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WO2000065351A1 - Procede de detection d'antigenes par luminescence - Google Patents

Procede de detection d'antigenes par luminescence Download PDF

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Publication number
WO2000065351A1
WO2000065351A1 PCT/EP2000/003576 EP0003576W WO0065351A1 WO 2000065351 A1 WO2000065351 A1 WO 2000065351A1 EP 0003576 W EP0003576 W EP 0003576W WO 0065351 A1 WO0065351 A1 WO 0065351A1
Authority
WO
WIPO (PCT)
Prior art keywords
biologically active
adhesive surface
test device
test
detected
Prior art date
Application number
PCT/EP2000/003576
Other languages
German (de)
English (en)
Inventor
Reinhard Geiger
Original Assignee
Intermedical S.A.H.
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 Intermedical S.A.H. filed Critical Intermedical S.A.H.
Priority to AU47498/00A priority Critical patent/AU4749800A/en
Publication of WO2000065351A1 publication Critical patent/WO2000065351A1/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
    • G01N33/58—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)
    • 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

Definitions

  • the invention relates to a method for the luminescent detection of biologically active bodies in the form of antigens, antibodies, haptens and the like.
  • the first radio immunoassays were developed a few decades ago in order to be able to determine the substances described. So-called enzyme immunoassays were later developed in which enzymes serve as markers. The enzymes are coupled with a ligand to form a ligand-enzyme conjugate. These ligands can be one of the substances listed above and thus an antibody, an antigen or a hapten.
  • an antibody labeled with an enzyme is reacted with its biochemical "counter body" (in the present example, an antigen), the ligand attaching to the antigen together with the enzyme.
  • biochemical "counter body” in the present example, an antigen
  • Free reaction product that can be determined analytically.
  • the amplification effect via the substrate turnover of the enzymes is used per unit of time.
  • the amount of the reaction product formed and thus also the amount of bound ligands labeled with an enzyme can thereby be determined qualitatively or quantitatively.
  • luminescent processes which use, for example, bioluminescence or chemiluminescence, have recently emerged as particularly sensitive processes.
  • Particularly advantageous substrates for the detection of ligands labeled with enzymes have been found to be luciferin derivatives, which are described, for example, in EP 0 243 429 A1, to which express reference is made, reference also being made to the references cited therein.
  • test strips are used in the description below.
  • This test strip has an adhesive surface for the biologically active body to be detected and can be handled in a simple manner.
  • the biologically active body in step a) is applied to the adhesive surface of this test strip, preferably in that the biologically active body or the biologically active bodies to be detected are "clipped" from a surface by means of the test strip by using the test strip with its adhesive surface is brought into contact with the place where the biologically active body to be detected is located and / or this place is covered with it.
  • the biologically active bodies located on the surface are transferred to the latter by brushing over this surface with the adhesive surface of the test strip and adhere there firmly to the adhesive surface.
  • the test strips can then be used immediately for examination.
  • the type of surface on which the biologically active bodies are located and from which they are quasi removed can be of any nature and of various configurations.
  • a human mucous membrane or a contaminated area on a piece of equipment can be mentioned as examples of such a surface, in order to list only a few possible variants.
  • the adhesive surface of the test strip is then simply immersed in the solution obtained. If the biologically active bodies are already in a liquid medium, all that is required to take a sample or to apply the biologically active bodies to the adhesive surface of the test strip is to immerse this adhesive surface in the medium or to pipette or drip on the medium onto the adhesive surface .
  • test strip used according to the invention virtually makes a smear at the location to be examined. When painted over, it absorbs the Adhesive surface of the biologically active body to be detected or to be verified.
  • the test strip with the biologically active body is then immersed in step b) with its adhesive surface in a so-called blocking solution, which is located, for example, in a tube.
  • This blocking solution contains various proteins and detergents that at least partially saturate free binding sites on the adhesive surface so that later no other proteins or other substances, for example the conjugate, can be non-specifically absorbed. In other words, the blocking solution thus blocks the free binding sites on the adhesive surface of the test strip.
  • BSA bovine serum albumin
  • Triton X-100 ionogenic, surface-active substance which is formed by reaction of the t-octylphenol with ethylene oxide
  • Tween 20 polyoxyethylene (20) sorbitan monolaurate
  • the adhesive surface of the test strip is preferably washed and, in step c), immersed in a conjugate solution, which is also preferably in a tube.
  • This conjugate is provided on the one hand with an enzyme and on the other hand with a biologically active counter-body which is specific for the biologically active body to be detected. If the biologically active body to be detected is, for example, an antigen, then the biologically active counterbody is expediently the specific or partially specific antibody. The same applies vice versa.
  • the biologically active body can couple to the biologically active counter body, so that a body-counter body-enzyme conjugate is formed.
  • the counter body represents the ligand mentioned at the beginning. However, not only an antigen but also an antibody or hapten etc. can function as the ligand of the ligand-enzyme conjugate.
  • the enzyme of this conjugate is one which can release a luminogenic compound from a substrate which can emit luminescent light in a subsequent reaction.
  • the biologically active body to be detected is an antigen
  • the antigen located on the adhesive surface of the test strip is coupled with its counterpart or counterpart, namely the antibody.
  • the enzyme is also coupled to the antigen via the antibody.
  • incubation is preferably carried out for a few minutes. Then the adhesive surface is removed and preferably washed.
  • the adhesive surface in stage d) is then immersed in a solution which is a substrate for the enzyme which is coupled to the adhesive surface of the test strip via the body-counter body bridge. It is preferably a bioluminogenic or chemiluminogenic substrate.
  • the enzyme releases a compound from the substrate that can emit luminescent light.
  • a so-called detection cocktail in stage e is dropped into the tube after removal of the test strip and this is introduced into a luminometer for light measurement.
  • the light measured is a measure of the amount of antigen to be detected.
  • the tube is inserted into a luminometer after removal of the test strip and sealed light-tight. Then a drop of a chemical minescence reagent in step e) dripped into the tube and the emitted light measured. In this case too, the measured light is a measure of the amount of the antigen to be detected.
  • the bioluminescent detection reaction can be carried out as follows, for example, using the bioluminescence of the firefly Photinus pyralis.
  • D-luciferin-O-phosphate is used as the bioluminogenic substrate.
  • the enzyme of the body-body conjugate is alkaline phosphatase.
  • the substrate D-luciferin-O-phosphate is reacted with the alkaline phosphatase of the body-body conjugate, then D-luciferin, among others, is formed.
  • the latter is implemented in a second step in the presence of ATP and Mg 2+ ions and the enzyme luciferase. This reaction produces light, which can be detected in a luminometer or quantified.
  • Step 1 Luciferin-O-Phosphate Alkaline Phosphatase, D-Luciferin + P
  • the method according to the invention is further explained below on the basis of detection of an antigen.
  • the antigen can be activated by an antibody or a hapten or another correspondingly biologically active ven body or substance to be replaced.
  • the reason for the explanation with reference to an antigen is for the purpose of simpler and clearer presentation.
  • bio-luminescence for the detection of the antigen according to the invention, the following solutions or materials can be used.
  • the blocking solution used in stage b) of the process according to the invention is one which effectively saturates free binding sites on the adhesive surface of the test strip.
  • the conjugate solution in stage c) of the process according to the invention is preferably one which contains 5 ng antibody-enzyme conjugate in 1 ml PBS (phosphate buffered salines).
  • the solution in step c) with the bioluminogenic substrate contains 0.5 mg LuPo in 1 ml water.
  • a detection cocktail which reacts with the released compound (here: D-luciferin) with light emission.
  • reaction sequence can be summarized schematically as follows:
  • AH chemiluminogenic substrate
  • a and H cleavage products of AH
  • R released chemiluminescent compound that can produce light.
  • chemiluminescence the same blocking solution can be used as for the bioluminescence described above.
  • an AE detection cocktail with the following composition is preferably used in step e): 10 mmol / l H 2 O 2 in 0.1 mmol / l NaOH. The measured light is then a measure of the amount of the antigen to be detected.
  • test strip used according to the invention, all materials can be used to which or on which biologically active bodies or counterparts adhere or can be immobilized. These are primarily membranes. Nitrocellulose membranes or nylon membranes or such materials are preferably used for this, which are also used for microtiter plate walls. All known materials suitable for the stated purposes can be used.
  • LM Listeria monocytogenes
  • a test strip with a nitrocellulose adhesive surface is spread over a surface / sample on which the LM are located.
  • the test strip with the nitrocellulose adhesive surface is immersed in a liquid sample containing the LM.
  • the LM adhere to the nitrocellulose adhesive surface of the test strip.
  • the test strip with the adhesive surface is then transferred to a test tube containing approximately 0.5 ml of blocking solution and incubated there for 15 minutes at room temperature.
  • the composition of this blocking solution can, for example, correspond to that of a Boehringer Blocking Reagent for Western blots as it is sold by Boehringer Mannheim.
  • test strip with its nitrocellulose adhesive surface is then immersed in a test tube containing 0.5 ml of Anti-Listeria monoc togenes-alkaline phosphatase conjugate (and / or ⁇ -galactosidase conjugate or another) and incubated for 5 min at room temperature.
  • concentration can be, for example, 10 ⁇ g / 0.5 ml.
  • test strip is then washed under running water or with the aid of distilled water and immersed in a test tube containing 0.5 ml of reagent solution (0.5 mg luciferin phosphate / 1 ml of water) and incubated for 2 min at room temperature. The test strip is then discarded.
  • 0.1 ml becomes 0.4 ml of a cocktail solution (41 mmol / l HEPES, 5 mmol / l MgCl 2 0, 2.6 mmol / l ATP, pH 7.75; contains 1 ⁇ g luciferase / ml Cocktail) and measured in a tube luminometer.
  • the measured light is a measure of the existing number of LM. In this way it is possible to determine amounts as small as 100 LM cells.
  • the invention also relates to a test device for carrying out the method according to the invention.
  • This test device is preferably a test strip or a test stick.
  • These test strips or test pins have an adhesive surface which is preferably arranged in the vicinity of one of their ends.
  • This adhesive surface can be the membranes described above and in particular a nitrocellulose membrane or a nylon membrane and materials which are used for microtiter plate walls. be applied.
  • a preferred embodiment is a test pen or a test stick, the adhesive surface of which has approximately the shape of a ball or which is flattened on one side and is made of polystyrene.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (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)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne un procédé de détection par luminescence caractérisé en ce que a) le corps biologiquement actif est appliqué sur la surface adhésive d'un dispositif de test se présentant sous la forme d'une bandelette test ou d'un bâton test, b) la surface adhésive du dispositif de test est plongée dans une solution de blocage où elle est incubée, c) la surface adhésive du dispositif de test est plongée dans une solution où se trouve un conjugué qui comprend une enzyme et un anticorps biologiquement actif spécifique d'un corps biologiquement actif à détecter, l'enzyme dudit conjugué pouvant libérer d'un substrat un composé luminogène qui peut émettre une lumière luminescente, puis ladite surface adhésive est incubée dans ladite solution, le corps biologiquement actif étant couplé à l'anticorps biologiquement actif spécifique, d) la surface adhésive du dispositif de test est plongée dans une solution qui contient le substrat pour l'enzyme, et est incubée dans cette solution de sorte que le composé luminogène soit libéré, et e) on fait réagir, de manière connue en soi, le composé luminogène libéré, de façon à provoquer l'émission d'une lumière qui est détectée.
PCT/EP2000/003576 1999-04-23 2000-04-19 Procede de detection d'antigenes par luminescence WO2000065351A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47498/00A AU4749800A (en) 1999-04-23 2000-04-19 Antigen luminescent detection method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE1999118636 DE19918636A1 (de) 1999-04-23 1999-04-23 Verfahren zum lumineszenten Nachweis von Antigenen
DE19918636.7 1999-04-23

Publications (1)

Publication Number Publication Date
WO2000065351A1 true WO2000065351A1 (fr) 2000-11-02

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/003576 WO2000065351A1 (fr) 1999-04-23 2000-04-19 Procede de detection d'antigenes par luminescence

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AU (1) AU4749800A (fr)
DE (1) DE19918636A1 (fr)
WO (1) WO2000065351A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0173375A1 (fr) * 1984-07-23 1986-03-05 Polaroid Corporation Nouveau produit pour faire les essais et procédé
WO1987002667A1 (fr) * 1985-10-24 1987-05-07 Reinhard Geiger Derives de d-luciferine et leur utilisation
EP0653637A2 (fr) * 1993-11-12 1995-05-17 Eastman Kodak Company Eléments sèches, dispositifs et trousses d'essais et méthodes pour la détection chimoluminescente d'analytes utilisant les réactifs marques par peroxidase
EP0675362A2 (fr) * 1994-03-30 1995-10-04 Johnson & Johnson Clinical Diagnostics, Inc. Minimiser l'interférence dans des analyses immunologiques chimiluminescentes en couches minces
WO1997009619A1 (fr) * 1995-09-01 1997-03-13 Johnson & Johnson Clinical Diagnostics, Inc. Element et procede analytique de determination d'un ligand specifique de liaison utilisant la bromoperoxydase de vanadium comme enzyme generatrice de signaux

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0173375A1 (fr) * 1984-07-23 1986-03-05 Polaroid Corporation Nouveau produit pour faire les essais et procédé
WO1987002667A1 (fr) * 1985-10-24 1987-05-07 Reinhard Geiger Derives de d-luciferine et leur utilisation
EP0243429A1 (fr) * 1985-10-24 1987-11-04 Reinhard Dr Geiger Derives de d-luciferine et leur utilisation.
EP0653637A2 (fr) * 1993-11-12 1995-05-17 Eastman Kodak Company Eléments sèches, dispositifs et trousses d'essais et méthodes pour la détection chimoluminescente d'analytes utilisant les réactifs marques par peroxidase
EP0675362A2 (fr) * 1994-03-30 1995-10-04 Johnson & Johnson Clinical Diagnostics, Inc. Minimiser l'interférence dans des analyses immunologiques chimiluminescentes en couches minces
WO1997009619A1 (fr) * 1995-09-01 1997-03-13 Johnson & Johnson Clinical Diagnostics, Inc. Element et procede analytique de determination d'un ligand specifique de liaison utilisant la bromoperoxydase de vanadium comme enzyme generatrice de signaux

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Publication number Publication date
DE19918636A1 (de) 2000-10-26
AU4749800A (en) 2000-11-10

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