US4376165A
Expired / Lapsed
US
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Method of preparing an enzyme-labeled ligand for use in specific binding assays and the labeled conjugate produced thereby
Assignee: Bayer Corp
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Filing: 1981-03-16
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Granted: 1983-03-08
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Expires: 2001-03-16
Abstract
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.
Inventors
- 👤 William E. Hornby
- 👤 David L. Morris
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 15:14
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/542; C; C12; C12N; C12N9/00; C12N9/96; Y; Y10; Y10S; Y10S435/00; Y10S435/964
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
86 words
An enzyme-labeled compound having the formula: 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.
Claim 2
Dependent Claim
86 words
An enzyme-labeled compound having the formula: 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.
Claim 3
Dependent Claim
13 words
The labeled compound of claim 1 wherein said conjugated enzyme is catalytically active.
Claim 4
Dependent Claim
28 words
The labeled compound of claim 1 wherein the binding constant for association of said apoenzyme and said prosthetic group is greater than about 10 6 molar -1 .
Claim 5
Dependent Claim
18 words
The labeled compound of claim 1 wherein said prosthetic group is flavin adenine dinucleotide, flavin mononucleotide or heme.
Claim 6
Dependent Claim
25 words
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.
Claim 7
Dependent Claim
30 words
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.
Claim 8
Dependent Claim
14 words
The labeled compound of claim 1 wherein said hapten is an iodothyronine thyroid hormone.
Claim 9
Dependent Claim
12 words
The labeled compound of claim 7 wherein said thyroid hormone is thyroxine.
Claim 10
Dependent Claim
25 words
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.
Claim 11
Independent Claim
93 words
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.
Claim 12
Independent Claim
93 words
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.
Claim 13
Dependent Claim
27 words
The method of claim 10 wherein the binding constant for association of said apoenzyme and said prosthetic group is greater than about 10 6 molar -1 .
Claim 14
Dependent Claim
19 words
The method of claim 10 wherein said conjugated enzyme in the resulting enzyme-labeled antigen or hapten is catalytically active.
Claim 15
Dependent Claim
17 words
The method of claim 10 wherein said prosthetic group is flavin adenine dinucleotide, flavin mononucleotide, or heme.
Claim 16
Dependent Claim
24 words
The method of claim 10 wherein said prosthetic group is flavin adenine dinucleotide or heme, and said apoenzyme is apoglucose oxidase or apoperoxidase, respectively.
Claim 17
Independent Claim
25 words
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.
Claim 18
Dependent Claim
13 words
The method of claim 10 wherein said prosthetic group is flavin adenine dinucleotide.
Claim 19
Dependent Claim
11 words
The method of claim 16 wherein said apoenzyme is apoglucose oxidase.
Claim 20
Dependent Claim
15 words
The method of claim 17 wherein said antigen or hapten is an iodothyronine thyroid hormone.
Claim 21
Dependent Claim
40 words
The method of claim 18 wherein flavin adenine dinucleotide is covalently coupled to said iodothyronine hormone through a linking group between the N 6 -amino group in the adenine nucleus of the former and the carboxyl group of the latter.
Claim 22
Dependent Claim
29 words
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.
Claim 23
Dependent Claim
11 words
The method of claim 20 wherein said iodothyronine hormone is thyroxine.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-03-07 | 1986-09-06 | 1987-03-08 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-03-07 | 1990-09-06 | 1991-03-08 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-03-07 | 1994-09-06 | 1995-03-08 | $7700.00 | Window Closed / Lapsed |