EP0460094A1 - Prevention de faux resultats positifs dans des procedes de test elisa - Google Patents

Prevention de faux resultats positifs dans des procedes de test elisa

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Publication number
EP0460094A1
EP0460094A1 EP19900904497 EP90904497A EP0460094A1 EP 0460094 A1 EP0460094 A1 EP 0460094A1 EP 19900904497 EP19900904497 EP 19900904497 EP 90904497 A EP90904497 A EP 90904497A EP 0460094 A1 EP0460094 A1 EP 0460094A1
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EP
European Patent Office
Prior art keywords
antibodies
iacs
antibody
indicator
serum
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP19900904497
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German (de)
English (en)
Inventor
Abdus S. Mia
Mary M. Tierney
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mallinckrodt Veterinary Inc
Original Assignee
Pitman Moore Inc
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 Pitman Moore Inc filed Critical Pitman Moore Inc
Publication of EP0460094A1 publication Critical patent/EP0460094A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • 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
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to the use of enzyme-linked iiranunosorbent assays to test for the presence of an antigen in a sample and particularly to the prevention of false positive results in enzyme-linked immunosorbent assays caused by specific reactions with interference antibodies.
  • Enzyme-linked immunosorbent assays are used as a diagnostic tool to test for the presence of an antigen in an animal tissue such as serum, plasma or blood.
  • an animal tissue such as serum, plasma or blood.
  • a capture antibody (12) specific for the test antigen (14) is immobilized (adsorbed) onto the surface of a solid support (10) such as a test tube or microliter well.
  • An aliquot of serum to be tested for the presence of antigen is reacted with the adsorbed capture antibody; the antigen, if present in the serum, will form an antibody-antigen complex with the capture antibody.
  • an enzyme-linked antibody also specific for the antigen is reacted with the antigen-antibody complex.
  • the enzyme-linked antibody contains a covalently bound enzyme (18) which can be used as an indicator; the antibody-enzyme conjugate effectively functions as an indicator antibody (16). Finally, non-reacted indicator antibody is washed away and a substrate is reacted with the enzyme on any indicator antibody that remains bound to the support via the antigen and the capture antibody. The presence of the enzyme product indicates the presence of the antigen in the serum - a positive result.
  • the capture and indicator antibodies are typically monoclonal antibodies which have antigen combining sites for different antigenic determinants on the antigen and are generally produced from the same species.
  • Enzyme-linked immunosorbent assays do not always function properly; a false positive result may be obtained when the indicator antibody becomes bound to the capture antibody even though no antigen is present in the test serum.
  • FIG. 2 One example of how a false positive result can be obtained is shown diagrammatically in Figure 2.
  • the capture antibody (22) is bound to the support (20).
  • the indicator antibody (26) is bound to the capture antibody through non-specific interactions (24) such as hydrophobic interactions, hydrogen bonds, Van der Waals forces, ionic interactions, and the like.
  • the indicator antibody remains bound to the support through non-specific interactions with the capture antibody.
  • Addition of the substrate for the covalently bound enzyme (28) will produce the enzyme product which indicates the presence of the antigen in the test sample - a false positive result.
  • Methods for avoiding false positive reactions due to the non-specific interactions of the capture antibody and the indicator antibody include the addition of excess protein to the test sample.
  • excess protein such as bovine serum albumin (BSA), animal serum from murine, bovine, ovine, human, and the like to prevent false positive results caused by these non-specific interactions.
  • BSA bovine serum albumin
  • the excess protein competes with the indicator antibody for the non-specific interaction sites on the capture antibody effectively lowering the incidence of non-specific binding between the capture and indicator antibodies thus preventing false positive results.
  • FIG. 3 A second example of how a false positive result can be obtained is shown diagrammatically in Figure 3.
  • the adsorbed capture antibody (32) is bound to the support (30).
  • the serum sample to be tested contains a interference antibody (32) which has an antigenic combining site specific for an antigenic determinant on the capture antibody and for an antigenic determinant on the indicator antibody (36).
  • the interference antibody binds to the capture antibody and to the indicator antibody; the indicator antibody is effectively bound to the support even though no antigen was present in the test serum.
  • Addition of the substrate for the covalently bound enzyme (38) will produce the enzyme product which indicates that the antigen was in the test sample - a false positive result.
  • the interference antibody is typically present in serum samples from animals that have had their immune system exposed to the immunoglobulins of the species used to prepare monoclonal antibodies which are used to produce the capture and indicator antibodies. These immunoglobulins act as an antigen which induce the formation of the interference antibody in the animal.
  • cats are often tested for the presence of Feline Leukemia Virus (FeLV) antigen using capture and indicator monoclonals prepared from mice or rats. If the cat to be tested has been chasing mice or rats and, through some mechanism, has had its immune system exposed to mouse or rat immunoglobulin, the cat will produce an interference antibody which will bind to the murine capture and indicator antibodies to produce a positive test for FeLV even though the cat serum does not contain FeLV antigen.
  • FeLV Feline Leukemia Virus
  • mice Similarly, cats exposed to rats whose immunoglobulins cross react with those of mice may cause false positive reactions in some instances. Also, animals can be exposed to immunoglobulins from another species through vaccines, e.g. murine immunoglobulin components of rabies vaccines for cats and other animals.
  • vaccines e.g. murine immunoglobulin components of rabies vaccines for cats and other animals.
  • Feline Leukemia Virus Feline Leukemia Virus is a cause of lymphosarcoma, the most commonly occurring feline neoplasm. It also induces non-regenerative anemia; a "panleukopenia-like" syndrome of dysentery and leukopenia; and a "fading kitten" syndrome resulting from atrophy of the thymus. Myeloid disorders, fetal resorptions and abortions are thought to be attributable to FeLV infection. However, all cats with these conditions may not be infected with FeLV. Sixty to 90% of cats with lymphosarcoma and 40-70% of cats with non-regenerative anemia have been found to be infected with FeLV.
  • FeLV predisposes infected cats to a variety of secondary diseases.
  • a great majority of cats suffering from hemobartonellosis, septicemia, feline infectious peritonitis, toxoplasmosis, glomerulonephritis, chronic oral ulcers and chronic skin conditions also have been found to be infected with FeLV. It has been observed that the incidence of leukemia was approximately 900% greater in FeLV-infected cats than in non-infected cats in the same households. The incidence of diseases other than leukemia was reported to be 400% greater in the FeLV-infected cats. Kittens may acquire FeLV in utero, but more commonly infection results from prolonged contact of susceptible cats with those having FeLV.
  • a FeLV enzyme-linked immunosorbent assay diagnostic test kit which determines if cats are infected with FeLV by detecting the presence of FeLV group-specific antigens in the cat's blood is sold by Pitman-Moore, Inc. of Terre Haute, IN under the trademark LEUKASSAY F ⁇ 202 .
  • the kit contains: (1) Three Anti-Feline Leukemia Virus group- specific antigen .
  • Immunoglobulin G-Coated Plates (5 x 3 Wells/Plate) (contains preservative); (2) One vial Wash Additive (Reagent A)-5.0 ml; (3) One vial Feline Leukemia Virus Negative Reference (Reagent N) - 1.0 ml (contains preservative); (4) One vial Feline Leukemia Virus Positive Reference, Killed Virus, (Reagent P) - 1.0 ml (contains preservative); (5) One vial Anti-Feline Leukemia Virus group specific antigen Peroxidase-Conjugated Immunoglobulin G (Reagent C) - 3.0 ml (contains preservative); (6) One vial Substrate I (Reagent SI) - 3.0 ml (contains Preservative); (7) One vial Substrate II (Reagent SII) - 3.0 ml (contains preservatives); and (8) One vial Developer (Rea
  • Additional common laboratory materials which may be needed to perform the FeLV test using the above kit include but are not limited to a system for delivering about 50 microliters (or equivalent drop) of the sample to the test well (typically a micropipet with disposable tip, bulb dropper, or capillary tube), distilled water, and a wash bottle.
  • group specific antigens are detectable in infected tissues, such as blood cells, and in plasma and serum.
  • the sample whole blood with heparin or EDTA, plasma or serum
  • the specific antibody-enzyme conjugate are added to one of the wells in the test plate.
  • the wells have been precoated with monoclonal antibody specific for FeLV group specific antigens.
  • 4,703,001 discloses a method for pre-treating a sample which is to be analyzed for the presence of serum analytes.
  • U.S. Patent No. 4,518,701 discloses a method for reducing non-specific interference in competitive protein binding assays. The assay evaluates a hydrophobic analyte and employs a reagent comprising the analyte in a florescent label conjugated to a water-soluble polysaccharide carrier.
  • U.S. Patent Nos. 4,666,831 and 4,698,299 describe lipid-dependent assays in which interfering anti-phospholipid antibodies or lipoproteins are removed by pretreatment of the sample of one or more specific lipid particles or phospholipids.
  • U.S. Patent Nos. 4,362,531 and 4,680,274 describe methods by which particles are used to inhibit non-specific immunoreactions.
  • references describe methods for reducing false positive reactions in assays in general and immunoassays in particular.
  • the methods are, however, directed to reducing non-specific binding and interfering background reactions or are not directed to preventing false-positive reactions caused by specific binding in enzyme-linked immunosorbent assays.
  • DESCRIPTION OF DRAWINGS Figure 1 is a graphic representation of an enzyme-linked immunosorbent assay showing a positive result due to antigen binding between capture and indicator antibodies.
  • Figure 2 is a graphic representation of an enzyme-linked immunosorbent assay showing a false positive result due to non-specific binding between capture and indicator antibodies.
  • Figure 3 is a graphic representation of an enzyme-linked immunosorbent assay showing a false positive result due to specific binding between an interference antibody and capture and indicator antibodies.
  • an object of the present invention to provide a method for preventing false positive results in enzyme-linked immunosorbent assays. It is another object of the present invention to provide a method for preventing false positive results in enzyme-linked immunosorbent assays used for detecting feline leukemia virus (FeLV) group-specific antigens in the tissue of infected cats.
  • FeLV feline leukemia virus
  • IACS interference antibody complexing serum
  • the reaction between the IACS immunoglobulins and the interference antibody in the enzyme-linked immunosorbent assay procedure can be accomplished by (1) adding the IACS to the solid phase before adding the test sample, (2) adding the IACS to the solid phase after adding the test sample, or (3) adding the IACS with the indicator antibody, preferably by incorporating the IACS in the indicator antibody solution.
  • the invention also comprises an article of manufacture in the form of an enzyme-linked immunosorbent assay (ELISA) diagnostic test kit having in separate containers in a single package (1) a solid support (e.g. microwell) containing the capture antibody adsorbed onto the surface of the suppor , (2) a wash solution for removing sample serum and other materials during various stages of the test, (3) a positive control for the antigen, (4) a negative control for the antigen, (5) an indicator antibody, (6) an interference antibody complexing amount of an interference antibody complexing serum (IACS) from the same species that was used to prepare the capture and indicator antibodies, and (7) a substrate which reacts with the enzyme on the indicator antibody to indicate a positive test.
  • ELISA enzyme-linked immunosorbent assay
  • the invention comprises an article of manufacture in the form of an enzyme-linked immunosorbent assay (ELISA) diagnostic test kit which contains components useful for preventing false positive results when used for detecting the presence of FeLV group-specific antigens in the tissue of infected cats.
  • ELISA enzyme-linked immunosorbent assay
  • capture antibody is defined herein to mean the antibody used in an enzyme-linked immunosorbent assay which is bound to the solid support.
  • indicator antibody is defined herein to mean the antibody-enzyme conjugate used in an enzyme-linked immunosorbent assay which binds to the antigen that is bound to the capture antibody.
  • interference antibody is defined herein to mean an antibody which is present in the sample to be tested for the presence of an antigen and which has antigenic combining sites which are specific for an antigenic determinant on the capture antibody and on the indicator antibody.
  • interference antibody complexing serum is defined herein to mean a serum, antibody or plasma sample, or purified subtraction thereof containing the agents which react with the interference antibody, taken from the same species of animal which was used to produce the capture and indicator antibodies; the term will be designated herein as IACS.
  • substrate is defined herein to mean the true substrate of the enzyme and the associated reactions required to produce a result indicative of a positive test from the enzyme-linked immunosorbent assay. Typically this includes the enzyme substrate and the compounds which react with the enzyme-substrate product to produce a color indicative of the positive result.
  • Enzyme-linked immunosorbent assays are typically performed using the following procedure: (1) a capture antibody adsorbed on the support is reacted with a test sample - a capture antibody-antigen complex will be formed if the test sample contains an antigen specific for the capture antibody; (2) a wash solution is used to remove unreacted test sample; (3) an indicator antibody specific for the antigen is reacted with the capture antibody-antigen complex (if any) - a complex of the capture antibody, antigen and indicator antibody will be bound to the support; (4) the wash solution is used to remove unreacted indicator antibody; and (5) a substrate is reacted with the enzyme which is part of the indicator antibody. The presence of the products produced by the action of the enzyme on the substrate indicates the presence of the antigen. However, if an interference antibody specific for antigenic sites on the capture and indicator antibodies is present is the sample, a complex of the capture antibody, interference antibody and indicator antibody will be bound to the support. Addition of the indicator will give a false positive result.
  • a method for preventing false positive results in enzyme-linked immunosorbent assays caused by specific interactions between interference antibodies, capture antibodies, and indicator antibodies.
  • the method provided comprises reacting an interference antibody complexing amount of an interference antibody complexing serum (IACS) with the interference antibody in the tissue sample which is to be tested for the presence of an antigen.
  • IACS interference antibody complexing serum
  • Immunoglobulins in the IACS react with the interference antibody in the test sample and prevent the interference antibody from reacting with and linking the capture and indicator antibodies. False positive results due to specific binding by interference antibodies are prevented since the indicator antibody cannot bind indirectly to the capture antibody; binding will occur only directly through linking of the capture and indicator antibodies by the antigen.
  • IACS can be reacted with the interference antibody and the test sample at different steps in the enzyme-linked immunosorbent assay procedure described above.
  • the IACS can be added to the solid support (microwell) before or after the test sample is reacted with the capture antibody or the IACS can be added with the indicator antibody.
  • the IACS is incorporated into the indicator antibody reagent thus avoiding any additional steps in the testing procedure.
  • the amount of IACS reacted with the test sample should be sufficient to form a complex with all the interference antibody in the sample. The amount will vary depending on the sample size, amount of indicator antibody, when the IACS is added in the test procedure, and the like. Typically, from about 0.05-0.5 ml of IACS is reacted with about 1.0 ml of test sample.
  • the capture and indicator monoclonal antibodies are prepared from murine spleen cells and myeloma cells using the HAT (hypoxanthine, aminopterin, thymidine) procedure known in the art.
  • HAT hyperxanthine, aminopterin, thymidine
  • the antibodies used in an enzyme-linked immunosorbent assay can, however, be prepared from practically any species capable of producing antibodies.
  • murine IACS is reacted with the test sample during enzyme-linked immunosorbent assay procedures using capture and indicator antibodies prepared from murine cells.
  • a method for preventing false positive results in enzyme-linked immunosorbent assays used to determine the presence of feline leukemia virus (FeLV) group-specific antigens in the tissue of infected cats.
  • the false positive results are caused by specific interactions between interference antibodies in the cat's tissue that were formed after exposure to mouse or rat immunoglobulins and the murine capture antibodies and indicator antibodies used to produce the enzyme-linked immunosorbent assays.
  • the preferred method comprises reacting an interference antibody complexing amount of mouse or a mixture of mouse and rat IACS, preferably a mixture of mouse and rat serum, with the interference antibody in the tissue sample which is to be tested for the presence of FeLV antigen.
  • Immunoglobulins in the IACS react with the interference antibody in the sample and prevent the interference antibody from reacting with and linking the capture and indicator antibodies. False positive results due to specific binding by rat or mouse interference antibodies are prevented since the indicator antibody cannot bind indirectly to the capture antibody; binding will occur only directly through linking of the capture and indicator antibodies by the FeLV antigen.
  • the mouse serum or a mixture of mouse and rat serum is incorporated into the indicator antibody reagent. This eliminates the need for an extra step in the test procedure while reducing the occurrence of false positive reactions.
  • an enzyme-linked immunosorbent assay diagnostic test kit for a particular antigen can be produced or purchased which contains (1) a solid support (e.g.
  • microwell containing the capture antibody adsorbed onto the surface of the support, (2) a wash solution for removing sample serum and other materials during various stages of the test, (3) a positive control for the antigen, (4) a negative control for the antigen, (5) an indicator antibody, and (6) a substrate which reacts with the enzyme on the indicator antibody and possibly a developer for the substrate to indicate the presence of the product and therefore a positive test.
  • a kit for testing for FeLV group-specific antigens in cats is sold by Pitman-Moore, Inc. of Terre Haute, IN under the trademark LEUKASSAY F ⁇ 202 .
  • an article of manufacture in the form of an enzyme-linked immunosorbent assay (ELISA) diagnostic test kit for determining the presence of a particular antigen in a tissue sample comprises in separate containers in a single package (1) a solid support (e.g. microwell) containing a capture antibody adsorbed onto the surface of the support, (2) a wash solution for removing sample serum and other materials during various stages of the test, (3) a positive control for the antigen, (4) a negative control for the antigen, (5) an indicator antibody, (6) an interference antibody complexing amount of an interference antibody complexing serum (IACS) from the same species that was used to prepare the capture and indicator antibodies, and (7) a substrate which reacts with the enzyme on the indicator antibody to indicate a positive test.
  • a solid support e.g. microwell
  • IACS interference antibody complexing amount of an interference antibody complexing serum
  • an article of manufacture in the form of an enzyme-linked immunosorbent assay (ELISA) diagnostic test kit for determining the presence of a particular antigen in a sample comprises in separate containers in a single package (1) a solid support (e.g. microwell) containing the capture antibody adsorbed onto the surface of the support, (2) a wash solution for removing sample serum and other materials during various stages of the test, (3) a positive control for the antigen, (4) a negative control for the antigen, (5) in combination, an indicator antibody and an interference antibody complexing amount of an interference antibody complexing serum (IACS) from the same species that was used to prepare the capture and indicator antibodies, and (6) a substrate which reacts with the enzyme on the indicator antibody to indicate a positive test.
  • IACS interference antibody complexing serum
  • an article of manufacture in the form of an enzyme-linked immunosorbent assay (ELISA) diagnostic test kit for determining the presence of feline leukemia virus (FeLV) group-specific antigens in the tissue of infected cats comprises in separate containers in a single package (1) anti-feline leukemia virus group-specific antigen immunoglobulin G coated plates, (2) a wash solution for removing sample serum and other materials during various stages of the test, (3) feline leukemia virus positive reference, (4) feline leukemia virus negative reference, (5) anti-feline leukemia virus group specific antigen peroxidase-conjugated immunoglobulin G, (6) an interference antibody complexing amount of an interference antibody complexing serum (IACS) from the same species that was used to prepare the capture and indicator antibodies, preferably mouse or rat IACS, and (7) a substrate system consisting of hydrogen peroxide and tetramethylbenzidine which reacts with the peroxidase enzyme and develops the substrate product to indicate a positive test.
  • IACS interference antibody complexing
  • an article of manufacture in the form of an enzyme-linked immunosorbent assay (ELISA) diagnostic test kit for determining the presence of feline leukemia virus (FeLV) group-specific antigens in the tissue of infected cats comprises in separate containers in a single package (1) anti-feline leukemia virus group-specific antigen immunoglobulin G coated plates, (2) a wash solution for removing sample serum and other materials during various stages of the test, (3) feline leukemia virus positive reference, (4) feline leukemia virus negative reference, (5) in combination, anti-feline leukemia virus group specific antigen peroxidase-conjugated immunoglobulin G and an interference antibody complexing amount of an interference antibody complexing serum (IACS) from the same species that was used to prepare the capture and indicator antibodies, preferably mouse or rat IACS, and (6) a substrate system consisting of hydrogen peroxide and tetramethylbenzidine which reacts with the peroxidase enzyme and develops the substrate product to indicate a positive test.
  • IACS interference antibody complex
  • the articles of manufacture of the present invention should contain sufficient IACS to react with all the interference antibody in a sample; typically about 0.05-0.5 ml of IACS to react with about 1.0 ml of tissue sample or serum.
  • a wash solution was prepared by adding 100 ml of distilled water and 1.0 ml (20-22 drops) of Wash
  • the sample wells were scored visually for color compared to the negative reference - when the color of the test sample is distinctly more intense than that of the negative reference well the sample is called positive, otherwise it is called negative;
  • Example 1 On day 1, a healthy cat (5F31) was injected subcutaneously with 0.5 ml of mouse blood. On day 15, the cat was injected subcutaneously with a booster consisting of 0.5 ml of mouse blood and 0.5 ml of rat blood. On day 31, the cat was injected subcutaneously with a booster consisting of 0.5 ml of mouse blood. Blood samples were taken from the cat before and after injections at different intervals. Serum was prepared from the blood samples and stored frozen for subsequent analysis. To prepare cat sera containing antimouse and/or antirat immunoglobulin activity, blood samples from a known FeLV negative healthy cat (15039) was collected in sufficient quantity and serum was separated from the samples.
  • FeLV negative healthy cat (5F31) became false FeLV positive after injection of mouse and rat blood.
  • FeLV negative cat serum (15039) became false FeLV positive after adding Antimouse and/or Antirat Antibody.
  • false positive results may be obtained due to Antimouse and/or Antirat activity in the test sample when monoclonal antibodies are used for both the "capture” or “coating antibody” and the "indicator or conjugating antibody” in ELISA test.
  • This false positive reaction is not due to nonspecific or physical binding usually found in many im unological test systems including ELISA test, but due to immunological binding of both the Capture antibody and the indicator antibody by the Antimouse antibody molecule instead of the specific antigen molecule for which the test is designed.
  • BSA Bovine Serum Albumin
  • Example 3 The three sera samples (15039, 10539 with antimouse IgG, and 361/372) mentioned in Example 2 were tested according to the test procedure described earlier except that a drop of A) 4% BSA in PBS tween 20 or B) a mixture of equal parts of mouse sera and rat sera were added to the well before or after the addition of the samples. The results are shown in Table 3a and Table 3b for adding A, B and C before and after adding the samples, respectively.
  • Example 4 The monoclonal antibody peroxidase conjugate (Reagent C) of the kit was modified by adding a) 4% BSA in PBS tween 20, b) Mouse sera, c) Mouse sere and unrelated monoclonal ascetic fluid, and d) Mixture of Mouse sera, unrelated Monoclonal antibody, ascetic fluid and Rat sera.
  • Example 2 The three samples described in Example 2 were tested according to the procedure described earlier. The results are shown in Table 4.
  • Example 5 A modified antibody enzyme conjugate (Reagent C) was prepared by diluting the concentrated antibody enzyme conjugate with 4% BSA in PBS tween 20 containing 4% mouse serum and 2% rat serum. The final concentration of the conjugate was made similar to that of the kit conjugate (Reagent C). Serum samples from field cats identified to give false positive results in FeLV ELISA tests due to antimouse activity were obtained from a University Diagnostic Laboratory. Those field serum samples with antimouse activity, the samples shown in Table 5, and other known positive and negative samples were tested according to the procedure stated earlier using the kit-conjugate and the modified conjugate. The results of the test are shown in Table 5.

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Abstract

On prévient les résultats positifs faux dans des analyses d'immunosorbant à liaison enzymatique, résultats provoqués par des interactions spécifiques entre des anticorps d'interférence, des anticorps de capture, ainsi que des anticorps indicateurs, en faisant réagir une faible quantité de sérum de formation de complexes d'anticorps d'interférence (IACS) provenant des espèces utilisées pour préparer l'anticorps de capture et l'anticorps indicateur, avec l'anticorps d'interférence dans l'échantillon que l'on veut tester afin de détecter la présence d'un antigène. Les immunoglobulines IACS réagissent avec l'anticorps d'interférence, empêchant ainsi l'anticorps d'interférence de lier l'anticorps de capture et l'anticorps indicateur. L'invention comprend également un article de fabrication se présentant sous la forme d'un kit de test de diagnostic par analyse d'immunosorbant à liaison enzymatique (ELISA), lequel contient des composants utiles pour la prévention de faux résultats positifs, de préférence lorsqu'on l'utilise pour détecter la présence d'antigènes spécifiques au groupe de virus de la leucémie féline (FeLV), dans les tissus de chats atteints de l'infection.
EP19900904497 1989-02-21 1990-02-21 Prevention de faux resultats positifs dans des procedes de test elisa Withdrawn EP0460094A1 (fr)

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US312489 1981-10-19
US31248989A 1989-02-21 1989-02-21

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EP0460094A1 true EP0460094A1 (fr) 1991-12-11

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AU (1) AU5185590A (fr)
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JP2716103B2 (ja) * 1991-07-26 1998-02-18 デイド・ケミストリイ・システムズ・インコーポレーテツド イムノアッセイにおける誤結果除去方法

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JPS6165162A (ja) * 1984-09-05 1986-04-03 Chemo Sero Therapeut Res Inst モノクロ−ナル抗体を用いた免疫学的測定方法における非特異反応吸収剤
DE3720983A1 (de) * 1987-06-25 1989-01-05 Hoechst Ag Immunometrisches bestimmungsverfahren

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