Specific binding assay with a prosthetic group as a label component
First Claim
1. 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, anddetermining the proportion of the flavin adenine dinucleotide label component in said two formed species by measuring glucose oxidase activity in said reaction mixture.
0 Assignments
0 Petitions
Accused Products
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.
-
Citations
66 Claims
-
1. 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. - View Dependent Claims (2, 3, 4, 5, 6)
-
-
7. 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.- View Dependent Claims (8, 9, 10, 11, 12, 13, 14, 15, 16)
-
17. 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.- View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25)
-
26. 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.
-
31. 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.
-
39. 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.
-
46. 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. - View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54)
-
-
55. 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.
Specification