels 32 are partially or completely covered, the depth of the channels is great enough so that liquid applied to the channels will be overlayed by air.
  • the membrane 34 of the removable cassette 28 preferably a nitrocellulose membrane generally cut to a rectangular or square shape, is affixed to the bottom surface of the top plate 30 with the wicking member 36, such as filter paper, optionally connected to the membrane 34 on the side of the membrane 34 opposite the membrane surface that is attached the top plate 30.
  • the wicking member 36 provides a more uniform distribution of liquid through the membrane.
  • the membrane 34 (and optionally the wicking member 36) may be affixed to the top plate 30 by welding, e.g., ultrasound or vibration welding. This welding provides a seal between the channels 32, such that a sample applied to one channel does not diffuse laterally through the membrane to a neighboring channel.
  • the membrane 34 contains resolved T. cruzi antigens alone, or optionally in conjunction with isolated T. cruzi and/or Leishmania sp. antigens and can also include channel numbers, letters, or other designations embossed, imprinted, stamped, or otherwise affixed thereon to assist the user in proper channel identification.
  • the top plate 30 of the removable cassette 28 may be embossed, imprinted, stamped, or otherwise designated to assist in identifying cassette channels.
  • the numbering or lettering scheme can include every channel, every other channel, or any other convenient system.
  • FIG. 3 presents an exploded view of a removable cassette 40 of a second preferred embodiment of the present invention.
  • This cassette 40 has a membrane 52 disposed between a top plate 44 and a bottom plate 46.
  • the top plate 44 and bottom plate 46 preferably made with acrylic, styrene plastic or polymethylpentene, each have 8 channels (48,50) that project perpendicularly from the top surface of a plate (43,45) through to the bottom surface of the respective plate (47,49).
  • a wicking member 42 may be placed between the membrane 52 and the top plate 44 or bottom plate 46.
  • Suitable fastening means are known in the art and include press-fit locating pins, tape, or a hinge and latch assembly, preferably located on flange portions of the plates.
  • FIG. 4 an exploded cross-sectional view is presented taken along lines 4-4 of the assay device 20 holding the removable cassette 40 of FIG. 3.
  • the cassette 40 may be disposed between the cover plate 24 and base plate 22 with the cover plate 24 having an aperture allowing the exposure of the top plate channels 48 of the removable cassette 40.
  • the cover plate 24 has a groove 56 that receives a flange portion 58 that skirts the channel portion of the top plate 44.
  • the base plate also has a groove 60 that receives a flange portion 62 that skirts a channel portion 63 of the bottom plate 46.
  • the base plate 22 has a central pocket 65 and vacuum/aspiration port 36 with tubing adaptor 38 that can be operatively linked to a vacuum source (not shown). With this assembled assay device 20, test samples and assay reagents may be applied to the membrane 42 through the multiple channels (48,50) of the removable cassette 40, and drawn through the membrane 42 under vacuum.
  • FIG. 5 shows an exploded cross-sectional view of FIG. 1 taken along lines 5-5.
  • the base plate 22 and cover plate 24 contain fasteners (64,66) that are used to lock the assembly 20.
  • the threaded end (68,70) of each fastener is inserted through apertures (72,74) in the cover plate 24, and threadably mated with recessed cavities (76,78) formed in the base plate 22.
  • locator pins (82,84) are provided that extend from the base plate 1—2 and that are received by alignment apertures (86,88) in the cover plate 24. It will, however, be appreciated that other means for fastening or clamping the plates (22,24) of the device 20 are readily available.
  • the heads of the fasteners (64,66) are preferably large enough to permit easy manipulation by hand and are also knurled, ridged, or otherwise textured to provide a good gripping surface.
  • the fasteners (64,66) can be solid or hollow, but are constructed of inert rigid materials which are resistant to the solutions and reagents used in the assay. Suitable materials include both metal and plastics, preferably acetal plastics.
  • the base plate 22 has a central pocket or cavity 118 that becomes a vacuum chamber when a cassette is placed over the cavity 118 so as to cover it.
  • a gasket 120 of soft rubber or other seal material is placed on the groove 122 in the base plate 22 that runs around the periphery of the cavity 118.
  • the flange portion of the removable cassette rests on this gasket 120 and pressure applied by the vacuum or cover plate effectively seals the cassette and base plate together.
  • the preferred embodiment utilizes a gasket material that is a sponged rubber sheet, however it can be seen that an 0-ring gasket or many other materials and configurations may be used to create the seal.
  • a seal may be created by lightly pressing on the cassette, with the subsequent application of the vacuum maintaining the seal during assaying. Once the chamber seal is created, application of the vacuum draws the applied liquids through the channels of the cassette. In order to assist in the collection of filtered liquid in the cavity 118, grooves 124 are placed in the cavity floor, effectively routing the collected liquid to the vacuum port for evacuation.
  • FIG. 7 illustrates an alternate embodiment of the present invention.
  • This cassette 90 is preferably used in an automated instrument as the components are ultrasonically welded to form a unitary cartridge.
  • the cassette 90 has a cassette top 92, membrane 94, optional wicking member 96, support 98, and cassette bottom 100.
  • the cassette top 92, support 98 and cassette bottom 100 are formed from a solution resistant material such as plastic, aluminum, stainless steel or other synthetic material.
  • the support 98 has a top side 104 with channels 106 corresponding to the channels 102 of the cassette top 92.
  • the support also has multiple footings 108 for operative contact with the cassette bottom 100 which has multiple grooves 110 aligned with the channels 102 of the cassette top 92.
  • the cassette bottom 100 also has a vacuum/aspiration port 112 for connection to a vacuum pump (not shown).
  • the membrane 94 is disposed between the cassette top 92 and support 98.
  • a wicking member 96 such as filter paper, may be included in the cassette 90, and is preferably inserted between the membrane 94 and support 98.
  • the support 98 is inserted into the cassette bottom 100, and all the components of the cassette are interlocked and sealed.
  • solutions are introduced into the channels of the cassette top contacting the membrane and analytes of interest (if present) will bind to the binding members immobilized on the membrane 94.
  • the binding members may also be pre- introduced to the membrane prior to operation.
  • an assay device having a removable cassette 114 can be supplied in a sealed plastic packaging 116.
  • This packaging 116 can include separate moisture-proof packaging to avoid contamination and permit pre-moistening of the membrane, thereby promoting longevity of the package contents.
  • the components of a cassette of the present invention can also be provided individually or as a complete assay device. It will also be appreciated that, although the various components of the apparatus of the invention can be cleaned and reused, the entire cassette unit or any of its components can be discarded after a single use, (i.e., the cassette is disposable).
  • the cassette can be removed from the base unit and disassembled and the membrane removed from the cassette.
  • the membrane can be dried to provide a permanent record or can be scanned even while in the cassette by a scanning device such as a CCD camera, which can then generate a permanent electronic record of the result.
  • Example 1 Rapid Flow-Through Assay for Detecting Trypanosma cruzi antibodies as an Indication of Chagas Disease.
  • cruzi antigen 2/D/E and Leishmania chagasi antigen rK39 were applied to the membrane at this point by striping with a pen.
  • the nitrocellulose membrane was 0.2 ⁇ pore size and was obtained from Schleicher & Schuell (Keene, NH).
  • the nitrocellulose membrane was cut to fit a cassette, 2" wide x 4" long.
  • the wet membrane was placed over an identically sized piece of dry filter paper (#320 paper, a 2.5 mm thick blotting paper, from Ahlstrom Filtration Inc., Mount Holly Springs, PA).
  • the membrane and filter paper were placed in the removable cassette, the cassette top and bottom were fixed together, and the membrane was allowed to dry. Drying of the membrane was preferable for packaging, since a wet membrane could support the growth of microbes during shipping and long-term storage, in the absence of sterilization.
  • the cassette was mounted on a base fixture and the screws were tightened, applying pressure to the cassette and thereby sealing the membrane against the cassette channels and preventing cross-channel leakage of solutes. Tightening also seals the cassette against the vacuum chamber, preventing vacuum leakage.
  • the entire unit was placed on a vertical rocking platform, and all subsequent steps (liquid additions, incubations and aspiration) were performed while the unit rocked at approximately 100 cycles/minute through an 8° angle.
  • a wetting solution consisting of phosphate-buffered saline (PBS) containing 0.5% Tween-20 detergent was introduced into the cassette channels 1-2 ml per channel.
  • PBS phosphate-buffered saline
  • Tween-20 detergent 0.5% Tween-20 detergent
  • test sample 20 ⁇ of a human serum was mixed with 600 ⁇ of PBS/0.5% Tween-20 before introduction into the cassette. Diluted serum specimens were incubated in the channels for 3 minutes, then the vacuum was turned on and solutions were drawn through the membrane in about 10 sec. The membrane was subjected to 3-4 washing steps. In each one, approximately 600 ⁇ of PBS/0.5% Tween was added per channel with the vacuum on constantly.
  • the detection reagent was then added.
  • an affinity purified goat anti-human IgG-alkaline phosphatase conjugate (commercially available from Jackson Immunoresearch, West Grove, PA) was diluted 1000-fold in PBS/0.5% Tween-20/0.05% Polyvinylpyrrolidone (MW 360,000), and 600 ⁇ were added per channel.
  • the vacuum was turned on and all conjugate solution aspirated through the membrane within about 10 sees.
  • the addition of polyvinylpyrrolidone to the conjugate dilution buffer increased the sensitivity of detection of bound antibody without a parallel increase in background noise; thereby functioning as a reaction "enhancer.”
  • the membrane was subjected to 3-4 washing steps. In each one, approximately 600 ⁇ of PBS/0.5% Tween-20 was added per channel with the vacuum on constantly. The membrane was then washed with about 600 ⁇ distilled water per channel.
  • the enzyme substrate a solution containing 4-chloro-3-bromo-2-indolyl phosphate and nitroblue tetrazolium (commercially available from Kierkegaard & Perry,
  • a Chagas'/Leishmania membrane immunoassay kit (QualiCode) was evaluated on panels of well-characterized human sera from patients with infections confirmed by culture, microscopy or radioimmunoprecipitation (RIP A).
  • the test incorporates both electrophoretically resolved native lysate antigens and recombinant antigens derived from T. cruzi and L. chagasi.
  • the sensitivity and specificity of detection of Chagas' antibodies were 96% and 95%, respectively, while for visceral leishmaniasis values for both were 100%.
  • the QualiCode immunoassay kit thus provides a highly accurate method for laboratory detection and discrimination of antibodies from Chagas' and visceral leishmaniasis patients.
  • the kit is a membrane immunoassay which incorporates electrophoretically resolved antigens of T. cruzi trypomastigotes in tandem with recombinant antigens of T. cruzi and L. chagasi. Test Procedure
  • Sera were tested for Chagas and Leishmania IgG antibodies following kit instructions. Briefly, sera were diluted 100-fold in sample dilution buffer and incubated individually with membrane strips for 30'. Following several buffer washes, strips were incubated with a goat-anti-human IgG alkaline phosphatase conjugate solution for 15'.
  • strips were incubated in a substrate solution containing BCIP/NBT (5'-bromo-4'-chloro-3'-indolyl phosphate/nitroblue tetrazolium). When bands were clearly visible, strips were rinsed in water and air dried.
  • BCIP/NBT 5'-bromo-4'-chloro-3'-indolyl phosphate/nitroblue tetrazolium
  • Footnotes ( 1 ) RIP A performed by The American Red Cross
  • Sensitivity of QualiCode Leishmaniasis assay increased to 100%> when Cutaneous Leishmaniasis samples are excluded from comparison.
  • the overall performance of the QualiCode test was calculated by combining results in the same category from both CDC and ARC panels. From these values, sensitivity, specificity and predictive value were calculated independently for the Chagas' and Leishmania portions of the QualiCode test (Table 3).
  • the QualiCode Chagas'/Leishmania test was designed to provide a serological method of confirmatory accuracy for laboratory diagnosis of the respective diseases.
  • the Chagas' test results correlated almost exactly with the reference methods, with the exception of one discrepancy in both positive and negative categories. With a sensitivity of 96%> and specificity of 95%, the Chagas' test approaches gold standard accuracy.
  • Two Leishmania-positive specimens were not detected by the QualiCode test Leishmania antigen; however, these were from cutaneous leishmaniasis patients, in which an immune response is frequently difficult to detect. In visceral leishmaniasis patient sera, both sensitivity and specificity of the QualiCode test were 100%>. Given the relative simplicity of the membrane immunoassay vs. culture, microscopy or RIP A analysis, it should prove useful in the laboratory diagnosis of Chagas 1 disease and visceral leishmaniasis.

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Abstract

L'invention concerne des analyses, des dispositifs et des trousses permettant d'identifier spécifiquement l'infection d'un échantillon par Trypanosoma Cruzi. Des antigènes résolus pour T. Cruzi sont placés sur un support (34) et l'échantillon peut entrer en contact avec le support (34). Des complexes antigène-anticorps sont ensuite détectés. Le support (34) fait partie d'une cassette dans un appareil de détection de T. Cruzi.
PCT/US1998/015888 1997-07-28 1998-07-28 ANALYSE ET DISPOSITIF PERMETTANT D'IDENTIFIER UNE INFECTION A $i(TRYPANOSOMA CRUZI) WO1999005528A1 (fr)

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US5395997P 1997-07-28 1997-07-28
US60/053,959 1997-07-28

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WO1999047928A3 (fr) * 1998-03-19 2000-03-23 Immunetics Inc Systemes et procedes de criblage rapide par buvardage
US7749717B2 (en) 2006-10-19 2010-07-06 Abbott Laboratories Methods for the detection and diagnosis of Trypanosoma cruzi infection
US8468680B2 (en) 2010-08-24 2013-06-25 Roche Diagnostics Operations, Inc. Biosensor test member and method for making the same
CN108593939A (zh) * 2018-07-18 2018-09-28 广州瑞博奥生物科技有限公司 一种全自动蛋白芯片及其应用
WO2023077043A3 (fr) * 2021-10-28 2023-09-07 Vida Pharmacal, Inc. Procédé et composition d'antigène pour la détection rapide de la maladie de chagas, de la leishmaniose et/ou du ver du coeur chez l'homme et l'animal

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047928A3 (fr) * 1998-03-19 2000-03-23 Immunetics Inc Systemes et procedes de criblage rapide par buvardage
US6194160B1 (en) 1998-03-19 2001-02-27 Immunetics, Inc. Systems and methods for rapid blot screening
US7749717B2 (en) 2006-10-19 2010-07-06 Abbott Laboratories Methods for the detection and diagnosis of Trypanosoma cruzi infection
US8468680B2 (en) 2010-08-24 2013-06-25 Roche Diagnostics Operations, Inc. Biosensor test member and method for making the same
US8793865B2 (en) 2010-08-24 2014-08-05 Roche Diagnostics Operations, Inc. Biosensor test member and method for making the same
US8920728B2 (en) 2010-08-24 2014-12-30 Roche Diagnostics Operations, Inc. Biosensor test member and method for making the same
CN108593939A (zh) * 2018-07-18 2018-09-28 广州瑞博奥生物科技有限公司 一种全自动蛋白芯片及其应用
CN108593939B (zh) * 2018-07-18 2023-11-14 瑞博奥(广州)生物科技股份有限公司 一种全自动蛋白芯片及其应用
WO2023077043A3 (fr) * 2021-10-28 2023-09-07 Vida Pharmacal, Inc. Procédé et composition d'antigène pour la détection rapide de la maladie de chagas, de la leishmaniose et/ou du ver du coeur chez l'homme et l'animal

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