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WO1991015769A1 - Procedes de test chromatographique lateral bidirectionnel - Google Patents

Procedes de test chromatographique lateral bidirectionnel Download PDF

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Publication number
WO1991015769A1
WO1991015769A1 PCT/US1991/002403 US9102403W WO9115769A1 WO 1991015769 A1 WO1991015769 A1 WO 1991015769A1 US 9102403 W US9102403 W US 9102403W WO 9115769 A1 WO9115769 A1 WO 9115769A1
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WO
WIPO (PCT)
Prior art keywords
sample
zone
tracer
reaction zone
reagent
Prior art date
Application number
PCT/US1991/002403
Other languages
English (en)
Inventor
Frederick C. Horstman
Julia A. Whiteside
Original Assignee
Disease Detection International
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 Disease Detection International filed Critical Disease Detection International
Publication of WO1991015769A1 publication Critical patent/WO1991015769A1/fr

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Classifications

    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography

Definitions

  • the invention relates to a diagnostic device for performing solid phase immunoassays to detect the presence of antigens or antibodies in biological or non-biological fluids.
  • the teachings of the invention are incorporated in a bi-directional lateral chromatographic device for use in solid phase immunoassays, or for the non-immunological detection or quan ita ion of proteins , or substances in biological or non-biological fluids.
  • the invention relates to devices and methods which utilize filter means for testing biological fluids to detect the presence of analytes, such as bacterial, viral, parasitic or fungal antigens, and immunoglobulins, hormones, serum proteins, drugs and the
  • U.S. Patent No. 4,623,461 discloses a filter body located in a housing having an opening therein for the reception of a suspension sample to permit the upper face of the filter to trap colored or particulate matter contained within the specimen, and prevent such matter from reaching the bottom face of the reaction zone, which has been previously treated with a suitable reactant.
  • the perimeter of the filter is engaged with a suitable reactant.
  • the perimeter of the filter is engaged with a suitable absorbent body, and the absorbent body is intended to receive the outward diffusion of liquids applied to the filter.
  • U.S. Letters Patent No. 3,825,410 discloses a disposable, combined storage and reaction cell for use in the performance of chemical and biological reactions which receives reactants dispensed therein and maintains the same in stored condition so that they remain stable. Reactants will not mutually react until such time as it is required to initiate the reaction.
  • the immobilization of the reactants is accomplished by such procedures as freeze drying, and. the reaction is initiated by the introduction of a sample to be analyzed, whereafter separation of bound and free ligand can be performed either within the unit itself or externally.
  • the reaction cell of the '410 patent may include a filter so that the entire process of separation can be completed within the reaction cell, and the filter can be removed from the reaction cell and submitted for radioactivity or other tracer counts.
  • U.S. Letters Patent No. 3,888,629 discloses a reaction cell for performing various types of assays which incorporates a matrix pad of absorbent material retaining the necessary reagents for the reaction and serving as a site in which the reaction totally occurs.
  • a separable, lower chamber incorporates absorbent material abutting the matrix pad to promote filtration through the pad after the reaction has taken place.
  • One of the objects of the invention is the provision of a diagnostic device which incorporates a planar filter body having sample application, separation and reaction zones, said filter body being configured in such a manner that the bulk of the solids in the suspension sample are retained in the sample application zone and the fluid is caused to migrate bilaterally through the interstices of the filter by the fluid communication of absorbent means with the application and reaction zones.
  • Another object of the invention is the provision of a diagnostic test device of the aforementioned character, wherein the planar filter incorporates a sample application zone which is relatively large, and which is connected to the reaction zone by a separation zone, the length of the separation zone being proportioned to the character of the suspension sample applied to the application zone; and the separation and application zones cooperate to retain the bulk of the solids or particulate matter in the application and separation zones so that the immuno-reagent or chemical test reagent deposited in the reaction zone, when subjected to the test procedures and analytes, will have a minimum of, or no, particulates embodied therein which could cause the emission of high background signals, thus creating a negative effect on the test readout.
  • test device of the invention is designed particularly for use in the field for the testing of various human and animal diseases, or for various chemical tests involving the utilization of blood samples from humans and animals; and it is capable of giving test results equal to laboratory results within a matter of minutes, depending upon the sample solution which is applied to the specific device.
  • Another object of the invention is the provision of a method of performing a test by the utilization of the device of the invention which incorporates a plurality of simple steps which can be carried forth by non-laboratory personnel in the field, and which can provide such personnel with an almost immediate readout of the presence or absence of the sought-after infection or drug, or the like.
  • Still another object of the invention is the provision of a simple two-step method of determining the presence or absence of an analyte in a sample fluid.
  • necessary reagents may be embedded within a filter matrix prior to use.
  • the chromatic eliciting substrate reagent should be added after application of the sample.
  • the two-step method requires only the application of the sample itself, followed by application of a washing solution, which may be a solution of a chromatic eliciting substrate reagent.
  • Figure 1 is a top plan view of a typical device of the invention
  • Figure 2 is a device similar to Figure 1, with the exception that it incorporates a plurality of application and reaction zones;
  • Figure 3 is a top plan view showing the housing of the device of Figure 2 with the cover removed therefrom to illustrate the location of and configuration of the filter and the relation thereof with the absorbent means and the particular design of the housing to encapsulate the filter and absorbent means:
  • Figure 4 is a view similar to Figure 3 illustrating removal of the filter to disclose the relationship of the absorbent means with the housing and the particular configuration thereof;
  • Figure 5 is a vertical sectional view taken on the broken line 5-5 of Figure 2 and illustrates the fluid relationship of the filter with the absorbent means and the filter and absorbent components with the specific design of the housing and cover therefor;
  • Figure 6 is an exploded view illustrating the various components of the test device. Description of the Preferred Embodiments of the Invention
  • the chromatic assay device of the present invention may be used to perform solid phase immunoassays for the detection of antigens or antibodies, hereinafter referred to as analytes, in biological and non-biological fluids.
  • the device may be non-immunologically used to identify and/or quantitate proteins or substances in biological and non-biological fluids.
  • the device may be utilized to perform assays such as competitive or non-competitive enzyme-linked immunoassays, enzyme-multiplied immunoassays, enzyme-inhibition assays, heterogeneous or homogeneous fluorescent immunoassays, chemiluminescent and bioluminescent assays, these assays utilizing various labelled probes, and the like.
  • a test device 10 constructed in accordance with the teachings of our invention which is incorporated in a housing 12 said housing consisting of a lower or bottom component 14 and a cover or closure 16.
  • the bottom component 14 of the housing may be fabricated by injection molding from suitable synthetic plastic materials such as polyethylene and, as will appear further hereinbelow, is specifically designed to receive a flat co- planar filter 20.
  • the closure 16 overlies the filter 20 and is secured to the housing by pressure-sensitive adhesive or other adhering means and incorporates an application port or opening 22 and a reaction port or opening 24, said ports communicating, respectively, with the application zone 26 and the reaction zone 28 of the filter.
  • the entire structure may be inverted so that the bottom component 14 forms the top of the structure, with the cover or closure 16 being on the underside.
  • the application and reaction ports 22, 24 are formed in bottom component 14, rather than the closure 16. Small wells may be formed around the ports to receive the liquid and divert it into the port.
  • This inverted embodiment is in all other respects identical to the embodiment of the invention having the closure 16 on top.
  • the test device 10 is designed for the performance of a single test, but, as best shown in Figure 2 of the drawings, a device 30 incorporating a multiplicity of application ports 22 and reaction ports 24 can be provided in a closure or cover 32 which overlies a suitably configured, as will be explained in greater detail below, lower housing portion 34 and filter 36.
  • the ports 22 and 24 respectively overlie application and reaction zones 38 and 40 of the filter 36.
  • the lower or bottom portion 34 of the housing of the test device 30 is configured, as best shown in Figures 4 and 5 of the drawings, to provide receptacles 56 for the application, separation and reaction zones of the filter 36 so that fluid flow is confined in the plane of the filter 36 because of the sandwich created between the closure 32 and the lower portion 34 of the housing.
  • the receptacles are defined by integrally molded lobes 58 in the body of the lower portion 34 of the housing and stringently confine the relevant portions of the filter in the receptacles 56.
  • Juxtaposed to the receptacles 56 is a first elongated rectangular well 60 for the reception of the first absorbent means 44, and a corresponding well 62 is provided for the reception of the second absorbent means 48.
  • the closure or cover 32 of the device 30 can be fabricated from vinyl or other plastic sheet material and may be adhesively or otherwise secured to the bottom portion of 34 of the housing of the test device 30.
  • the application, separation and reaction zones are contiguous within the co-planar surfaces of the glass fiber matrix.
  • a sample or samples applied to the sample application zones 38 will migrate laterally by capillary and chromatographic action.
  • the fundamental result achieved by the test devices constructed in accordance with the teachings of the invention is bilateral flow of the fluid component of suspensions applied to the application zones 38.
  • particulate matter present within the sample volume i.e., cellular components of whole blood, salt crystals of urine or protein aggregates of serum or plasma, etc.
  • particle size exclusion dictated by the mean pore size of the glass fiber matrix. Since the mean pore size of the glass fiber matrix is not an absolute value, but, rather, represents a Poisson distribution of a range of pore sizes, the length and width of the separation zone will be influenced and dictated by the mean porosity of the glass fiber matrix.
  • the length and width of the separation zone between the reaction and sample application zones must be carefully established empirically in order to position the reaction zone at a proper distance from the sample application zone to prohibit an inhibitory quantum of particulates from entering the reaction zone. If the separation zone length is too short, some particulates may enter the reaction zone; if too long, the volume of filtered sample fluid containing the desired analyte to be detected may be insufficient for optimal detection.
  • Bilateral migration of the fluid portion of the applied sample is also channeled in a direction 180 degrees away from the separation zone and, subsequently, the reaction zone, by the tapered constriction in the lateral boundaries of the bulbous or lobular-shaped glass fiber matrix.
  • This constriction of the glass fiber matrix favors migration of the sample through the separation zone in the direction of the reaction zone, yet still allows for some migration of fluid away from the separation and reaction zones, facilitating removal of unwanted or interfering debris (particulates, protein aggregates, unreacted test reagents) from the reaction zone upon subsequent application of wash solution and/or test reagents to the reaction zone.
  • this design functions as a valve and reduces or eliminates back-washing of unreacted components into the reaction zone which may cause high background signals.
  • the bilateral flow through the separation zones 42 is facilitated by the location of the reaction zones 40 adjacent to a relatively large area of the filter 36, shown, in the particular embodiment of the test device, as generally rectangular in configuration and overlying the second absorbent means 48.
  • this relatively large area of the filter 36 may be sufficiently absorbent to serve as the second absorbent means by itself without providing any underlying absorbent material-
  • bilateral flow established in this manner reduces the hydraulic pressure in the application zones 38 and causes rapid settling of particulates or other inclusions in the sample suspension, thus causing rapid settling out of the particulates or other detritus before reaching the reaction zones 40.
  • the second absorbent means 48 is of much larger dimensions than the absorbent means 44, causing more rapid absorption of the excess fluid of the sample and causing the accentuation of the bilateral flow phenomenon achieved by the filter design and its association with the first and second absorbent means.
  • the configuration of the application zones 38 can be readily altered to accommodate the needs of the particular samples being tested by the devices 20 and 30 and, furthermore, as specified hereinabove, the length and width of the separation zones be empirically established to conform to the bilateral flow patterns to be established for the particular sample.
  • the relative dimensions and depth of the absorbent means 44 and 48 can be altered to establish greater or lesser fluid communication between the application zones 38 and reaction zones 40, respectively.
  • a typical glass fiber matrix filter has its source in Eaton-Dikeman Division of Filtration Sciences, Mount Holly Springs, Pennsylvania.
  • the weight is 71 gm/m 2 ; the depth is 0.43 mm; the mean pore size is 0.6 micron (u) ; the mean fiber diameter is 0.7u (0.25u to l.5u) ; and the composition is borosilicate glass.
  • the dimensions of the application zone are 8 mm in diameter, and the separation zone 4 mm X 9 mm.
  • the filters 20 and 36 are described as fabricated in accordance with the previously set forth specifications, it will be obvious to those skilled in the art that the filter means can be made of any porous material capable of drawing liquid through its structure by capillary action.
  • the pores of the filter matrix should, obviously, be sufficiently small to accomplish filter separation of the non-solubilized components of the test sample from solubilized components.
  • the filter may be composed of such materials as glass fiber filter paper, nitrocellulose, plastic, synthetic polymer, cellulose, cellulose acetate, and various other equivalent materials having the qualities and characteristics described hereinabove.
  • test devices 10 and 30 have their respective reaction zones 28 and 40 treated with specific analyte reactants. Localized regions of the respective filters 20 and 36 are treated to provide the reaction zones 28 and 40 to prepare the test devices 20 and 30 for use with a predetermined test specimen without any preparatory additions to the test devices.
  • a binding protein could be placed in the reaction zones to which an antibody is bound, which antibody is immunologically reactive with a specific antigen.
  • An i muno-reagent, or chemical test reagent in the case of a biochemical test, complimentary to the test analyte or an analog thereof, conjugated with an enzyme or other suitable tracer, such as a radionuclide or fluorescent dye, may optionally be embedded in the application zones 38.
  • an enzyme or other suitable tracer such as a radionuclide or fluorescent dye
  • the absorbent material utilized in the first and second absorbent means 44 and 48 may be of any suitable material, such as hydrophilic polymers, particulate absorbents, glass fiber, carbon fiber, cellulose fiber, wood pulp or sponge material.
  • the size and shape of the respective absorbent means 44 and 48 is dictated by the volumetric considerations applicable to the specific test for which the test devices 20 and 30 are designed, and corresponding diminishment or enhancement of the absorptive capacity of the first and second absorbent means 44 and 48 result from empirical calculations of the needs for the establishment of greater or lesser bilateral flow of the fluid components of the test specimen.
  • test devices of the present invention may incorporate a variety of different control tests for use with methods of the present invention.
  • one or more of the reaction zones of a test device 30 having a multiplicity of application ports 22 may be provided which provide a positive control to verify that all reagents are performing correctly. JThis is especially important where false negative results are particularly troubling, such as a test for human chorionic gonadotropin (hCG) as an indication of pregnancy.
  • An exemplary positive control may have the analyte to be tested immobilized to the filter matrix of the reaction zone 40.
  • reaction zones 40 may be provided which have the analyte being tested immobilized in known quantities.
  • a comparison between the amount of tracer reagent bound to the control reaction zone and a test reaction zone will give an indication of the amount of analyte in the unknown.
  • This technique is especially useful where a cut-off level of analyte indicates a positive test.
  • the level of antibody providing resistance to infection with rubella virus has been determined to be a hemagglutination inhibition titer of 1:8.
  • a positive test result for rubella resistance would show an equal or greater amount of tracer reagent bound to the test reaction zone than to a positive control providing a level of tracer reagent bound for a titer of 1:8.
  • negative controls can also be provided.
  • An exemplary negative control may have a reaction zone 40 to which all of the steps of immobilizing the antibody are performed, however, without the presence of antibody.
  • Such a negative control test would be useful in determining the amount of non-specific binding of analytes or interfering components to the filter matrix. Negative control tests guard against false positive test results. In many instances it may be desirable to provide both negative and positive tests to guard against both false negatives and false positives.
  • a suitable volume of sample is applied directly to the sample application zone of the glass fiber matrix.
  • a suitable volume of wash reagent is then applied tp the same area of the sample application zone.
  • no wash reagent may be necessary if a sufficient volume of sample is added to move sample components into the reaction zone.
  • Capillary and chromatographic forces within the body of the glass fiber matrix draw the fluid portion of the sample primarily in the direction of the separation zone but, also, secondarily in the opposite direction.
  • the bilateral flow is defined by the lateral boundaries of the glass fiber matrix and the fluid communication of the application and reaction zones with their respective absorbent means.
  • particles larger than the mean pore size of the glass fiber matrix are restricted in their lateral migration toward the reaction zone, so that only the fluid portion of the sample reaches and flows into and through the reaction zone.
  • the analyte, contained within the fluid portion of the sample reacts and binds with the specific complimentary immuno-reagent (antigen or antibody) or chemical test reagents, which have been immobilized to the glass fiber matrix in the area of the reaction zone.
  • wash reagent is applied directly to the reaction zone. This washes away unreacted sample components which may interfere with subsequent steps, in bilateral directions, again defined by the lateral boundaries of the glass fiber matrix. Alternatively, this step of applying wash reagent may be omitted, with the required wash being accomplished by the subsequent application of a chromatic-eliciting substrate reagent or other solution in sufficient volume to wash away unreacted sample components.
  • Unreacted sample components are carried away by the bilateral flow of the washing step, whether accomplished by a separate wash reagent or by subsequent application of another solution.
  • the two wash directions of the bilateral flow are l) away from the separation and sample application zones, and 2) toward the separation and sample application zones, reversing the original direction of flow. Wash in the latter direction inhibits or prevents previously filtered particulates from reaching the reaction zone and actually acts as a "counter current" to back flush potential interfering particulates present in the original sample away from the reaction zone.
  • the analyte present in the fluid portion of the sample is bound to the complimentary immuno-reagent or chemical test reagent immobilized to the glass fiber matrix at the reaction zone site.
  • an immuno-reagent, or chemical test reagent in the case of a biochemical test complimentary to the test analyte or an analog thereof, conjugated with an enzyme or other suitable tracer, such as a radionuclide or fluorescent dye, is applied directly to the reaction zone.
  • Unbound immuno-reagent conjugate or chemical test reagent may optionally be washed from the reaction zone in the lateral bi-directional mode outlined above by the application of a suitable wash volume applied directly to the reaction zone.
  • the complimentary reagent-tracer conjugate may be embedded in the application zone prior to application of the sample, as described above in connection with the description of fiber matrix of the test device.
  • the conjugate is embedded in a manner which permits the conjugate to be hydrated and solubilized during the application of the sample.
  • the conjugate when sample is applied to the sample application zone, the conjugate is hydrated and will react with any analyte found in the sample.
  • an analyte-complimentary reagent-tracer conjugate will be formed.
  • the bilateral flow of the test device will then carry the analyte-complimentary reagent-tracer conjugate and all other components through the separation zone and into the reaction zone.
  • the reaction zone has an immobilized reagent complementary to the analyte present in the analyte-complimentary reagent-tracer conjugate.
  • the analyte was present in the sample, it will be sandwiched between the immobilized complimentary reagent and the enzyme conjugate immuno-reagents within the reaction zone.
  • a suitable substrate or chromogen may then be added to the reaction zone if the tracer is an enzyme requiring such a substrate to develop color.
  • the enzyme conjugated to the analyte bound immuno-reagent acts upon the substrate or chromogen to produce a colored product within the reaction zone which may be viewed or measured with an instrument.
  • the tracer-complimentary reagent conjugate is applied to the application zone in molar excess of the sample to assure binding of the sample.
  • the substrate solution serves also to wash away unreacted components from the reaction zone.
  • a wash reagent may be added prior to addition of the substrate solution to wash away unreacted components. In either case, the unreacted components are washed from the reaction zone in a bi-directional mode.
  • a tracer requiring no substrate to be detected such as a radionuclide or fluorescent dye
  • no substrate need be added.
  • a suitable wash reagent should be added to wash away unreacted components from the reaction zone.
  • Inactivated Rubella virus antigen is immobilized onto the reaction zone of the glass fiber matrix. This is followed by the addition of a blocking agent such as 2.0% bovine serum albumin or 0.5% non-fat milk suspension to the same area and allowed to dry.
  • a blocking agent such as 2.0% bovine serum albumin or 0.5% non-fat milk suspension
  • a blocking agent decreases the non-specific binding of extraneous proteins present in the fluid (serous) portion of whole blood to the reaction zone of the glass fiber matrix.
  • the whole blood specimen contains antibodies to the Rubella virus
  • the antibodies in the serous portion of the blood sample will bind to the Rubella virus antigens immobilized within the reaction zone of the glass fiber matrix.
  • 60 microliters of an affinity purified rabbit anti-human IgG conjugated to alkaline phosphatase is applied to the reaction zone. This will bind to the antibody of the Rubella virus which may be present in the blood sample and will be trapped by the immobilized antigen located in the reaction zone of the glass fiber matrix. Unreacted enzyme conjugate is washed away as described above.
  • a suitable substrate chromogen may be applied to the reaction zone. Appearance of a colored product at the reaction zone is evidence of enzyme activity and, therefore, indicative of antibody to Rubella virus present in the whole blood sample.
  • a polypeptide hormone human choriogonadotropin (hCG)
  • hCG human choriogonadotropin
  • the antibody of hCG is immobilized to the reaction zone of the glass fiber matrix.
  • a blocking protein is then applied to the reaction zone as described in the previous example.
  • a few drops of the specimen are applied to the sample application zone of the glass fiber matrix of the device. This is followed by a sufficient volume of a wash solution applied to the same area to cause the sample to migrate through the separation zone toward and through the reaction zone of the glass fiber matrix which contains the immobilized antibody to hCG. If hCG is present in the sample, it will bind to the immobilized antibody located within the reaction zone.
  • sufficient volume of urine may be applied to the sample application zone to cause the sample to chromatograph through the reaction zone without the use of a wash. In either case, migration of the urine sample through the separation zone will filter out urine particulates which may interfere in subsequent testing steps.
  • a suitable volume of a washing solution is applied to the reaction zone.
  • bi-directional flow of the wash solution will carry unreacted urine components away from the reaction zone, i.e., away from the separation and sample application zones as well as toward the separation and sample application zones, reversing the original direction of flow. Movement toward the reaction zone of wash fluid in the latter direction prohibits further movement of unwanted particulates by counter flow forces. Indeed, subsequent addition of any wash or test reagent to the area of the reaction zone will force any trapped particulates or debris located within the separation zone away from the reaction zone.
  • an appropriate enzyme labeled antibody to hCG (either polyclonal or monoclonal) will bind to the hCG of the sample which has been trapped by the immobilized antibody bound to the reaction zone of the glass fiber matrix.
  • a wash solution is applied, as indicated above, to wash away, in a lateral, bi-directional mode, any unreacted enzyme conjugated antibody.
  • a suitable substrate chromogen solution to the reaction zone will indicate the presence of enzyme and, therefore the presence of hCG, by the development of a colored product at the reaction zone.
  • the method described above in this example is typical of a "sandwich technique," whereby the analyte, hCG in this case, is sandwiched between two antibodies, one immobilized to the glass fiber matrix of the reactipn zone, the other conjugated to an enzyme or other suitable label.
  • the presence of the hCG analyte is indicated by the development of color within the reaction zone.
  • test devices of the invention are not limited to "sandwich” methodology, but may be applied to competitive inhibition techniques as described by the following example.
  • Antibody immobilization, sample application and washing methods and separation/chromatographic principles are as described in the previous example.
  • an antibody enzyme conjugate one may apply to the reaction zone an enzyme conjugate of the analyte, i.e., hCG coupled to an appropriate enzyme.
  • hCG If hCG is present in the sample, it will bind to a finite and limited number of available antibody binding sites located and immobilized within the reaction zone of the glass fiber matrix. If the sample contains substantial amounts of hCG, then all available antibody binding sites in the reaction zone will be saturated. Upon subsequent application of an enzyme conjugated to hCG (instead of enzyme conjugated to an anti-hCG antibody) , all available immobilized antibody binding sites are saturated with the hCG from the sample and will not bind to the enzyme-hCG conjugate. When a suitable wash solution is applied, the enzyme-hCG conjugate will be washed away from the reaction zone in a lateral, bi-directional fashion.
  • hCG enzyme conjugate will bind to the available immobilized hCG binding sites and will not be washed away with subsequent washing steps.
  • the absence of color development in the reaction zone is indicative of the presence of the analyte (hCG) in the sample, while the presence of color development in the reaction zone indicates little or no analyte (hCG) in the sample.
  • the device can also be used for competitive immuno ⁇ assays of low molecular weight analytes, such as thyroid hormones, therapeutic drugs, steroids and other low molecular weight analytes.
  • enzyme labeled antibody to an epitope on the ⁇ chain of hCG (anti- ⁇ hCG) is applied and allowed to dry, but not immobilized.
  • the enzyme portion of the enzyme labeled antibody is alkaline phosphatase.
  • An antibody to an epitope on the ⁇ chain of hCG (anti- ochCG) is immobilized to one reaction zone of the glass fiber matrix and blocking protein applied as in Example 2.
  • This* reaction zone, to which anti- ⁇ hCG has been applied is the test reaction zone.
  • the positive control reaction zone antibody reactive with the anti-,5hCG embedded in the application zones (anti-anti- ⁇ hCG) is immobilized. Both reaction zones are included within a test device of the present invention.
  • a few drops of the sample are applied to the sample application zone of both application zones of the device.
  • the application of sample rehydrates the enzyme labeled antibody present in the application zone. If hCG is present in the sample, it will bind to the antibody portion of the enzyme labelled antibody.
  • a sufficient volume of sample is applied to both application zones to cause the sample to migrate through the respective separation zones toward and into the reaction zones. Thus, any hCG-enzyme labeled antibody complexes are carried into the reaction zones.
  • hCG-antibody complexes carried into the test reaction zone, these complexes will bind to the immobilized antibody found therein.
  • enzyme labeled antibody which is carried into the control reaction zone will bind to the anti-anti / 3hCG found therein regardless of the presence of hCG in the sample.
  • chromogen substrate is then added to each reaction zone in order to elicit color.
  • the volume of chromogen solution applied to each application zone is sufficient so that lateral, bi-directional flow of the sample will occur to carry unreacted urine components and enzyme labeled antibody away from the reaction zones, i.e., away from the separation and sample application zones as well as toward the separation and sample application zones, reversing the original direction of flow. Movement toward the reaction zones of fluid in the latter direction prohibits further movement of unwanted particulates by counter flow forces, as in the step of applying wash reagent of Example 2.
  • the chromogen solution indicates the presence of the enzyme at the reaction zone, and therefore the presence of hCG in the test sample, by the development of a colored product at the reaction zone.
  • the positive control reaction zone shows the strong presence of a colored product in all cases where the reagents are performing correctly.
  • the test zone indicates a colored product only if hCG is present in the sample.
  • the antibody to Strep-A antigen is immobilized to the reaction zone of a glass fiber matrix and blocking protein applied. Enzyme labeled antibody is applied, without immobilization, to the application zone.
  • a throat swab extract sample contains Strep-A antigen
  • a few drops of the extract sample are applied to the sample application zone.
  • Strep-A antigens will bind to the enzyme labeled antibody present in the application zone and be carried into the reaction zone.
  • Example 6 Detection of Glucose in Whole Blood
  • the device may be used to perform assays to indicate the presence or quantitation of analytes without employing immunological methods and principles. For example, one may detect the presence of glucose in whole blood by standard enzyme analytical techniques. In this instance, a mixture of the enzymes glucose oxidase and horseradish peroxidase is immobilized to the reaction zone of the device. Whole blood is then applied to the sample application zone.
  • a suitable wash solution is then applied to the sample application zone to effect the bi-directional lateral chromatographic separation of the fluid portion of the sample from the cellular components as described previously to introduce the fluid portion containing glucose into the reaction zone.
  • the immobilized oxidase acts upon the glucose of the sample to produce D-glucono- ⁇ -lactone and hydrogen peroxide.
  • the horseradish peroxidase also immobilized within the reaction zone, catalyses the hydrogen peroxide in situ as it is generated.
  • a suitable chromogen test reagent will react with the products of catalysis to produce a colored product, the intensity of which is proportional to the amount of glucose present in the original sample.
  • the intensity of color development may be observed visually or detected by the use of suitable instrumentation. It is evident from this example that the device of the invention accomplishes other than immunoassays with equal effectiveness.
  • test devices manufactured in accordance with the teachings of this invention provides both more effective and less time-consuming testing of various suspensions in the field by relatively inexperienced personnel.
  • the bilateral migration of the fluid components of the various samples applied to the application zones attributable to the unique construction of the test devices prevents the contamination of the reaction zones by the particulates in the suspension samples and also facilitates the migration of the fluid component of the sample to the reaction zones.
  • test devices manufactured in accordance with the teachings of the invention and incorporating single application, separation and reaction zones may be snapped together or otherwise associated on a mounting board or the like to permit a series of different tests to be accomplished by juxtaposition of the single test devices.

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  • Analytical Chemistry (AREA)
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Abstract

Dispositif de test chromatique (10, 30) comportant un corps fibreux blanc unitaire (20, 36) comprenant une zone d'application d'échantillon (26, 38), une zone de séparation (42) ainsi qu'une zone de réaction (28, 40), et procédés d'utilisation du dispositif. La zone d'application (26, 38) du dispositif est en communication fluide avec un premier moyen absorbant (44) et la zone de réaction (28, 40) est en communication fluide avec un second moyen absorbant (48) afin d'établir un écoulement bidirectionnel de constituant fluide d'un échantillon appliqué au corps de filtre fibreux.
PCT/US1991/002403 1990-04-09 1991-04-09 Procedes de test chromatographique lateral bidirectionnel WO1991015769A1 (fr)

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US506,914 1983-06-22
US50691490A 1990-04-09 1990-04-09

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WO1991015769A1 true WO1991015769A1 (fr) 1991-10-17

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Cited By (12)

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Publication number Priority date Publication date Assignee Title
EP0553770A3 (en) * 1992-01-31 1993-08-25 Boehringer Mannheim Gmbh Analytical element for immunoassays
WO1995017676A1 (fr) * 1993-12-23 1995-06-29 Orgenics International Holdings B.V. Appareil de separation, concentration et detection de molecules cibles dans un echantillon liquide
WO1995027081A1 (fr) * 1994-03-31 1995-10-12 E.I. Du Pont De Nemours And Company Methode de detection de fragments d'acide nucleique
EP0600929A4 (en) * 1991-07-31 1996-01-17 Idexx Lab Inc Reversible flow chromatographic binding assay.
EP0634016A4 (fr) * 1992-03-27 1997-06-04 Abbott Lab Controle de verification de dosage pour methodes d'analyse.
FR2759782A1 (fr) * 1997-02-14 1998-08-21 Unilever Nv Dispositif de dosage
US6007999A (en) * 1991-07-31 1999-12-28 Idexx Laboratories, Inc. Reversible flow chromatographic binding assay
US6037127A (en) * 1994-03-31 2000-03-14 E. I. Du Pont De Nemours And Company Method for detection of non-denatured nucleic acid fragments
US6143510A (en) * 1994-07-29 2000-11-07 Iatron Laboratories Inc. Measuring method using whole blood sample
WO2011057025A2 (fr) 2009-11-04 2011-05-12 Buchanan Thomas M Procédés et dispositifs pour augmenter la sensibilité et évaluer l'adéquation de l'échantillon et la réactivité du réactif dans les dosages immunologiques rapides à écoulement latéral
CN105987547A (zh) * 2015-02-11 2016-10-05 浙江三花股份有限公司 双向干燥过滤器
WO2017035389A1 (fr) * 2015-08-27 2017-03-02 Quidel Corporation Dispositif d'analyse par dosage immunologique comportant deux trajets d'écoulement de fluide pour la détection et la différentiation d'au moins deux analytes

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US4857453A (en) * 1987-04-07 1989-08-15 Syntex (U.S.A.) Inc. Immunoassay device
US4900663A (en) * 1985-09-13 1990-02-13 Environmental Diagnostics, Inc. Test kit for determining the presence of organic materials and method of utilizing same

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Publication number Priority date Publication date Assignee Title
US4235601A (en) * 1979-01-12 1980-11-25 Thyroid Diagnostics, Inc. Test device and method for its use
US4435504A (en) * 1982-07-15 1984-03-06 Syva Company Immunochromatographic assay with support having bound "MIP" and second enzyme
US4678757A (en) * 1985-04-11 1987-07-07 Smithkline Diagnostics, Inc. Device and method for whole blood separation and analysis
US4900663A (en) * 1985-09-13 1990-02-13 Environmental Diagnostics, Inc. Test kit for determining the presence of organic materials and method of utilizing same
US4761381A (en) * 1985-09-18 1988-08-02 Miles Inc. Volume metering capillary gap device for applying a liquid sample onto a reactive surface
US4857453A (en) * 1987-04-07 1989-08-15 Syntex (U.S.A.) Inc. Immunoassay device

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5726010A (en) * 1991-07-31 1998-03-10 Idexx Laboratories, Inc. Reversible flow chromatographic binding assay
US6007999A (en) * 1991-07-31 1999-12-28 Idexx Laboratories, Inc. Reversible flow chromatographic binding assay
EP0600929A4 (en) * 1991-07-31 1996-01-17 Idexx Lab Inc Reversible flow chromatographic binding assay.
US5750333A (en) * 1991-07-31 1998-05-12 Idexx Laboratories, Inc. Reversible flow chromatographic binding assay
US5726013A (en) * 1991-07-31 1998-03-10 Idexx Laboratories, Inc. Reversible flow chromatographic binding assay system, kit, and method
US5424220A (en) * 1992-01-31 1995-06-13 Boehringer Mannheim Gmbh Analysis element and method for determination of an analyte in a liquid sample
EP0553770A3 (en) * 1992-01-31 1993-08-25 Boehringer Mannheim Gmbh Analytical element for immunoassays
EP0634016A4 (fr) * 1992-03-27 1997-06-04 Abbott Lab Controle de verification de dosage pour methodes d'analyse.
US5698395A (en) * 1993-12-23 1997-12-16 Orgenics Ltd Apparatus for separation, concentration and detection of target molecules in a liquid sample
WO1995017676A1 (fr) * 1993-12-23 1995-06-29 Orgenics International Holdings B.V. Appareil de separation, concentration et detection de molecules cibles dans un echantillon liquide
WO1995027081A1 (fr) * 1994-03-31 1995-10-12 E.I. Du Pont De Nemours And Company Methode de detection de fragments d'acide nucleique
US6037127A (en) * 1994-03-31 2000-03-14 E. I. Du Pont De Nemours And Company Method for detection of non-denatured nucleic acid fragments
US6143510A (en) * 1994-07-29 2000-11-07 Iatron Laboratories Inc. Measuring method using whole blood sample
FR2759782A1 (fr) * 1997-02-14 1998-08-21 Unilever Nv Dispositif de dosage
WO2011057025A2 (fr) 2009-11-04 2011-05-12 Buchanan Thomas M Procédés et dispositifs pour augmenter la sensibilité et évaluer l'adéquation de l'échantillon et la réactivité du réactif dans les dosages immunologiques rapides à écoulement latéral
CN102782495A (zh) * 2009-11-04 2012-11-14 托马斯·M·布坎南 用于在快速侧流免疫测定中增强灵敏度以及评估样品充足性和试剂反应性的方法和装置
EP2496941A4 (fr) * 2009-11-04 2013-11-06 Thomas M Buchanan Procédés et dispositifs pour augmenter la sensibilité et évaluer l'adéquation de l'échantillon et la réactivité du réactif dans les dosages immunologiques rapides à écoulement latéral
CN102782495B (zh) * 2009-11-04 2014-12-31 托马斯·M·布坎南 用于在快速侧流免疫测定中增强灵敏度以及评估样品充足性和试剂反应性的方法和装置
CN105987547A (zh) * 2015-02-11 2016-10-05 浙江三花股份有限公司 双向干燥过滤器
WO2017035389A1 (fr) * 2015-08-27 2017-03-02 Quidel Corporation Dispositif d'analyse par dosage immunologique comportant deux trajets d'écoulement de fluide pour la détection et la différentiation d'au moins deux analytes
US11131670B2 (en) 2015-08-27 2021-09-28 Quidel Corporation Immunoassay test device with two fluid flow paths for detection and differentiation of two or more analytes
US11846637B2 (en) 2015-08-27 2023-12-19 Quidel Corporation Immunoassay test device with two fluid flow paths for detection and differentiation of two or more analytes
EP4538705A3 (fr) * 2015-08-27 2025-07-02 Ortho-Clinical Diagnostics, Inc. Dispositif d'essai immunologique avec deux voies d'écoulement de fluide pour la détection et la différenciation de deux analytes ou plus

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