Enzyme immunoassays for beta-adrenoreceptor blocking agent, befunolol and its main metabolite, M1

Enzyme immunoassays for befunolol (BFE-60) and its main metabolite, M1, were established. These assays were based on the double antibody solid phase principle. Specific antisera to these agents were raised in rabbits by immunization with N-succinyl-BFE-60-human serum albumin (HSA) conjugate and N-succinyl-M1-HSA conjugate, respectively. Antibody was incubated with a mixture of beta-D-galactosidase (beta-Gal) labelled hapten and unlabelled standard or sample drug which had been succinylated prior to assay. The antibody-bound drug was separated from free using anti-rabbit IgG immobilized on a polystyrene ball, and activity of the enzyme on the solid phase was determined fluorometrically. Succinylation of both BFE-60 and M1 resulted in 100-fold increased sensitivity with reduced cross-reactivity. The present immunoassays allow detection of 0.4 pg of BFE-60 (10 pg/ml) and 3 pg of M1 (75 pg/ml). The time courses of plasma levels of BFE-60 and M1 were examined after a single intravenous administration (1 mg/kg) or a single topical instillation (0.2, 1.0 and 5.0%: 50 microliters) to albino rabbits. BFE-60 and M1 in biological fluids required acetone-extraction before assay.