WO1987003691A1 - Determination de substances de test - Google Patents

Determination de substances de test Download PDF

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Publication number
WO1987003691A1
WO1987003691A1 PCT/US1986/002681 US8602681W WO8703691A1 WO 1987003691 A1 WO1987003691 A1 WO 1987003691A1 US 8602681 W US8602681 W US 8602681W WO 8703691 A1 WO8703691 A1 WO 8703691A1
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WO
WIPO (PCT)
Prior art keywords
labelled
test
indicator
enzyme
color change
Prior art date
Application number
PCT/US1986/002681
Other languages
English (en)
Inventor
Francis Cole
Original Assignee
Dennison Manufacturing Company
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 Dennison Manufacturing Company filed Critical Dennison Manufacturing Company
Publication of WO1987003691A1 publication Critical patent/WO1987003691A1/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
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • This invention relates to testing for substances, particularly by "assays” and more particularly by “immunoassays.
  • An assay is a procedure for determining whether or not a test substance is present or absent, and, where necessary, in what amount.
  • the testing involves the "immunological" agents, which are produced by or cause a response in, the immune system of a living body.
  • the agents produced by the immune system are “antibodies”. They result from the reaction of the body against foreign substances, like viruses, or may be generated in response to a physical change in body chemistry, like pregnancy.
  • Antibodies are the response to the presence of "antigens”. For example, during pregnancy, the body produces elevated levels of a hormone known as human chorionic gonadotrophin (HCG) , so called because it originates in the reproductive organs. A non-human body reacts to HCG exposure by producing anti ⁇ bodies known as anti-HCG. Another example is the gonococcus (GC) bacteria that produces the venereal desease known as gonorrhea. The body reacts by producing antibodies known as anti-GC.
  • HCG human chorionic gonadotrophin
  • the correspon ⁇ ding antibody can be labelled with an indicator.
  • the labelled antibody is then applied to a test sample which may contain the complementary antigen. If the antigen is present, the labelled antibody binds to the antigen and the label or indicator now bound to the antigen can be detected by a variety of methods. Since these techniques employ antibodies and antigens associated with the immune system, they are known as immuno- assays. Originally, the labels indicators were radioactive and/or fluorescent materials. Thus, when the antigen, and an antibody labelled with a radioactive or fluorescent material, were brought together, the presence of the label permitted detection of a binding reaction between the antibody and the antigen.
  • Radioimmunoassays are objectionable in that they involve the potential hazards of radioactive materials. Fluoroimmunoassays, in turn, have the objection of requiring fluorescent dyes (fluorochromes such as fluorscein and rhoda ine) . These can be detected only by fluorescent microscopes, which are expensive and not widely available.
  • fluorescent dyes fluorochromes such as fluorscein and rhoda ine
  • Radio and Fluoroimmunoassays thus were primarily for laboratories and not suitable for home diagnosis.
  • enzymes have been used as the labelling material.
  • the anibody or antigen that is to be detected is used with its labelled counterpart. Where detection is to be made of an antigen the labelled counterpart is an enzyme labelled antibody. All that is necessary is to use an indicator which can react with an enzyme and thus indicate its presence.
  • antibodies that are specific to a test antigen are first absorbed in excess on a solid surface such as a plastic tube.
  • a test solution containing the antigen is then added, and the antigen binds to the antibody.
  • the "solid phase”, composed of all material bound to the antibody, is then thoroughly washed to remove unbound components. The test then proceeds by identifying the bound antigen.
  • an enzyme labelled second antibody preferably having binding sites different from those of the first antibody, is added and reacts with specific determinant sites on the bound antigen.
  • the enzyme labelled second antibody is added in excess to assure that all antigen in the solid phase, and bound to the. first antibody, will also be bound to enzyme labelled second antibody.
  • the enzyme labelled second antibody molecules will bind to each antigen molecule in a fixed ratio depending on the specific available binding sites at the antigen.
  • the solid phase is again washed to remove excess second antibody and any other unbound constituents.
  • An enzyme "substrate”, i.e. a substance which reacts with the enzyme, is then added in solution in excess amounts to make contact with the bound solid phase.
  • the substrate may be a solution of hydrogen peroxide and a chromogenic material.
  • the constituents of the indicator material which are commonly hydrogen peroxide and a chromogenic material, must be kept apart until the time for the assay. If the indicator materials are mixed prematurely there is a decay and the resultant solution is not satisfactory for use after a delay interval. Indeed, the chromogenic reagent and hydrogen peroxide must be mixed in a fresh batch just prior to each use because they tend to oxidize and become spontaneously colored when left in storage for as little as one hour.
  • a related object is to facilitate immunoassay, particularly enzyme immunoassay, procedures.
  • Another object of the invention is to simplify the rinsing requirements of the prior art.
  • a related object is to completely eliminate rinsing after formation of a test "sandwich”.
  • Still another object of the invention is to reduce the amount of time required in enzymatic assays.
  • Yet another object of the invention is to eliminate the need for combining a chromogen with an activator such as hydrogen peroxide.
  • a related object is to eliminate any adverse consequences from the premature combination of a chromogen with an activator.
  • the invention provides for determining whether or not a test substance is present by combining a sample, which may contain the test substance, with a first-labelled material. The result of this combination is then combined with a second labelled material and a substance is applied to react with one of the labelled materials to produce a reagent that is able, in turn, to react with the other of the labelled materials.
  • an indicator reacts with the reagent and one of the labels to produce a color change.
  • the indicator is a leuco dye, desirably tetramethylbenzidine, or a derivative, and the labels are different enzymes.
  • One of the enzymes can be an oxidase which reacts with a substance, such as glucose or cholesterol to produce a peroxide.
  • the other of the enzymes can be peroxidase which reacts with a peroxide and tetramethylbenzidine to produce a color change.
  • the second labelled material is bound to a porous support.
  • the test substance When the test substance is present, it binds jointly to the first labelled material and the second labelled material.
  • Peroxide produced by one of the labels reacts with the other label and an indicator, such as tetramethylbenzidine, to produce a color change indicative of the presence of the test substance.
  • an indicator such as tetramethylbenzidine
  • the first labelled material is unbound, i.e., when the test substance is absent, the amounts of reagent, indicator and second label having been preadjusted such that the unbound first reagent is carried away before a color change occurs.
  • a test kit including a first labelled material, a second labelled material, means for permitting a first reaction of the first material test sample, means for permitting a reaction of the second material with the result of the first reaction, and means for applying an indicator to the further reaction.
  • the first material is desirably labelled with a first enzyme
  • the second material is desirably labelled with a second enzyme.
  • the indicator is desirably a benzidine or a derivative of benzidine.
  • the first material is supported by a first vessel and the second material is supported by a second vessel.
  • the first vessel is a test tube on which the first material is liquid or lyophilized preparation and the second vessel is a container for a porous support for the second material. Consequently, when the complement of the second material is present in a test sample, it is able to bind to the second material on the support. Since it is bound to the support along with the first labelled complement, a reaction such as a color change that takes place on the support can provide an indication of whether or not the test substance is present. For this purpose it is important that the support be visible from the exterior of the second vessel or housing.
  • a structure for indicating the presence or absence of a test substance is produced by providing a housing, including a porous membrane within the housing, using the porous membrane to support a labelled material, and overlying the porous member with the porous membrane.
  • the labelled material desirably has an enzyme label which is an oxidase, such as glucose oxidase.
  • the porous membrane is fitted in the mouth of the housing and is visible when the mouth is uncovered in order to provide an indication of color change when a test substance is present.
  • the porous membrane that supports the labelled material desirably has a lesser degree of porosity than the porous member that supports the membrane.
  • Fig. 1 is a test housing in accordance with the invention:
  • Fig. 2 is an illustration of a test assay in accordance with the invention making use of the test housing of Fig. 1 and auxiliary test components
  • Fig. 1 shows a test kit 10 which includes three basic constituents: a test housing 20, a preliminary mixing vial 30, and an indicator vial 40.
  • the test housing is shown with a part of its side wall broken away to reveal the realtionship of interior constituents 21 and 22 which are respectively a porous support and an overlying porous membrane.
  • the housing 20 includes a mouth 23 which exposes a substantial portion of the surface of the membrane 22 and permits the application of fluids to the housing 21.
  • the upper portion of the housing has retaining walls 21-4 to 24-4 which are fitted above the base 25.
  • the mixing vial 30 has an interior wall 31 that is coated with a first labelled material, such as an enzyme labelled antibody.
  • a first labelled material such as an enzyme labelled antibody.
  • the enzyme labelled antibody is designated as AB-E,.
  • the remaining constituent of the kit 10 is the indicator vial 40.
  • the indicator vial 40 contains a chromogen such as tetramethylbenzidine or one of its derivatives, and a "substrate" for an enzyme.
  • the "substrate” is so designated - 7 -
  • the enzyme in question is linked to an antibody that is on the porous membrane 22.
  • the enzyme labelled antibody is designated as AB-E 2 .
  • test procedure in accordance with the invention, is illustrated in Fig. 2.
  • a test sample which possibly contains a test substance such as an antigen (AG) is applied to the vial 31. If antigen is present in the test sample, it binds to the enzyme labelled antibody in vial 30 in accordance with equation 1 (1) :
  • the result of this first step is to produce in the test sample, when the test substance is present, an enzyme-linked antibody that is in turn bound to the complementary antigen.
  • the contents of vial 30 are then deposited as indicated by the arrow for step No. 2 on the membrane 12 that contains the second enzyme linked antibody. If the test antigen is linked to the first antibody as in accordance with equation 1, the presence of this material with the enzyme linked antibody on the membrane 22 produces the reaction shown in equation (2) below:
  • the next step is to apply the contents of the indicator vial 40 as indicated by the arrow No. 3. Since the indicator vial contains a "substrate" that reacts with the enzyme E 2 , this produces a reagent, for example, hydrogen peroxide as shown in equation (3) :
  • equation (3) produces a reaction with the first enzyme E-. as shown in equation (4) :
  • glucose oxidase in various concentrations is immobilized while keeping the level of co-immobilized antibody constant. This procedure is commonly referred to as a titration. There are several dilutions that range from a concentration level of 30 mg. per illiliter in a stock solution of 1.4 mg. per ml. of monoclonal antibody (LH262G9H10) to 1.8 micrograms per ml.
  • dextrose is diluted in citrate phosphate buffer having a pH of 5.0. Concentrations of this substance are 50, 12.5, 3.12, 0.78 and 0.195 grams per liter. Additionally, 0.9 milligrams of Igepal CA 720 are added to each of the solutions to form a volume of 14.9 milliliters.
  • the foregoing immobilization procedure is begun by applying 3.0 milliliters of AB-GO mixture per membrane and allowing a 10 second drying time.
  • a blocking phase is completed by putting through a carnation miller PBS buffer under vacuum and drying the membranes in a desiccator.
  • the assay steps are as follows:
  • 0.25 milliliters of lyophilized conjugate is reconstituted with 0.25 milliliters of sample that can contain LH hormone.
  • the liquid is poured over the membrane, containing the immobilized AB and GO of the assembled test device.
  • This device consists of from top to bottom, a membrane, a semi-rigid porous disc and an absorbent matrix.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Détermination de la présente ou de l'absence d'une substance de test, telle qu'un antigène de grossesse (AG), par combinaison (1) de la substance de test (AG) avec une première substance marquée, par exemple un premier anticorps marqué par une enzyme (ABA-E1) dans une éprouvette (30). Le produit résultant est ajouté (2) à une deuxième substance marquée, par exemple un deuxième anticoorps marqué par une enzyme (ABA-E1), fixé sur une membrane (22) recouvrant un organe poreux (21) à l'intérieur d'un logement (10), un substrat étant appliqué (3) à partir de l'éprouvette (40) pour qu'il réagisse avec l'un des marqueurs et produise un réactif réagissant à son tour avec un indicateur et avec l'autre marqueur pour fournir une indication du test, tel qu'un changement de couleur.
PCT/US1986/002681 1985-12-12 1986-12-12 Determination de substances de test WO1987003691A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US80793685A 1985-12-12 1985-12-12
US807,936 1985-12-12

Publications (1)

Publication Number Publication Date
WO1987003691A1 true WO1987003691A1 (fr) 1987-06-18

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ID=25197479

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1986/002681 WO1987003691A1 (fr) 1985-12-12 1986-12-12 Determination de substances de test

Country Status (3)

Country Link
EP (1) EP0250557A4 (fr)
JP (1) JPS63501983A (fr)
WO (1) WO1987003691A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0315364A2 (fr) * 1987-11-02 1989-05-10 Bioventures, Inc. Dosage immunologique simultané pour la détermination des antigènes et des anticorps
GB2270158A (en) * 1992-08-03 1994-03-02 Marconi Gec Ltd Immunoassay using two detectable species
US5723304A (en) * 1992-08-03 1998-03-03 Gec-Marconi Limited Immunological detection using two detectable labels

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05227995A (ja) * 1992-02-17 1993-09-07 Sanyo Chem Ind Ltd 過酸化物分解性物質測定用指示薬および試薬キット

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3888629A (en) * 1971-09-08 1975-06-10 Kenneth Dawson Bagshawe Performance of chemical or biological reactions within an absorbent matrix pad
US4366241A (en) * 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4407943A (en) * 1976-12-16 1983-10-04 Millipore Corporation Immobilized antibody or antigen for immunoassay
US4486530A (en) * 1980-08-04 1984-12-04 Hybritech Incorporated Immunometric assays using monoclonal antibodies
US4540659A (en) * 1981-04-17 1985-09-10 Syva Company Simultaneous calibration heterogeneous immunoassay

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4269938A (en) * 1979-03-08 1981-05-26 Eastman Kodak Company Assay of peroxidatively active materials
FR2455320B1 (fr) * 1979-04-25 1986-01-24 Cii Honeywell Bull Dispositif de recyclage de supports d'enregistrement identifiables a l'aide de donnees d'identification et composes de memoires monolithiques non volatiles effacables
DE3049607C3 (de) * 1980-12-31 2003-07-17 Gao Ges Automation Org Verfahren zur Herstellung von Ausweiskarten und Vorrichtung zu dessen Durchführung
JPS5975380A (ja) * 1982-10-21 1984-04-28 Dainippon Printing Co Ltd Ｉｃカ−ド
US4632901A (en) * 1984-05-11 1986-12-30 Hybritech Incorporated Method and apparatus for immunoassays
GB8505899D0 (en) * 1985-03-07 1985-04-11 Boots Celltech Diagnostics Assay reagents

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3888629A (en) * 1971-09-08 1975-06-10 Kenneth Dawson Bagshawe Performance of chemical or biological reactions within an absorbent matrix pad
US4407943A (en) * 1976-12-16 1983-10-04 Millipore Corporation Immobilized antibody or antigen for immunoassay
US4486530A (en) * 1980-08-04 1984-12-04 Hybritech Incorporated Immunometric assays using monoclonal antibodies
US4366241A (en) * 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4366241B1 (fr) * 1980-08-07 1988-10-18
US4540659A (en) * 1981-04-17 1985-09-10 Syva Company Simultaneous calibration heterogeneous immunoassay

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0250557A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0315364A2 (fr) * 1987-11-02 1989-05-10 Bioventures, Inc. Dosage immunologique simultané pour la détermination des antigènes et des anticorps
EP0315364A3 (fr) * 1987-11-02 1990-03-07 Bioventures, Inc. Dosage immunologique simultané pour la détermination des antigènes et des anticorps
GB2270158A (en) * 1992-08-03 1994-03-02 Marconi Gec Ltd Immunoassay using two detectable species
GB2270158B (en) * 1992-08-03 1997-03-19 Marconi Gec Ltd Detection
US5723304A (en) * 1992-08-03 1998-03-03 Gec-Marconi Limited Immunological detection using two detectable labels

Also Published As

Publication number Publication date
EP0250557A4 (fr) 1990-01-11
EP0250557A1 (fr) 1988-01-07
JPS63501983A (ja) 1988-08-04

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