US4238565A
Expired / Lapsed
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Specific binding assay with a prosthetic group as a label component
Assignee: Bayer Corp
•
Filing: 1979-06-04
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Granted: 1980-12-09
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Expires: 1999-06-04
Abstract
Abstract A specific binding assay for determining a ligand in a liquid medium employing an organic prosthetic group residue, such as a residue of flavin adenine dinucleotide, flavin mononucleotide, or heme, as a label component in the labeled conjugate. Preferably, the label component is the prosthetic group residue alone or is a holoenzyme residue comprising such prosthetic group residue combined with an apoenzyme in the form of a holoenzyme complex. In the former case, the label component preferably is monitored in the assay by adding an apoenzyme after the binding reaction has been initiated and measuring the resultant holoenzyme activity. In the latter case, the label component is monitored simply by measuring holoenzyme activity. The assay method may follow conventional homogeneous and heterogeneous schemes. Preferred apoenzymes for use in the assay are apoglucose oxidase and apoperoxidase. The assay offers the advantages of colorimetric read-out and of being readily adaptable to automated techniques. Also disclosed are prosthetic group-labeled and enzyme-labeled conjugates for use in the assay, as well as a method for preparing the latter conjugates.
Inventors
- 👤 William E. Hornby
- 👤 David L. Morris
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 08:27
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/536; G01N33/542; C; C12; C12N; C12N9/00; C12N9/96; G01N33/74; G01N33/78; Y; Y10; Y10S; Y10S435/00; Y10S435/975
Claims Analysis
Total Claims: 74
Claim 1
Independent Claim
153 words
A homogeneous specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: forming a reaction mixture by contacting said liquid medium with (a) reagent means including a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to a binding component, such contact producing a binding reaction system in which a bound-species and a free-species of said labeled conjugate are formed, the proportion of the flavin adenine dinucleotide label component in said two formed species being a function of the presence of said ligand in said liquid medium, and (b) apoglucose oxidase, the ability of the flavin adenine dinucleotide label component to combine with the apoglucose oxidase to produce the holoenzyme glucose oxidase being different in activity in said two formed species, and determining the proportion of the flavin adenine dinucleotide label component in said two formed species by measuring glucose oxidase activity in said reaction mixture.
Claim 2
Dependent Claim
153 words
A homogeneous specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: forming a reaction mixture by contacting said liquid medium with (a) reagent means including a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to a binding component, such contact producing a binding reaction system in which a bound-species and a free-species of said labeled conjugate are formed, the proportion of the flavin adenine dinucleotide label component in said two formed species being a function of the presence of said ligand in said liquid medium, and (b) apoglucose oxidase, the ability of the flavin adenine dinucleotide label component to combine with the apoglucose oxidase to produce the holoenzyme glucose oxidase being different in activity in said two formed species, and determining the proportion of the flavin adenine dinucleotide label component in said two formed species by measuring glucose oxidase activity in said reaction mixture.
Claim 3
Dependent Claim
20 words
The method of claim 1 wherein said liquid medium is contacted with apoglucose oxidase after contact with said reagent means.
Claim 4
Dependent Claim
20 words
The method of claim 1 wherein said liquid medium is contacted with said reagent means and apoglucose oxidase substantially simultaneously.
Claim 5
Dependent Claim
21 words
The method of claim 3 wherein said reagent means and apoglucose oxidase are contacted with said liquid medium as separate entities.
Claim 6
Dependent Claim
38 words
The method of claim 1 wherein said ligand to be determined is an antigen or an antibody thereto; a hapten or an antibody thereto; or a hormone, vitamin, metabolite or drug, or a receptor or binding substance therefor.
Claim 7
Dependent Claim
18 words
The method of claim 1 wherein said ligand is a hapten of molecular weight between 100 and 1000.
Claim 8
Dependent Claim
113 words
A homogeneous specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: forming a reaction mixture by combining said liquid medium with reagent means including (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to said ligand or a binding analog thereof, (2) a specific binding partner of said ligand, and (3) apoglucose oxidase, wherein the binding by said binding partner of said ligand or analog thereof in said labeled conjugate inhibits the ability of the flavin adenine dinucleotide label component to combine with the apoglucose oxidase to produce the holoenzyme glucose oxidase; and measuring the resulting glucose oxidase activity in said reaction mixture.
Claim 9
Dependent Claim
113 words
A homogeneous specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: forming a reaction mixture by combining said liquid medium with reagent means including (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to said ligand or a binding analog thereof, (2) a specific binding partner of said ligand, and (3) apoglucose oxidase, wherein the binding by said binding partner of said ligand or analog thereof in said labeled conjugate inhibits the ability of the flavin adenine dinucleotide label component to combine with the apoglucose oxidase to produce the holoenzyme glucose oxidase; and measuring the resulting glucose oxidase activity in said reaction mixture.
Claim 10
Dependent Claim
23 words
The method of claim 7 wherein said reaction mixture is incubated for a predetermined period of time prior to measuring glucose oxidase activity.
Claim 11
Dependent Claim
23 words
The method of claim 7 wherein said liquid medium is contacted with said labeled conjugate, said binding partner and apoglucose oxidase substantially simultaneously.
Claim 12
Dependent Claim
34 words
The method of claim 7 wherein said liquid medium is combined first with said labeled conjugate and said binding partner, and after a predetermined incubation period, with apoglucose oxidase to form said reaction mixture.
Claim 13
Dependent Claim
15 words
The method of claim 7 wherein glucose oxidase activity is measured by a colorimetric method.
Claim 14
Dependent Claim
25 words
The method of claim 7 wherein said ligand to be determined is an antigen or a hapten and said binding partner is an antibody thereto.
Claim 15
Dependent Claim
18 words
The method of claim 7 wherein said ligand is a hapten of molecular weight between 100 and 1000.
Claim 16
Dependent Claim
11 words
The method of claim 13 wherein said ligand is a drug.
Claim 17
Dependent Claim
14 words
The method of claim 7 wherein said ligand is an antigenic protein or polypeptide.
Claim 18
Dependent Claim
11 words
The method of claim 15 wherein said ligand is an antibody.
Claim 19
Dependent Claim
102 words
A homogeneous specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: forming a reaction mixture by combining said liquid medium with reagent means including (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to a specific binding partner of said ligand and (2) apoglucose oxidase, wherein the binding of said binding partner in said labeled conjugate to said ligand inhibits the ability of the flavin adenine dinucleotide label component to combine with the apoglucose oxidase to produce the holoenzyme glucose oxidase; and measuring the resulting glucose oxidase activity in said reaction mixture.
Claim 20
Dependent Claim
102 words
A homogeneous specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: forming a reaction mixture by combining said liquid medium with reagent means including (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to a specific binding partner of said ligand and (2) apoglucose oxidase, wherein the binding of said binding partner in said labeled conjugate to said ligand inhibits the ability of the flavin adenine dinucleotide label component to combine with the apoglucose oxidase to produce the holoenzyme glucose oxidase; and measuring the resulting glucose oxidase activity in said reaction mixture.
Claim 21
Dependent Claim
23 words
The method of claim 17 wherein said reaction mixture is incubated for a predetermined period of time prior to measuring glucose oxidase activity.
Claim 22
Dependent Claim
20 words
The method of claim 17 wherein said liquid medium is contacted with said labeled conjugate and apoglucose oxidase substantially simultaneously.
Claim 23
Dependent Claim
30 words
The method of claim 17 wherein said liquid medium is combined first with said labeled conjugate, and after a predetermined incubation period, with apoglucose oxidase to form said reaction mixture.
Claim 24
Dependent Claim
15 words
The method of claim 17 wherein glucose oxidase activity is measured by a colorimetric method.
Claim 25
Dependent Claim
11 words
The method of claim 17 wherein said ligand is an antibody.
Claim 26
Dependent Claim
19 words
The method of claim 22 wherein said binding partner is a hapten of molecular weight between 100 and 1000.
Claim 27
Dependent Claim
12 words
The method of claim 22 wherein said binding partner is a drug.
Claim 28
Dependent Claim
15 words
The method of claim 22 wherein said binding partner is an antigenic protein or polypeptide.
Claim 29
Dependent Claim
56 words
In a reagent means for determining a ligand in a liquid medium, which means includes a conjugate having a label component and a binding component, the improvement wherein said label component comprises flavin adenine dinucleotide and said means includes apoglucose oxidase, which combines with the flavin adenine dinucleotide label component to produce the holoenzyme glucose oxidase.
Claim 30
Dependent Claim
56 words
In a reagent means for determining a ligand in a liquid medium, which means includes a conjugate having a label component and a binding component, the improvement wherein said label component comprises flavin adenine dinucleotide and said means includes apoglucose oxidase, which combines with the flavin adenine dinucleotide label component to produce the holoenzyme glucose oxidase.
Claim 31
Dependent Claim
21 words
The reagent means of claim 26 wherein said binding component in said conjugate is said ligand or a binding analog thereof.
Claim 32
Dependent Claim
39 words
The reagent means of claim 26 wherein said ligand to be determined is an antigen or an antibody thereto; a hapten or an antibody thereto; or a hormone, vitamin, metabolite or drug, or a receptor or binding substance therefor.
Claim 33
Dependent Claim
19 words
The reagent means of claim 26 wherein said ligand is a hapten of molecular weight between 100 and 1000.
Claim 34
Dependent Claim
15 words
The reagent means of claim 26 wherein said ligand is an antigenic protein or polypeptide.
Claim 35
Dependent Claim
59 words
A test kit for determining a ligand in a liquid medium, comprising, (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to said ligand or a binding analog thereof, (2) a specific binding partner of said ligand, and (3) apoglucose oxidase, which combines with the flavin adenine dinucleotide label component to produce the holoenzyme glucose oxidase.
Claim 36
Dependent Claim
59 words
A test kit for determining a ligand in a liquid medium, comprising, (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to said ligand or a binding analog thereof, (2) a specific binding partner of said ligand, and (3) apoglucose oxidase, which combines with the flavin adenine dinucleotide label component to produce the holoenzyme glucose oxidase.
Claim 37
Dependent Claim
20 words
The test kit of claim 31 wherein said one or more containers additionally hold reagents for measuring glucose oxidase activity.
Claim 38
Dependent Claim
27 words
The test kit of claim 31 comprising at least two separate containers, one holding said labeled conjugate and the other holding said binding partner and apoglucose oxidase.
Claim 39
Dependent Claim
26 words
The test kit of claim 31 wherein said ligand to be determined is an antigen or a hapten and said binding partner is an antibody thereto.
Claim 40
Dependent Claim
19 words
The test kit of claim 31 wherein said ligand is a hapten of molecular weight between 100 and 1000.
Claim 41
Dependent Claim
12 words
The test kit of claim 35 wherein said ligand is a drug.
Claim 42
Dependent Claim
15 words
The test kit of claim 31 wherein said ligand is an antigenic protein or polypeptide.
Claim 43
Dependent Claim
12 words
The test kit of claim 37 wherein said ligand is an antibody.
Claim 44
Dependent Claim
51 words
A test kit for determining a ligand in a liquid medium, comprising, (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to a specific binding partner of said ligand, and (2) apoglucose oxidase, which combines with the flavin adenine dinucleotide label component to produce the holoenzyme glucose oxidase.
Claim 45
Dependent Claim
51 words
A test kit for determining a ligand in a liquid medium, comprising, (1) a labeled conjugate comprising, as label component, flavin adenine dinucleotide coupled to a specific binding partner of said ligand, and (2) apoglucose oxidase, which combines with the flavin adenine dinucleotide label component to produce the holoenzyme glucose oxidase.
Claim 46
Dependent Claim
20 words
The test kit of claim 39 wherein said one or more containers additionally hold reagents for measuring glucose oxidase activity.
Claim 47
Dependent Claim
23 words
The test kit of claim 39 comprising at least two separate containers, one holding said labeled conjugate and the other holding apoglucose oxidase.
Claim 48
Dependent Claim
12 words
The test kit of claim 39 wherein said ligand is an antibody.
Claim 49
Dependent Claim
20 words
The test kit of claim 42 wherein said binding partner is a hapten of molecular weight between 100 and 1000.
Claim 50
Dependent Claim
13 words
The test kit of claim 42 wherein said binding partner is a drug.
Claim 51
Dependent Claim
16 words
The test kit of claim 42 wherein said binding partner is an antigenic protein or polypeptide.
Claim 52
Dependent Claim
168 words
A specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: contacting said liquid medium with reagent means including (1) a labeled conjugate comprising (a) a complex of an organic prosthetic group and an apoenzyme which combine to form a holoenzyme and (b) said ligand or a binding analog thereof coupled to said complex through said prosthetic group, and (2) a specific binding partner of said ligand, such contact producing a binding reaction in which are formed a bound-species of said labeled conjugate in which said coupled ligand or analog in said conjugate is bound to said binding partner and a free-species of said labeled conjugate in which said coupled ligand or analog in said conjugate is not bound to said binding partner, the proportion of said label component in said two formed species being a function of the presence of said ligand in said liquid medium, and determining said proportion by measuring holoenzyme activity in said bound-species or free-species, or both.
Claim 53
Dependent Claim
168 words
A specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: contacting said liquid medium with reagent means including (1) a labeled conjugate comprising (a) a complex of an organic prosthetic group and an apoenzyme which combine to form a holoenzyme and (b) said ligand or a binding analog thereof coupled to said complex through said prosthetic group, and (2) a specific binding partner of said ligand, such contact producing a binding reaction in which are formed a bound-species of said labeled conjugate in which said coupled ligand or analog in said conjugate is bound to said binding partner and a free-species of said labeled conjugate in which said coupled ligand or analog in said conjugate is not bound to said binding partner, the proportion of said label component in said two formed species being a function of the presence of said ligand in said liquid medium, and determining said proportion by measuring holoenzyme activity in said bound-species or free-species, or both.
Claim 54
Dependent Claim
35 words
The method of claim 46 wherein the holoenzyme activity of said complex in the labeled conjugate is different in activity when said labeled conjugate is in said bound-species from when it is in said free-species.
Claim 55
Dependent Claim
22 words
The method of claim 47 of the homogeneous type wherein said holoenzyme activity is measured in said bound-species and said free-species combined.
Claim 56
Dependent Claim
34 words
The method of claim 46 of the heterogeneous type wherein said complex has holoenzyme activity and wherein said bound-species and said free-species are physically separated and said holoenzyme activity is measured in one thereof.
Claim 57
Independent Claim
30 words
The method of any of claims 46-49 wherein said prosthetic group is flavin adenine dinucleotide or heme, and said apoenzyme with which it combines is apoglucose oxidase or apoperoxidase, respectively.
Claim 58
Independent Claim
25 words
The method of any of claims 46-49 wherein said prosthetic group is flavin adenine dinucleotide and said apoenzyme with which it combines is apoglucose oxidase.
Claim 59
Dependent Claim
13 words
The method of claim 46 wherein said ligand is an iodothyronine thyroid hormone.
Claim 60
Dependent Claim
24 words
The method of claim 46 wherein said ligand to be determined is an antigen or hapten and said binding partner is an antibody thereto.
Claim 61
Dependent Claim
18 words
The method of claim 53 wherein said ligand is a hapten of molecular weight between 100 and 1000.
Claim 62
Dependent Claim
63 words
In a reagent means for determining a ligand in a liquid medium, which means includes a conjugate having a label component and a binding component, the improvement wherein said label component is a residue of a conjugated enzyme formed of an organic prosthetic group residue combined with an apoenzyme, said binding component being coupled to said conjugated enzyme residue through said prosthetic group.
Claim 63
Dependent Claim
63 words
In a reagent means for determining a ligand in a liquid medium, which means includes a conjugate having a label component and a binding component, the improvement wherein said label component is a residue of a conjugated enzyme formed of an organic prosthetic group residue combined with an apoenzyme, said binding component being coupled to said conjugated enzyme residue through said prosthetic group.
Claim 64
Dependent Claim
37 words
The reagent means of claim 55 wherein the binding constant for the association of said apoenzyme and the prosthetic group whose residue is comprised in the labeled conjugate is greater than about 10 6 molar -1 .
Claim 65
Dependent Claim
14 words
The reagent means of claim 55 wherein said conjugated enzyme complex exhibits holoenzyme activity.
Claim 66
Dependent Claim
22 words
The reagent means of claim 55 wherein said binding component in said labeled conjugate is said ligand or a binding analog thereof.
Claim 67
Dependent Claim
49 words
The reagent means of claim 58 wherein said ligand, or analog thereof, and said prosthetic group are coupled at a site on the ligand or analog away from its specific binding locus and at a site on the prosthetic group away from its active binding locus for the apoenzyme.
Claim 68
Dependent Claim
22 words
The reagent means of claim 55 wherein said prosthetic group residue is a residue of flavin adenine dinucleotide, flavin mononucleotide, or heme.
Claim 69
Dependent Claim
33 words
The reagent means of claim 55 wherein said prosthetic group residue is a residue of flavin adenine dinucleotide or heme, and said apoenzyme with which it combines is apoglucose oxidase or apoperoxidase, respectively.
Claim 70
Dependent Claim
18 words
The reagent means of claim 55 wherein said prosthetic group residue is a residue of flavin adenine dinucleotide.
Claim 71
Dependent Claim
19 words
The reagent means of claim 62 wherein said apoenzyme with which said prosthetic group residue combines is apoglucose oxidase.
Claim 72
Dependent Claim
14 words
The reagent means of claim 63 wherein said ligand is an iodothyronine thyroid hormone.
Claim 73
Dependent Claim
38 words
The reagent means of claim 55 wherein said ligand to be determined is an antigen or an antibody thereto; hapten or an antibody thereto; or a hormone, vitamin, metabolite or drug, or a receptor or binding substance therefor.
Claim 74
Dependent Claim
19 words
The reagent means of claim 55 wherein said ligand is a hapten of molecular weight between 100 and 1000.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1983-12-09 | 1984-06-09 | 1984-12-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1987-12-09 | 1988-06-09 | 1988-12-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1991-12-09 | 1992-06-09 | 1992-12-09 | $7700.00 | Window Closed / Lapsed |