Method and apparatus for assaying whole blood

Abstract

A method of assaying a sample of whole blood for one member of a specific binding pair, the method involving treating the sample with salt to alter the red blood cells in the sample to decrease their ability to pass through filter media, exposing the resulting sample to a filter which retains the red blood cells but allows the passage therethrough of filtrate of the sample containing the member of the specific binding pair, and measuring the member of the filtrate.

Claims

I claim: 1. A method for assaying a sample of whole blood for a first member of a specific binding pair, said method comprising treating the sample with a salt to alter the red blood cells in the sample to decrease the ability of the red blood cells to pass through filter media, exposing the treated sample to a filter which retains the altered red blood cells but allows passage therethrough of a sample filtrate, and contacting any of the first member in the filtrate with a second member of the specific binding pair to permit binding of the first member with the second member, whereby said first member is determined. 2. The method of claim 1 wherein said treating substantially increases the rigidity of the red blood cells in the sample. 3. The method of claim 1 wherein the treating step comprises mixing the sample with a hypertonic solution of said salt having an ionic strength at least 0.5M. 4. The method of claim 1 wherein, prior to the step of exposing the sample to a filter, a non-ionic detergent is added to the sample. 5. The method of claim 1 wherein said contacting step comprises contacting the filtrate with a solid support to affix the first specific binding pair member to said solid support. 6. The method of claim 5 wherein said solid support and said filter are in mutual contact when the sample is exposed to said filter. 7. The method of claim 1 wherein the first specific binding pair member is a virus or an antibody against a virus. 8. The method of claim 7 wherein the virus is HTLV-3. 9. A method for assaying feline leukemia antigen in a sample of whole blood, comprising treating the sample with a salt to alter the red blood cells in the sample to decrease the ability of the red blood cells to pass through filter media, exposing the treated sample to a filter which retains the altered red blood cells but allows passage therethrough of a sample filtrate containing the antigen, and contacting any of the first member in the filtrate with a second member of the specific binding pair to permit binding of the first member with the second, whereby said first member is determined. 10. The method of claim 9 wherein the treating step comprises mixing the sample with a hypertonic solution having an ionic strength at least 0.5M. 11. The method of claim 10 wherein said hypertonic solution comprises KCl and sodium citrate. 12. The method of claim 11 wherein said hypertonic solution further comprises nonionic detergent. 13. The method of claim 12 wherein said solution further comprises bovine serum albumin and non-fat dry milk. 14. A kit for assaying a sample of whole blood for one member of a specific binding pair, said kit comprising a first container holding a hypertonic solution, a second container for mixing said hypertonic solution with the sample, a filter positioned with respect to said second container so as to permit a filtrate of said mixture of the sample with said hypertonic solution to pass from said second container through said filter, said filter being capable of retaining the red blood cells in said mixture, and means for collecting the filtrate. 15. The kit of claim 14 wherein said collecting means comprises a solid support. 16. A method for assaying a sample of whole blood for a first member of a specific binding pair, said method comprising: (a) first forming a solution comprising the sample and sufficient salt to alter red blood cells in the sample to decrease the ability of the red blood cells to pass through a filter media; (b) exposing said solution to a filter selected retain the altered red blood cells and to allow passage therethrough of a filtrate, said filter being able to pass unaltered red blood cells; and (c) contacting any of the first member in the filtrate with a second member of the specific binding pair to permit binding of the first member with the second member, whereby the first member is determined. 17. The method of claim 1 or claim 16 in which said filter is wetted with a buffer before sample is exposed to the filter. 18. The method of claim 1 in which said filter is selected to retain the altered red blood cells, to allow passage of a filtrate, and able to pass unaltered red blood cells therethrough.
BACKGROUND OF THE INVENTION This invention relates to the performance of immunoassays on whole blood. Immunoassays are carried out to detect a particular antigen, hapten, or antibody in blood. Since red blood cells interfere with such assays, they are normally removed from the blood prior to performing the assay, for example, by centrifugation or filtration. The resulting plasma solution can then be used in any standard immunoassay, e.g., as described by Gerber et al. U.S. Pat. No. 4,503,143, hereby incorporated by reference, which describes a colorimetric method of detection of bindable substances such as antigens, using chromogenic agents such as tetramethyl benzidine. SUMMARY OF THE INVENTION In general, the invention features a method of assaying a sample of whole blood for one member of a specific binding pair, involving treating the sample with a salt to alter the red blood cells in the sample to decrease their ability to pass through filter media, exposing the resultant sample to a filter which retains the red blood cells but allows the passage therethrough of filtrate containing the member of the specific binding pair being assayed, and measuring the member of the filtrate. As used herein, the term "specific binding pair" means any pair of molecules which bind to each other but do not bind to a significant degree to other molecules; examples of such pairs are antigens and antibodies specific therefor. It is believed that the salt causes the red blood cells in the sample to exhibit a change in physical properties, e.g., deformability, so that they are unable to pass through filter media which would have permitted their passage prior to treatment with the salt. A filter having an average pore size small enough to retain untreated red blood cells would become clogged and prevent passage of liquid as well. According to the invention, a filter can be used which has an average pore size (nominal size 0.3-0.6 microns) large enough to prevent clogging, but which still retains the treated red blood cells. In preferred embodiments of the method, the step of treating the sample with a salt involves mixing the sample with a hypertonic solution to form a dilute solution, and following the exposing of the sample to the filter, the filtrate is deposited on a solid support on which the measuring of the member of the specific binding pair is carried out; preferably, the solid support and the filter are in contact with one another when the sample is exposed to the filter. Preferably, the hypertonic solution contains a salt of ionic strength of at least 0.5M. The method of the invention is preferably carried out using a kit composed of a first container holding a hypertonic solution, a second container for holding the hypertonic solution and mixing it with the sample, a filter positioned with respect to the second container so as to permit the mixture of the sample and the hypertonic solution to pass from the container through the filter, such filter being capable of retaining the treated red blood cells in the sample, and means e.g., a solid support, for collecting the sample after it has passed through the filter. The invention allows the rapid and simple assay of whole blood samples for any immunologically detectable compound, without prior removal of red blood cells. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. DESCRIPTION OF THE PREFERRED EMBODIMENT The FIGURE is a diagrammatic representation of an assay filter assembly of the invention. STRUCTURE Referring to the FIGURE, cylindrical membrane assembly 10 includes filter holder 12 fitted with filter 14 to form fluid chamber 11, and an assay membrane holder 16 fitted with assay membrane 18. Membrane holder 16 and filter holder 12 may be constructed of any suitable nonporous material, for example, PVC. Filter holder 12 has an annular groove which mates with annular ridge 22 on assay membrane holder 16 so that filter 14 is positioned in intimate and homogeneous contact with assay membrane 18, which is supported by porous support disc 19, which in turn overlies absorbent bed 21. Assay membrane 18 bears immunologically reactive material 24 which consists, for example, of latex microspheres 1 micron in diameter coated with a suitable immunologically reactive compound, for example, an antibody, or of a bioreactive zone prepared by binding the immunologically reactive compound directly to a solid support consisting of either an activated surface or an inert surface capable of irreversibly binding the immunologically reactive compound. Assembly 10 is of a diameter suitable for holding membrane 18, for example, 1 cm. Membrane 18 and filter 14 are attached to their respective holders by glue, or other means which prevents leakage between the holder and membrane. Filter 14 is capable of retaining red blood cells in a mixture of whole blood and salt while allowing passage therethrough of red blood cell-free liquid. Filter 14 can e.g., be a filter having fibers lying on top of one another with a thickness of 1/4 to 1/2 mm and a nominal pore size rating of 0.4 microns, for example, a glass AE filter (Gelman). Assay membrane 18 can be any standard assay membrane, e.g., a glass AE filter. The salt (not shown) is provided in any suitable container, e.g. a cylindrical PVC container, preferably as a buffer solution. The buffer is hypertonic, preferably with an ionic strength greater than 0.5M. In order to reduce the background level of reactivity of the assay membrane with blood solutions, the buffer also contains proteins unreactive with the assay membrane, for example, bovine serum albumin and non-fat dry milk. Preferably, the buffer also contains nonionic detergents, e.g., Tween 80 or Triton X-100 which increase the sensitivity of the assay. ASSAY In the first step of the assay, filter 14 is wetted with buffer. Whole blood from the animal or human patient to be tested (approximately 3 drops (0.1 ml)) is gently mixed with 0.5 ml of buffer and the resulting treated whole blood solution is poured into the assembled membrane filter assembly having filter 14 in contact with assay membrane 18. The red blood cells in the blood are retained by the filter, and the red cell-free liquid containing the component to be detected passes through the filter to the assay membrane. After the filtration Process, which takes approximately 1-5 minutes, is complete the filter assembly 12 is removed from the assay membrane and the assay membrane is processed using standard technique to detect the blood component being assayed. The following example is illustrative. EXAMPLE A: Detection of Feline Leukemia Antigen Antibodies to feline leukemia antigen (commercially available) are bound to latex microspheres by standard techniques and then spotted onto an assay membrane (Gelman AE filter) fixed to a membrane holder as in the FIGURE. A Gelman AE filter is mounted above and in contact with membrane 18, as shown in the FIGURE. Blood is drawn from a cat by a standard procedure and 3 drops (approximately 0.1 ml) mixed with 0.5 ml of a buffer containing 0.25 molar sodium citrate, one molar KCl, 5% bovine serum albumin, 2.5% non-fat dry milk, and 0.1% Tween 80. The resulting dilute whole blood solution is then poured into the assembly and given 3 minutes to pass through the filter onto the assay membrane. The filter assembly is then removed and the assay membrane treated to determine the presence or absence of feline leukemia antigen using conventional techniques. Generally, after the leukemia antigen has bound to anti-leukemia antibody on the membrane, enzyme-labeled anti-leukemia antibody is added to form a "sandwich", and color is developed using an enzyme substrate and colormetric indicator. Other embodiments are within the following claims. For example, although it is preferred that the red cell-free liquid be collected on a solid support as described above, the liquid can also be collected in a container and assayed using, e.g., a homogeneous assay method. Any hypertonic solution can be used which causes the required change in shape and deformability of the red cells in the blood, e.g., other salts, and sugar solutions. Any member of a specific binding pair can be measured, for example the assay can be used to screen whole blood samples for HTLV-3 virus or antibodies to this virus to test for Acquired Immune Deficiency Syndrome (AIDS).

Description

Topics

Download Full PDF Version (Non-Commercial Use)

Patent Citations (23)

    Publication numberPublication dateAssigneeTitle
    EP-0140337-A2May 08, 1985EASTMAN KODAK COMPANY (a New Jersey corporation)Multizone element and method for analysis of whole blood
    EP-0143574-A1June 05, 1985Syntex (U.S.A.) Inc.Bestimmungsmethode und Testsatz für Proben von Vollblut
    EP-0159727-A2October 30, 1985Fuji Photo Film Co., Ltd.Elément de test pour analyse d'un échantillon de sang entier
    EP-0190488-A1August 13, 1986Becton Dickinson and CompanySéparation de lymphocytes et monocytes d'échantillons de sang
    FR-2344842-A1October 14, 1977Ortho DiagnosticsTraitement de cellules biologiques indicatrices
    GB-2105210-AMarch 23, 1983Michael Peter Entis, Phyllis EntisFiltration apparatus
    US-3552925-AJanuary 05, 1971Miles LabWhole blood separation method and test using same
    US-3763879-AOctober 09, 1973Boehringer Mannheim GmbhCombination column
    US-3825410-AJuly 23, 1974K BagshawePerformance of routine chemical reactions in compartmentalized containers
    US-3929583-ADecember 30, 1975Canadian Patents DevApparatus for enumerating microorganisms
    US-3963119-AJune 15, 1976Lucaks And Jacoby AssociatesSerum separating apparatus
    US-3972812-AAugust 03, 1976Becton, Dickinson And CompanyBlood serum separation filter disc
    US-4090850-AMay 23, 1978E. R. Squibb & Sons, Inc.Apparatus for use in radioimmunoassays
    US-4111807-ASeptember 05, 1978Gelman Instrument CompanyMouth filter for use with pipettes
    US-4301010-ANovember 17, 1981Spectrum Medical Industries, Inc.Vacuum filter
    US-4397955-AAugust 09, 1983Phyllis Entis, Entis Michael PTransfer apparatus for microorganisms
    US-4406786-ASeptember 27, 1983Carl Schliecher & Schuell Gmbh & Co., KgPressure filtering device
    US-4424279-AJanuary 03, 1984QuidelRapid plunger immunoassay method and apparatus
    US-4503143-AMarch 05, 1985Btc Diagnostics Limited PartnershipEnzyme immunoassay with two-part solution of tetramethylbenzidine as chromogen
    US-4594327-AJune 10, 1986Syntex (U.S.A.) Inc.Assay method for whole blood samples
    US-4632901-ADecember 30, 1986Hybritech IncorporatedMethod and apparatus for immunoassays
    US-4727019-AFebruary 23, 1988Hybritech IncorporatedMethod and apparatus for immunoassays
    WO-7901120-A1December 27, 1979Department Of CommerceAppareil de filtration pour la separation de suspensions liquides contenant des cellules sanguines

NO-Patent Citations (9)

    Title
    Brodsky et al. (1982), J. Food Protect., 45:292 96.
    Brodsky et al. (1982), J. Food Protect., 45:301 04.
    Chem. Abstracts (1985), vol. 102, p. 484, Abstract No. 22174m.
    Entis et al. (1982), Appl. and Env. Microbiol., 43:261 68.
    Entis et al. (1982), J. Food Protect, 45:8 11.
    G. Arterioscler. (1983) (Suppl. 2), pp. 87 94 (AR ).
    Innovative Products for Separation Science Ad.
    New Brunswick Scientific Co. Ad The Iso Grid System .
    Snyder et al., Activation of Complement by Blood Transfusion Filters , Vox Sang., 45:288 293 (1983).

Cited By (26)

    Publication numberPublication dateAssigneeTitle
    US-2003153094-A1August 14, 2003Board Of Trustees Of Michigan State UniversityConductimetric biosensor device, method and system
    US-2004058316-A1March 25, 2004Jensen Wayne A., Lappin Michael R., Rosen David K., Andrews Janet S.Use of recombinant antigens to determine the immune status of an animal
    US-2008108517-A1May 08, 2008Board Of Trustees Of Michigan State UniversityConductimetric biosensor device, method and system
    US-2008286295-A1November 20, 2008Jensen Wayne A, Lappin Michael R, Rosen David K, Andrews Janet SUse of recombinant antigens to determine the immune status of an animal
    US-2008305963-A1December 11, 2008Board Of Trustees Of Michigan State UniversityConductimetric biosensor device, method and system
    US-2008314766-A1December 25, 2008Board Of Trustees Of Michigan State UniversityElectrically-active ferromagnetic particle conductimetric biosensor test kit
    US-2009123939-A1May 14, 2009Board Of Trustees Of Michigan State UniversityBiologically enhanced electrically-active magnetic nanoparticles for concentration, separation, and detection applications
    US-5073344-ADecember 17, 1991Porex Technologies Corp.Diagnostic system employing a unitary substrate to immobilize microspheres
    US-5084240-AJanuary 28, 1992Cirrus Diagnostics Inc.Centrifuge vessel for automated solid-phase immunoassay
    US-5098845-AMarch 24, 1992Cirrus Diagnostics, Inc.Device and procedure for automated solid-phase immunoassay
    US-5166051-ANovember 24, 1992Genesis Labs, Inc.Membranes, membrane overlays for exclusion of erythrocytes, and method for immunoassay of whole blood analytes
    US-5256372-AOctober 26, 1993Idexx CorporationDipstick test device including a removable filter assembly
    US-5258309-ANovember 02, 1993Cirrus Diagnostics, Inc.Procedure for automated solid-phase immunoassay using a centrifuge tube
    US-5318748-AJune 07, 1994Cirrus Diagnostics, Inc.Centrifuge vessel for automated solid-phase immunoassay having integral coaxial waste chamber
    US-5389338-AFebruary 14, 1995Orgenics Ltd.Apparatus for dry chemical analysis of fluids
    US-5846838-ADecember 08, 1998Smithkline Diagnostics, Inc.Opposable-element assay device employing conductive barrier
    US-5869345-AFebruary 09, 1999Smithkline Diagnostics, Inc.Opposable-element assay device employing conductive barrier
    US-6344337-B1February 05, 2002Board Of Trustees Of Michigan State UniversityAntigen test to detect equine protozoal myeloencephalitis in horse serum and cerebrospinal fluid
    US-6458528-B1October 01, 2002Idexx Laboratories, Inc.Diagnosis of feline immunodeficiency virus infection using ENV/GAG polypeptide markers
    US-6517593-B1February 11, 2003Larry Don Robertson, Robert Allen GarrisonMBI vortex bioaerosol cassette insert
    US-6632271-B2October 14, 2003Larry Don Robertson, Robert Allen GarrisonMBI bioaerosol vortex cassette
    US-8287810-B2October 16, 2012Board Of Trustees Of Michigan State UniversityElectrically-active ferromagnetic particle conductimetric biosensor test kit
    US-8859297-B2October 14, 2014Board Of Trustees Of Michigan State UniversityDetection of conductive polymer-labeled analytes
    US-8936946-B2January 20, 2015Board Of Trustees Of Michigan State UniversityBiologically enhanced electrically-active magnetic nanoparticles for concentration, separation, and detection applications
    US-9448236-B2September 20, 2016Board Of Trustees Of Michigan State UniversityBiologically enhanced electrically-active magnetic nanoparticles for concentration, separation, and detection applications
    US-9488650-B2November 08, 2016Board Of Trustees Of Michigan State UniversityDetection of conductive polymer-labeled analytes