Methods and reagent combinations are provided for competitive protein binding assays for determining a member of an immunological pair (ligand and receptor) whereby an enzyme-ligand conjugate is employed in combination with an enzyme inhibitor, conveniently an antibody to said enzyme. When ligand is the analyte, receptor for ligand is also included in the assay medium, while supplemental amounts of receptor need not be added when receptor is the analyte. The assay is carried out in an aqueous buffered medium, normally at constant temperature, by combining in the assay medium the unknown sample suspected of containing the analyte, enzyme-bound-ligand, ligand receptor (antiligand), enzyme inhibitor (antienzyme), and enzyme substrates, and the enzymatic activity in the assay medium determined. By comparing the observed enzymatic activity with an unknown to the enzymatic activity observed in an assay medium with a known amount of analyte, the amount of analyte can be quantitatively determined.
Kits are provided having matched amounts of enzyme-bound-ligand, antienzyme and, when appropriate, antiligand for use in the subject assay.