EP0005271A2
Expired / Lapsed
EP
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Method and kit for enzyme immunoassay of biological substances
Assignee: Wellcome Foundation Ltd
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Filing: 1979-05-03
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Granted: —
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Expires: 1999-05-03
Abstract
Abstract A sample. e.g. serum, containing an antigen, hapten or other biological substance is mixed with antibodies raised against that substance which have been tagged with biotin, and with a known amount of that substance labelled with an enzyme. After the competitive complexation of the antibody with the labelled substance and the substance to be detected, avidin, immobilised on an inert support is added. The avidin binds to the biotin and causes the complex to be precipitated. The solid and liquid phases are separated by centrifugation and the amount of biological substance in the original sample is determined by measuring the activity of the enzyme in either phase.
Inventors
- 👤 Indu Parikh
- 👤 Pedro Cuatrecasas
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 05:55
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/537; G01N33/531; G01N33/532; G01N33/535; G01N33/538; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S435/975; Y10S436/00; Y10S436/807; Y10S436/808
Claims Analysis
Total Claims: 11
Claim 1
Independent Claim
82 words
A process for the detection and/or determination of a biological substance in a test sample charac-- terised in that the process comprises admixing the test sample, a pre-determined quantity of a soluble enzyme-labelled form of the biological substance and a pre-determined quantity of a soluble biotin-tagged antibody raised against the biological substance and allowing these to come to equilibrium, then adding insolubilised avidin, separating the resulting solid phase from the liquid phase and determining the enzyme activity of either of these phases.
Claim 2
Dependent Claim
82 words
A process for the detection and/or determination of a biological substance in a test sample charac-- terised in that the process comprises admixing the test sample, a pre-determined quantity of a soluble enzyme-labelled form of the biological substance and a pre-determined quantity of a soluble biotin-tagged antibody raised against the biological substance and allowing these to come to equilibrium, then adding insolubilised avidin, separating the resulting solid phase from the liquid phase and determining the enzyme activity of either of these phases.
Claim 3
Dependent Claim
44 words
A process as claimed in claim 1 characterised in that the enzyme is selected from malate dehydrogenase, staphylococcal nuclease, delta-S-ketosteroid isomerase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase, alpha glycerophosphate dehydrogenase, triose phosphate isomerase, horse radish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase and ribonuclease.
Claim 4
Dependent Claim
61 words
A process as claimed in claim 1 or claim 2 characterised in that the avidin is insolubilised by covalent binding to a macromolecular insoluble carrier such as agarose, polyacrylamide, nylon, cross-linked dextran or filter paper or by physical coupling to insoluble carriers such as glass beads, plastic objects or to the inside of test tubes made from either plastic or glass.
Claim 5
Dependent Claim
21 words
A process as claimed in any one of claims 1 to 3 characterised in that the biological substance is an antigen.
Claim 6
Dependent Claim
25 words
A process as claimed in any one of claim 1 to 3 characterised in that the biological substance is a hapten, preferably digoxin or codeine.
Claim 7
Dependent Claim
124 words
A kit for use in the process of claim 1 characterised in that the kit comprises, in separate containers:- a) a pre-selected quantity of enzyme-labelled biological substance; b) a pre-selected quantity of biotin-tagged antibody raised against the biological substance; c) a pre-selected quantity of insolubilised avidin; and d) a substrate or substrates and co-factors for the determination of the activity of the enzyme employed; together with instructions to admix the enzyme- labelled biological substance, the biotin-tagged antibody raised against the biological substance, and a test sample, allowing the mixture to come to equilibrium, then adding the insolubilised avidin, separating the resulting solid phase from the liquid, and determining the enzyme activity of either of these phases using the enzyme substrate or substrates and co-factors.
Claim 8
Dependent Claim
44 words
A kit as claimed in claim 6 characterised in that the enzyme is selected from malate dehydrogenase, staphylococcal nuclease, delta-5-ketosteroid isomerase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase, alpha glycerophosphate dehydrogenase, triose phosphate isomerase, horse radish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase and ribonuclease.
Claim 9
Dependent Claim
61 words
A kit as claimed in claim 6 or claim 7 characterised in that the avidin is insolubilised by covalent binding to a macromolecular insoluble carrier such as agarose, polyacrylamide, nylon, cross-linked dextran or filter paper or by physical coupling to insoluble carriers such as glass beads, plastic objects or to the inside of test tubes made from either plastic or glass.
Claim 10
Dependent Claim
22 words
A kit as claimed in any one of claims 6 to 8 characterised in that the biological substance is digoxin or codeine.
Claim 11
Dependent Claim
19 words
A biotin-tagged antibody for use in the process of claim l.and for incorporation into the kit of claim 6.
References Cited (Backward Citations)
No citation records indexed.
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