PCT No. PCT/EP89/01612 Sec. 371 Date Aug. 7, 1991 Sec. 102(e) Date Aug. 7, 1991 PCT Filed Dec. 22, 1989 PCT Pub. No. WO90/07714 PCT Pub. Date Jul. 12, 1990.The invention concerns a method of homogeneous immunoassay of a ligand in a liquid test sample which comprises: a)incubating a mixture of (1) the liquid test sample; (1i) where the ligand under assay has only one epitope, a covalent conjugate of the ligand with any mono- or poly-epitope molecule having at least one epitope distinct from the epitope of the ligand; (2) a labile enzyme; (3) a first bispecific monoclonal antibody protecting the activity of the encyme through binding to a first epitope on the encyme and capable of binding the ligand under assay; and (4) a second bispecific monoclonal antibody capable of bonding said enzyme and a second distinct epitope on the ligand under assay or an epitope of the said covalent conjugate; whereby a quaternary immunocomplex is formed; b) after incubation, subjecting the mixture to conditions whereby any enzyme not present in such a quaternary immunocomplex is inactivated; and c) determining the amount of detectable encyme, the determination being related to the amount, if any, of ligand in the test sample. Kits for use in carrying out the method are also described.
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