Heterogenous specific binding assay employing a cycling reactant as label
First Claim
1. In a heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:
- (a) contacting said liquid medium with reagent means including a labeled conjugate comprising a specific binding substance coupled to a labeling substance, said reagent means and the ligand forming a binding reaction system producing(1) a bound-phase of the labeled conjugate in which the specific binding substance therein is bound by a specific binding partner thereto and(2) a free-phase of the labeled conjugate in which the specific binding substance therein is not bound by a specific binding partner thereto,(b) separating said bound-phase and said free-phase; and
(c) determining said labeling substance in said bound-phase or said free-phase as a function of the amount of said ligand in said liquid medium;
the improvement wherein said labeling substance is a participant in a cyclic chemical reaction of the following type;
##STR57## wherein said labeling substance is a non catalytic material selected from the group consisting of reactants A, reactants B, and said cycled reactant.
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Abstract
An improved heterogenous specific binding assay method which employs a substance having reactant activity, i.e., a reactant, as a labeling substance in the detection of a ligand in a liquid medium. The method is carried out using reagent means which comprises, as its labeled constituent, a conjugate formed of a specific binding substance coupled to the reactant. The reactant advantageously is an enzymatic reactant such as an enzyme substrate or coenzyme. The activity of the conjugated reactant as a constituent of a predetermined reaction system is utilized as means for monitoring the extent of binding of the labeled constituent in conventional heterogenous specific binding assay schemes. The presence of a ligand in a liquid medium may be determined following conventional competitive binding manipulative techniques. After the necessary separation of the bound-and free-phases resulting in the specific binding reaction system, the extent of binding of the labeled constituent is determined by contacting either phase with the necessary materials to form the predetermined monitoring reaction system in which the labeling substance is active and assessing reactant activity therein.
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Citations
41 Claims
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1. In a heterogeneous specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of:
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(a) contacting said liquid medium with reagent means including a labeled conjugate comprising a specific binding substance coupled to a labeling substance, said reagent means and the ligand forming a binding reaction system producing (1) a bound-phase of the labeled conjugate in which the specific binding substance therein is bound by a specific binding partner thereto and (2) a free-phase of the labeled conjugate in which the specific binding substance therein is not bound by a specific binding partner thereto, (b) separating said bound-phase and said free-phase; and (c) determining said labeling substance in said bound-phase or said free-phase as a function of the amount of said ligand in said liquid medium; the improvement wherein said labeling substance is a participant in a cyclic chemical reaction of the following type;
##STR57## wherein said labeling substance is a non catalytic material selected from the group consisting of reactants A, reactants B, and said cycled reactant. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. In a reagent means for use in a heterogeneous specific binding assay method for determining a ligand in a liquid medium, which means includes (i) a labeled conjugate comprising a specific binding substance coupled to a labeling substance having a predetermined characteristic, which means and the ligand form a binding reaction system producing a bound-phase and a free-phase of the labeled conjugate, one of such phases being insoluble and the other being soluble, the amount of said predetermined characteristic of the labeling substance in said bound-phase or said free-phase being a function of the amount of the ligand present in the liquid medium under assay, and (ii) a specific binding partner of said ligand which binding partner is in a form which is insoluble in said liquid medium,
the improvement wherein said labeling substance is a participant in a cyclic chemical reaction of the following type: - ##STR58## wherein said labeling substance is a non catalytic material selected from the group consisting of reactants A, reactants B, and said cycled reactant, wherein the conjugate and the immobilized specific binding partner are present in amounts sufficient to determine said ligand.
- View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
Specification