Method of preparing an enzyme-labeled ligand for use in specific binding assays and the labeled conjugate produced thereby

• US 4,376,165 A
• Filed: 03/16/1981
• Issued: 03/08/1983
• Est. Priority Date: 06/22/1978
• Status: Expired due to Fees

• Current Assignee: Miles Laboratories, Inc. (Bayer AG)
• Original Assignee: Miles Laboratories, Inc. (Bayer AG)
• Inventors: Morris, David L., Hornby, William E.
• Primary Examiner(s): Wiseman, Thomas G.
• Application Number:
  US06/244,047
• Time in Patent Office: 722 Days
• Field of Search: 435/7, 435/177, 435/188, 435/810, 435/189, 435/190, 260/112 R, 260/121, 536/4, 536/23, 536/24, 23/230 B, 424/8, 424/12
• US Class Current: 435/188
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 435/964 including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage

Abstract

A method of preparing an enzyme-labeled ligand for use in specific binding assays for determining said ligand or a specific binding partner thereof, by covalently coupling said ligand or a binding analog thereof to an organic prosthetic group and combining the resulting coupled product with an apoenzyme to form a conjugated enzyme complex with said prosthetic group in said coupled product. Preferably, the conjugated enzyme complex in the resulting labeled conjugate exhibits holoenzyme activity which is affected by binding of said labeled conjugate by a specific binding partner of said ligand. Preferred prosthetic groups are flavin mononucleotide, heme, and, especially, flavin adenine dinucleotide, with preferred apoenzymes being apoperoxidase and, especially, apoglucose oxidase.

Claims

1. An enzyme-labeled compound having the formula:

space="preserve" listing-type="equation">Apo-Pr-R-L

wherein Pr represents an organic prosthetic group, Apo is an apoenzyme combined with said prosthetic group forming its conjugated enzyme, R is a linking group, and L is a hapten of molecular weight between 100 and 1000;

wherein said hapten and said prosthetic group are coupled through said linking group at a site on the hapten away from its specific binding site and at a site on the prosthetic group away from its active binding site for the apoenzyme.

2. The labeled compound of claim 1 wherein said conjugated enzyme is catalytically active.

3. The labeled compound of claim 1 wherein the binding constant for association of said apoenzyme and said prosthetic group is greater than about 10⁶ molar^-1.

4. The labeled compound of claim 1 wherein said prosthetic group is flavin adenine dinucleotide, flavin mononucleotide or heme.

5. The labeled compound of claim 1 wherein said prosthetic group is flavin adenine dinucleotide or heme, and said apoenzyme is apoglucose oxidase or apoperoxidase, respectively.

6. The labeled compound of claim 1 wherein said linking group is a chain containing 1 to 14 carbon atoms and 0 to 5 heteroatoms selected from nitrogen, oxygen and sulfur.

7. The labeled compound of claim 1 wherein said hapten is an iodothyronine thyroid hormone.

8. The labeled compound of claim 7 wherein said thyroid hormone is thyroxine.

9. The labeled compound of any one of claims 7 and 8, wherein said prosthetic group is flavin adenine dinucleotide and said apoenzyme is apoglucose oxidase.

10. A method of preparing an enzyme-labeled antigen or hapten for use in specific binding assays for determining said antigen or hapten or a specific binding partner thereof, comprising the steps of:

covalently coupling said antigen or hapten, at a site thereon away from its specific binding locus, to an organic prosthetic group, at a site thereon away from its active binding locus for an apoenzyme, and

combining the resulting coupled product with said apoenzyme to form a conjugated enzyme with said prosthetic group in said coupled product yielding an enzyme-labeled antigen or hapten.

11. The method of claim 10 wherein the binding constant for association of said apoenzyme and said prosthetic group is greater than about 10⁶ molar^-1.

12. The method of claim 10 wherein said conjugated enzyme in the resulting enzyme-labeled antigen or hapten is catalytically active.

13. The method of claim 10 wherein said prosthetic group is flavin adenine dinucleotide, flavin mononucleotide, or heme.

14. The method of claim 10 wherein said prosthetic group is flavin adenine dinucleotide or heme, and said apoenzyme is apoglucose oxidase or apoperoxidase, respectively.

15. The method of any one of claims 10 and 11-14 wherein said antigen or hapten is a hapten of molecular weight between 100 and 1000.

16. The method of claim 10 wherein said prosthetic group is flavin adenine dinucleotide.

17. The method of claim 16 wherein said apoenzyme is apoglucose oxidase.

18. The method of claim 17 wherein said antigen or hapten is an iodothyronine thyroid hormone.

19. The method of claim 18 wherein flavin adenine dinucleotide is covalently coupled to said iodothyronine hormone through a linking group between the N⁶ -amino group in the adenine nucleus of the former and the carboxyl group of the latter.

20. The method of claim 19 wherein said linking group is a chain containing 1 to 14 carbon atoms and 0 to 5 heteroatoms selected from nitrogen, oxygen and sulfur.

21. The method of claim 20 wherein said iodothyronine hormone is thyroxine.