EP0040224A1 - Procede ameliore d'analyse immunologique d'enzymes non homogenes - Google Patents

Procede ameliore d'analyse immunologique d'enzymes non homogenes

Info

Publication number
EP0040224A1
EP0040224A1 EP80902222A EP80902222A EP0040224A1 EP 0040224 A1 EP0040224 A1 EP 0040224A1 EP 80902222 A EP80902222 A EP 80902222A EP 80902222 A EP80902222 A EP 80902222A EP 0040224 A1 EP0040224 A1 EP 0040224A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
analogue
ligand
antibody
analog
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Withdrawn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
EP80902222A
Other languages
German (de)
English (en)
Other versions
EP0040224A4 (fr
Inventor
Patrick Duffy
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.)
PRINCE CHARLES HOSPITAL DEVELOPMENT CENTRE TRUST
Original Assignee
PRINCE CHARLES HOSPITAL DEVELOPMENT CENTRE TRUST
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 PRINCE CHARLES HOSPITAL DEVELOPMENT CENTRE TRUST filed Critical PRINCE CHARLES HOSPITAL DEVELOPMENT CENTRE TRUST
Publication of EP0040224A1 publication Critical patent/EP0040224A1/fr
Publication of EP0040224A4 publication Critical patent/EP0040224A4/fr
Withdrawn legal-status Critical Current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

Definitions

  • AN IMPROVED METHOD OF NON HOMOGENOUS ENZYME IMMUNOASSAY This invention relates to an improved method of the technique known as non homogenous enzyme immunoassa , described, for example, in “Quantitative Enzyme Immunoassay", Ed. E. Engvall and A.J. Pesce, Blackwood Scientific Publications (Scand. J. Immunol. 8, Suppl. 7, 1978) .
  • non homogenous enzyme immunoassay is based on the competition for the active site of an anti ⁇ body between a ligand and a covalently coupled derivative of that same ligand to an enzyme. With separation of the bound and free enzyme ligand phases, enzyme assay of either fraction can then be made. To aid in the separation of the phases, it is often convenient to bind the antibody to an insoluble particle such as the wall of the reaction vessel itself, or a polysaccharide particle. Plotting of the percent bound against the ligand concentration, results in a typical immunoassay curve.
  • the enzyme immunoassay system is also capable of simple modification to provide for the measurement of serum antibody levels by making enzyme-anti- body derivatives and using a solid phase ligand to assist in the separation step.
  • the antibody characteristics required for the development of a successful non homogenous enzyme immunoassay for a particular ligand are those of high specificity for the ligand, as well as a high apparent affinity constant (as 1/mol) for the ligand.
  • an antibody demonstrating a high specificity and a high affinity constant towards a particular ligand will also bind substances which are closely similar in chemical structure to the ligand itself. Such substances are herein referred to as "analogues of the ligand”.
  • the affinity constant of the antibody observed with respect to the analogue may differ markedly from that observed for the antibody with the ligand itself. It is the object of
  • the present invention provides a method of non homogenous enzyme immunoassay including the steps of: (i) binding an analogue of a ligand to an enzyme to form an enzyme-ligand analogue complex; (ii) contacting the enzyme-ligand analogue complex with antibody for the ligand so as to bind the complex to the antibody;
  • a suitable analogue of the ligand is selected such that it is bound specifically by the antibody, though to a much lower affinity than is the case for the original ligand itself.
  • Such an analogue is then covalently joined to an appropriat enzyme by known methods of chemistry.
  • preparations of the antib are linked by appropriate means to an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead.
  • an insoluble particle which may be the surface of the inner wall of the small tube or may be a small insoluble particle, such as a polysaccharide or a glass bead.
  • Such a "formed solid phase” antibody is required to retain its function as an antibody.
  • the enzyme-labelled analogue is then brought into contact with the solid phase antibody, such that all the binding sites of the solid phase antibody are occupied by the enzyme-labelled analogue. Such an.
  • enzyme-labelled analogue may then be displaced from the binding site of the solid phase antibody by addition under appropriate conditions of the original ligand to which the antibody has been derived, or a different analogue having a higher affinity for the antibody than the enzyme-labelled analogue.
  • liberated enzyme analogue By separation of the liberated enzyme analogue from the enzyme analogue remaining bound to the solid phases , it is found that the quantity of liberated enzyme analogue, as measured by whatever means, bears an analytical relation ⁇ ship to the quantity of antigen or other ligand which causes the displacement.
  • the amount of enzyme analogue liberated by known concentration of ligand in appropriate standards, the measurement of an unknown concen ⁇ tration of the ligand in a sample can be effected.
  • known amounts of the enzyme-labelled analogue bound to the antibody can be dispensed into vials and subjected to the process known as freeze-drying.
  • freeze-drying By adding a required volume of water to the vial to reconstitute the active enzyme-analogue-antibody-complex, the assay can then be performed directly.
  • appropriate precautions to maintain stability of the reagent it would be possible to utilise a previously defined calibration curve from which the amount of ligand in a sample can be calculated without the necessity of so performing a calibration curve at that time. This method thus has application in times of emergency when speed
  • an assay for the anti- epilepsy drug dilantin, in the patients taking this drug is described.
  • Antibodies were raised to dilantin in goats by appropriate means and the antibodies demonstrated an affinity for dilantin of 10 1/mole, while having an affini for meth l-dilantin of 10 1/mole.
  • Methyl dilantin was the covalently joined to bacterial beta-galactosidase using water soluble carbo-dii ide, and the antibody covalently bound to sepharose beads. Aliquots of the sepharose antibo methyl dilantin-beta-galactosidase complexes were formed an all available antibody binding sites saturated.
  • Tube No. Dilantin Cont b-Galactosidase activity (Mg/L) (A405/min/ml)
  • ligand or ligand analogue such as the systems phenobarbitone (meta-nitro phenobarbitone) theophylline (l-methyl-3-carboxy butyl xanthine) thyroxine (3,3' 5 - triiodo-5 '-bromo thyronine) and triiodothyronine (3,5 - diiodo-3'-bromo thyronine).
  • phenobarbitone metal-nitro phenobarbitone
  • theophylline l-methyl-3-carboxy butyl xanthine
  • thyroxine l-methyl-3-carboxy butyl xanthine
  • thyroxine l-methyl-3-carboxy butyl xanthine
  • thyroxine l-methyl-3-carboxy butyl xanthine
  • thyroxine l-methyl-3-carboxy butyl x

Abstract

Procede d'analyse immunologique d'enzymes non homogenes dont l'etape initiale consiste a lier un analogue de liant a une enzyme pour former un complexe enzyme-liant. Ce complexe est ensuite mis en contact d'un anticorps du liant de maniere a y lier le complexe. L'analogue d'enzyme peut alors etre deplace des sites de liaison de l'anticorps par adjonction du liant de depart pour lequel l'anticorps a ete derive ou d'un analogue different possedant une specificite plus elevee pour l'anticorps que l'analogue marque par l'enzyme. Ensuite, l'analogue d'enzyme libere est separe de l'analogue lie a l'anticorps et une certaine quantite d'analogue enzymatique liberee est mesuree determinant ainsi la quantite de liant dans un echantillon par comparaison de la quantite d'analogue d'enzyme liberee avec des concentrations connues de liant.
EP19800902222 1979-11-19 1980-11-19 Procede ameliore d'analyse immunologique d'enzymes non homogenes. Withdrawn EP0040224A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPE137479 1979-11-19
AU1374/79 1979-11-19

Publications (2)

Publication Number Publication Date
EP0040224A1 true EP0040224A1 (fr) 1981-11-25
EP0040224A4 EP0040224A4 (fr) 1982-09-09

Family

ID=3768341

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19800902222 Withdrawn EP0040224A4 (fr) 1979-11-19 1980-11-19 Procede ameliore d'analyse immunologique d'enzymes non homogenes.

Country Status (3)

Country Link
EP (1) EP0040224A4 (fr)
JP (1) JPS56501583A (fr)
WO (1) WO1981001414A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3626468A1 (de) * 1986-08-05 1988-02-11 Hoechst Ag Verfahren und testkit zur bestimmung freier wirkstoffe in biologischen fluessigkeiten

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2184371A5 (fr) * 1972-05-11 1973-12-21 Akzo Nv
GB1401297A (en) * 1971-05-14 1975-07-16 Syntex Energy Res Enzyme aplification assay
US3966764A (en) * 1972-07-10 1976-06-29 Syva Company Ligand determination of spin labeled compounds by receptor displacement-amphetamine analogs
FR2348493A1 (fr) * 1976-04-15 1977-11-10 Technicon Instr Immunotest pour la diphenyl-hydantoine
GB1575610A (en) * 1976-04-15 1980-09-24 Technicon Instr Immunoassay for diphenylhydantoin
EP0026103A1 (fr) * 1979-09-24 1981-04-01 AMERSHAM INTERNATIONAL plc Procédé de détermination de la proportion libre des substances dans les fluides biologiques

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3673410A (en) * 1969-06-25 1972-06-27 John H Waite Method of examination of cell samples using a radioactively tagged dye
US3817837A (en) * 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
US3852157A (en) * 1971-05-14 1974-12-03 Syva Corp Compounds for enzyme amplification assay
US3905871A (en) * 1971-05-14 1975-09-16 Syva Co Lactam conjugates to enzymes
DE2128670B2 (de) * 1971-06-09 1977-06-30 Merck Patent Gmbh, 6100 Darmstadt Immunologische isoenzym-bestimmungsmethode
US3975237A (en) * 1972-11-06 1976-08-17 Syva Company Compounds for enzyme amplification assay - - ecgonine analogs
US3966556A (en) * 1972-11-06 1976-06-29 Syva Company Compounds for enzyme amplification assay methadone analogs
IN142734B (fr) * 1975-04-28 1977-08-20 Miles Lab
JPS5925183B2 (ja) * 1978-07-05 1984-06-15 ダイナボット株式会社 エラスタ−ゼ−１の測定方法
US4273866A (en) * 1979-02-05 1981-06-16 Abbott Laboratories Ligand analog-irreversible enzyme inhibitor conjugates and methods for use

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1401297A (en) * 1971-05-14 1975-07-16 Syntex Energy Res Enzyme aplification assay
GB1401298A (en) * 1971-05-14 1975-07-16 Syntex Energy Res Ligands bonded to enzymes
FR2184371A5 (fr) * 1972-05-11 1973-12-21 Akzo Nv
GB1433783A (en) * 1972-05-11 1976-04-28 Akzo Nv Process for the detection and determination of haptens
US3966764A (en) * 1972-07-10 1976-06-29 Syva Company Ligand determination of spin labeled compounds by receptor displacement-amphetamine analogs
FR2348493A1 (fr) * 1976-04-15 1977-11-10 Technicon Instr Immunotest pour la diphenyl-hydantoine
GB1575610A (en) * 1976-04-15 1980-09-24 Technicon Instr Immunoassay for diphenylhydantoin
EP0026103A1 (fr) * 1979-09-24 1981-04-01 AMERSHAM INTERNATIONAL plc Procédé de détermination de la proportion libre des substances dans les fluides biologiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO8101414A1 *

Also Published As

Publication number Publication date
EP0040224A4 (fr) 1982-09-09
WO1981001414A1 (fr) 1981-05-28
JPS56501583A (fr) 1981-10-29

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PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

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Inventor name: DUFFY, PATRICK