EP0235224A1 - Method for carrying out immunoassays by using enzyme labels - Google Patents

Method for carrying out immunoassays by using enzyme labels

Info

Publication number
EP0235224A1
EP0235224A1 EP19860905303 EP86905303A EP0235224A1 EP 0235224 A1 EP0235224 A1 EP 0235224A1 EP 19860905303 EP19860905303 EP 19860905303 EP 86905303 A EP86905303 A EP 86905303A EP 0235224 A1 EP0235224 A1 EP 0235224A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
separation liquid
added
substrate
separation
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Withdrawn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
EP19860905303
Other languages
German (de)
French (fr)
Inventor
Hannu Harjunmaa
Juhani Luotola
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.)
Thermo Fisher Scientific Oy
Original Assignee
Labsystems Oy
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 Labsystems Oy filed Critical Labsystems Oy
Publication of EP0235224A1 publication Critical patent/EP0235224A1/en
Withdrawn legal-status Critical Current

Links

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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates

Definitions

  • the present invention concerns a fluorometric or photometric immunoassay method in which the bottom or the walls of the measurement vessel are used as the solid phase.
  • the separation of the solid phase from the reaction solution includes washing of the solid phase, which at present requires manual operations or operations whose automation is difficult.
  • both antibodies and antigens can be determined.
  • the basic idea of the invention can be applied both to photometric measurement and to fluorometric measurement.
  • a separation liquid heavier than the reaction solution is used, which said separation liquid is administered into the reaction vessel after the antigen or antibody immobilized on the bottom or walls of the vessel, on one hand, and the antibody or antigen to be determined and also the antibody or antigen labelled with the tracer, on the other hand, have been allowed to react with each other.
  • the separation liquid is administered preferably as two layers, of which only the layer to be placed as the bottom layer contains a substrate of the enzyme used as the tracer, whose colour is changed by the enzyme.
  • Substances that may be concerned are, e.g., alkaline or acid phosphatase as the enzyme, paranitrophenylphosphate as the substrate, and paranitrophenol as the coloured final product.
  • the separation liquid is most appropriately a substance which absorbs the excitation light or the emission light (or both) strongly and contains a substrate of the enzyme used as the tracer, which said substrate is made fluorescent by the enzyme.
  • Figure 1 shows a measurement vessel, whose inside face is covered with an antigen 1 and which also acts as the reaction vessel.
  • the sample has been administered, which includes the antibody 2 to be determined and, moreover, the cor ⁇ responding antibody 3 labelled with an enzyme.
  • labelled antibody and unlabelled antibody have adhered to the antigen present on the bottom and walls of the measurement vessel by means of an immunological bond in the proportion in which they are present in the reaction solution.
  • a separation liquid 4 heavier than the sample solution has been added into the mea ⁇ surement vessel shown in Fig. 1, which said separation liquid 4 further includes such a substrate 5 of the enzyme tracer as is made fluorescent by the enzyme.
  • a possible enzyme concerned may be, e.g., alkaline or acid phosphatase, and the substrate may be methylum- belliferylphosphate, whereby methylumbelliferone is liberated as the fluorescent substance.
  • the separation liquid has displaced the sample solution and allowed only the molecules adhering by means of the immunological bond to remain therein.
  • a suitable separation liquid is, e.g., a 20...60-% saccharose solution.
  • the fluorescence is measured from below by directing an excitation light 6 of suitable colour into the fluorescent layer present above the bottom of the cuvette and by measuring the amount of fluorescent light 7 emitted from same.
  • the fluorescent substance present in the top portion of the separation liquid is not seen in the measurement, because the separation liquid acts as an optical shutter.
  • the absorption factor of the separation liquid at the excitation and emission wavelengths of the fluorescence is chosen so that it does not form a substantial - obstacle for the measurement of the fluorescent agent in the layer in direct contact with the bottom or wall of the cuvette but that, when measured through the whole separation layer, it forms such a high optical density that access of the excitation light or of the emission light (or both) through the whole thickness of the separation liquid is substantially prevented.
  • the separation liquid it is possible to use, e.g., potassium chromate or methyl orange (or a mixture of same) as the colouring agent.
  • antigen 1 has been immobilized onto the partition wall in the measurement vessel, and antibody 2 and enzyme-labelled antibody 3 have been administered into the vessel.
  • a colourless isolating separation liquid 9 with no substrate and heavier than the sample layer has been administered, and then, preferably without removing the dosage tip in between, a still heavier colourless separation liquid 10 has been administered, which additionally contains such a colourless substrate 11 as is converted by the enzyme to a coloured, photometrica ly measurable substance.
  • the absorbance of the coloured substance is measured by means of a beam of light 12 that passes vertically.
  • the enzyme it is possible to use, e.g., alkaline or acid phosphatase, and as the substrate paranitrophenylphosphate, in which case paranitrophenyl is liberated as the coloured compound.
  • the embodiment shown in Figures 3 and 4 may also be accomplished so that, at the final stage of the administration of one and the same separation liquid, the substrate is added to the separation liquid, preferably into the liquid duct out of a side duct opening close to the dosage tip.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Réalisation d'immuno-analyses en utilisant un enzyme indicateur dans un récipient sur la face intérieure duquel est immobilisé un antigène de l'anticorps à déterminer. On ajoute dans le récipient la solution échantillon à étudier ainsi qu'un anticorps correspondant marqué par un enzyme. Après la réaction immunologique, on ajoute un liquide de séparation (4) qui est plus lourd que la solution échantillon et qui contient un substrat (5) de l'enzyme que ce dernier convertit afin qu'il puisse être détecté par un procédé fluorométrique ou photométrique.Performing immunoassays using an indicator enzyme in a container on the inside of which is immobilized an antigen of the antibody to be determined. The sample solution to be studied and a corresponding antibody labeled with an enzyme are added to the container. After the immunological reaction, a separation liquid (4) is added which is heavier than the sample solution and which contains a substrate (5) of the enzyme which the latter converts so that it can be detected by a fluorometric method or photometric.

Description

Method for carrying .out im unoassays by using enzyme labels
The present invention concerns a fluorometric or photometric immunoassay method in which the bottom or the walls of the measurement vessel are used as the solid phase.
In prior art, methods are known which are based on immobilization of an antibody or antigen on an antigen or antibody in advance placed on a solid face as well as on the use of an antibody or antigen labelled with a tracer. Such methods are, e.g., RIA (Radioimmuno- assay) and SP-FIA (Solid Phase Fluoroimmunoassay) , which latter one is to be dealt with in the present invention. In all of these methods, the solid face on which the immunological reaction has taken place and the reaction solution must be separated from each other before the signal of the tracer is measured in order that the excess tracer present in the reaction solution should not cover the signal of the tracer present in the antibody or antigen immobilized on the solid phase. The signal con¬ cerned may be, e.g., radioactivity (RIA), fluorescence (FIA) , or even enzyme activity (EIA) .
As a rule, the separation of the solid phase from the reaction solution includes washing of the solid phase, which at present requires manual operations or operations whose automation is difficult.
By means of the method of the present invention both antibodies and antigens can be determined. The basic idea of the invention can be applied both to photometric measurement and to fluorometric measurement. In both cases a separation liquid heavier than the reaction solution is used, which said separation liquid is administered into the reaction vessel after the antigen or antibody immobilized on the bottom or walls of the vessel, on one hand, and the antibody or antigen to be determined and also the antibody or antigen labelled with the tracer, on the other hand, have been allowed to react with each other. In a photometric method, the separation liquid is administered preferably as two layers, of which only the layer to be placed as the bottom layer contains a substrate of the enzyme used as the tracer, whose colour is changed by the enzyme. Substances that may be concerned are, e.g., alkaline or acid phosphatase as the enzyme, paranitrophenylphosphate as the substrate, and paranitrophenol as the coloured final product. In a fluorometric method, the separation liquid is most appropriately a substance which absorbs the excitation light or the emission light (or both) strongly and contains a substrate of the enzyme used as the tracer, which said substrate is made fluorescent by the enzyme.
A fluorometric embodiment and a photometric embodiment of the invention will be illustrated in the following by means of the accompanying figures. Herein a so-called competitive method is described, but other methods may also be used.
Figure 1 shows a measurement vessel, whose inside face is covered with an antigen 1 and which also acts as the reaction vessel. Into the measurement vessel, the sample has been administered, which includes the antibody 2 to be determined and, moreover, the cor¬ responding antibody 3 labelled with an enzyme. In the immunological reaction, labelled antibody and unlabelled antibody have adhered to the antigen present on the bottom and walls of the measurement vessel by means of an immunological bond in the proportion in which they are present in the reaction solution.
In Figure 2, a separation liquid 4 heavier than the sample solution has been added into the mea¬ surement vessel shown in Fig. 1, which said separation liquid 4 further includes such a substrate 5 of the enzyme tracer as is made fluorescent by the enzyme. A possible enzyme concerned may be, e.g., alkaline or acid phosphatase, and the substrate may be methylum- belliferylphosphate, whereby methylumbelliferone is liberated as the fluorescent substance. The separation liquid has displaced the sample solution and allowed only the molecules adhering by means of the immunological bond to remain therein. A suitable separation liquid is, e.g., a 20...60-% saccharose solution.
The fluorescence is measured from below by directing an excitation light 6 of suitable colour into the fluorescent layer present above the bottom of the cuvette and by measuring the amount of fluorescent light 7 emitted from same. The stronger the fluores¬ cence is, the less has there been of the antibody to be determined in the sample, because, then, a large quantity of the antibody labelled with the enzyme has adhered. The fluorescent substance present in the top portion of the separation liquid is not seen in the measurement, because the separation liquid acts as an optical shutter. The absorption factor of the separation liquid at the excitation and emission wavelengths of the fluorescence is chosen so that it does not form a substantial - obstacle for the measurement of the fluorescent agent in the layer in direct contact with the bottom or wall of the cuvette but that, when measured through the whole separation layer, it forms such a high optical density that access of the excitation light or of the emission light (or both) through the whole thickness of the separation liquid is substantially prevented.
When the fluorescence of methylumbelliferone is measured, in the separation liquid it is possible to use, e.g., potassium chromate or methyl orange (or a mixture of same) as the colouring agent.
Also, in the embodiment shown in Figures 3 and 4, antigen 1 has been immobilized onto the partition wall in the measurement vessel, and antibody 2 and enzyme-labelled antibody 3 have been administered into the vessel. After the immunological reaction, into the vessel, to the proximity of the bottom, through a dosage tip 8 and without stirring the sample, first a colourless isolating separation liquid 9 with no substrate and heavier than the sample layer has been administered, and then, preferably without removing the dosage tip in between, a still heavier colourless separation liquid 10 has been administered, which additionally contains such a colourless substrate 11 as is converted by the enzyme to a coloured, photometrica ly measurable substance. The absorbance of the coloured substance is measured by means of a beam of light 12 that passes vertically.
As the enzyme it is possible to use, e.g., alkaline or acid phosphatase, and as the substrate paranitrophenylphosphate, in which case paranitrophenyl is liberated as the coloured compound.
The embodiment shown in Figures 3 and 4 may also be accomplished so that, at the final stage of the administration of one and the same separation liquid, the substrate is added to the separation liquid, preferably into the liquid duct out of a side duct opening close to the dosage tip.

Claims

WHAT IS CLAIMED IS:
1. Method for carrying out immunoassays by using an enzyme tracer, wherein a sample solution to be
-5 studied as well as a corresponding enzyme-labelled antibody are added into a measurement vessel provided with a transparent window, onto whose inside face the antigen (1) of the antibody to be determined has been immobilized, c h a r a c t e r i z e d in that, aftur 0 the immunological reaction, a separation liquid (4, 10) heavier than the sample solution is added into the measurement vessel, which said separation liquid con¬ tains such a substrate (5, 11) of an enzyme as is con¬ verted by the enzyme so that it becomes fluorometrically 5 or photometrically detectable, and that the quantity of the detectable compound present in the separation layer is measured fluorometrically or photometrically.
2. Method as claimed in claim 1 , c h a r ¬ a c t e r i z e d in that, after the immunological 0 reaction, a separation liquid (4) is added which con¬ tains a substrate which is made by the enzyme fluoro¬ metrically detectable and that the quantity of the detectable compound is measured fluorometrically so that both the excitation radiation (6) is passed into the 5 separation layer and the emission radiation (7) is passed out of the separation layer through the measure¬ ment window.
3. Method as claimed in claim 2, c h a r ¬ a c t e r i z e d in that a separation liquid (4) is 0 added which absorbs strongly at the excitation wave¬ length or at the emission wavelength.
4. Method as claimed in claim 1, c h a r ¬ a c t e r i z e d in that, after the immunological reaction, a separation liquid (10) is added which con- 5 tains a substrate (11) that is made by the enzyme photometrically detectable and that the amount of the detectable compound is measured by means of a beam of measurement (12) passing vertically.
5. Method as claimed in claim 4, c h a r ¬ a c t e r i z e d in that, before the separation liquid (10) , first an isolating separation liquid (9) is added which is heavier than the sample solution and which does not contain substrate (11) of the enzyme.
6. Method as claimed in claim 5, c h a r ¬ a c t e r i z e d in that an isolating separation liquid (9) is added which is less heavy than the separation liquid (10) proper.
EP19860905303 1985-09-13 1986-09-10 Method for carrying out immunoassays by using enzyme labels Withdrawn EP0235224A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI853523A FI853523A0 (en) 1985-09-13 1985-09-13 FOERFARANDE FOER UTFOERING AV IMMUNOBESTAEMNINGAR MED HJAELP AV ENZYMSTAEMPEL.
FI853523 1985-09-13

Publications (1)

Publication Number Publication Date
EP0235224A1 true EP0235224A1 (en) 1987-09-09

Family

ID=8521340

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860905303 Withdrawn EP0235224A1 (en) 1985-09-13 1986-09-10 Method for carrying out immunoassays by using enzyme labels

Country Status (4)

Country Link
EP (1) EP0235224A1 (en)
JP (1) JPS63500962A (en)
FI (2) FI853523A0 (en)
WO (1) WO1987001810A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5073341A (en) * 1985-08-21 1991-12-17 Biotope, Inc. Devices for conducting specific binding assays
EP0344276B1 (en) * 1987-12-01 1993-06-23 Boehringer Mannheim Corporation Methods and devices for conducting assays
JPH0731196B2 (en) * 1989-03-28 1995-04-10 技術研究組合医療福祉機器研究所 Cell identification method in complex antigen-antibody reaction system and cell immobilization apparatus used in this method
DE4041300A1 (en) * 1989-12-22 1991-06-27 Syntex Inc METHOD FOR SEPARATING COMPONENTS IN A MIXTURE

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1566098A (en) * 1975-11-14 1980-04-30 Nat Res Dev Separation of solid and liquid components of mixtures
SE7812237L (en) * 1978-11-28 1980-05-29 Pharmacia Diagnostics Ab DETERMINATION OF CONCENTRATION OF SUBSTANCES WITH THE ABILITY OF BIOSPECIFICALLY BINDING MOLECULES OF BIOLOGICAL ORIGIN
GB2078370A (en) * 1980-06-13 1982-01-06 Nat Res Dev Binding assays
US4804625A (en) * 1984-09-27 1989-02-14 Amoco Corporation Assay procedures

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8701810A1 *

Also Published As

Publication number Publication date
JPS63500962A (en) 1988-04-07
FI872078A (en) 1987-05-11
FI853523A0 (en) 1985-09-13
WO1987001810A1 (en) 1987-03-26
FI872078A0 (en) 1987-05-11

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Inventor name: HARJUNMAA, HANNU

Inventor name: LUOTOLA, JUHANI