Specific binding assay with an enzyme modulator as a labeling substance
First Claim
1. In a specific binding assay method for determining a ligand in a liquid medium,wherein said liquid medium is combined with reagent means including a conjugate of a labeling substance and a binding component to form a binding reaction system having a bound-species and a free-species of said conjugate;
- andwherein the amount of ligand present in said liquid medium is determined as a function of the amount of labeling substance present in said bound-species or said free-species;
the improvement which comprises employing as said labeling substance a reversibly binding enzyme inhibitor, the binding constant for association of said inhibitor and an enzyme whose activity is inhibited thereby being less than about 1011 molar-1 ;
adding said enzyme whose activity is affected by said enzyme inhibitor to said bound-species or said free-species; and
determining the amount of said enzyme inhibitor therein by measuring the resulting activity of said enzyme.
0 Assignments
0 Petitions
Accused Products
Abstract
A specific binding assay method employing, as a labeling substance, a reversibly binding enzyme modulator for the detection of a ligand in a liquid medium. The method follows conventional specific binding assay techniques of either the homogeneous or heterogeneous type wherein the liquid medium to be assayed is combined with reagent means that includes a labeled conjugate to form a binding reaction system having a bound-species and a free-species of the conjugate. The amount of conjugate resulting in the bound-species or the free-species is a function of the amount of ligand present in the liquid medium assayed. In the present invention, the labeled conjugate comprises a reversibly binding enzyme modulator covalently linked to a binding component of the binding reaction system. The distribution of the conjugate between the bound-species and the free-species is determined by addition of an enzyme whose activity is affected, either decreased or increased, by said modulator and measuring the resulting activity of the enzyme. The enzyme modulator may be a conventional enzyme inhibitor, preferably of the coompetitive type, or an allosteric effector.
173 Citations
19 Claims
-
1. In a specific binding assay method for determining a ligand in a liquid medium,
wherein said liquid medium is combined with reagent means including a conjugate of a labeling substance and a binding component to form a binding reaction system having a bound-species and a free-species of said conjugate; - and
wherein the amount of ligand present in said liquid medium is determined as a function of the amount of labeling substance present in said bound-species or said free-species; the improvement which comprises employing as said labeling substance a reversibly binding enzyme inhibitor, the binding constant for association of said inhibitor and an enzyme whose activity is inhibited thereby being less than about 1011 molar-1 ;
adding said enzyme whose activity is affected by said enzyme inhibitor to said bound-species or said free-species; and
determining the amount of said enzyme inhibitor therein by measuring the resulting activity of said enzyme. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
- and
-
9. In a reagent means for use in determining a ligand in a liquid medium, which means includes a conjugate of a labeling substance and a binding component, which means and the ligand form a binding reaction system having a bound-species and a free-species of said conjugate,
the improvement wherein said labeling substance is a reversibly binding enzyme inhibitor and said means comprises additionally an enzyme whose activity is affected by said enzyme inhibitor, the binding constant for association of said inhibitor and said enzyme being less than about 1011 molar-1.
Specification