Biotinylated enzyme inhibitorsorbent assay: A specific method for quantitating enzyme and its inhibitor

A biotinylated enzyme inhibitorsorbent assay (BEISA) for quantitating enzym e and its inhibitor has been developed. The assay is based on the competiti on between unlabeled enzyme and biotin-labeled enzyme for binding by an imm obilized inhibitor or between free inhibitor and the immobilized inhibitor for binding by a biotin-labeled enzyme followed by reaction of the biotin-b ound complex with a streptavidin-alkaline phosphatase conjugate. The amount of enzyme or inhibitor can be determined from the intensity of color produ ced by the alkaline phosphatase acting on its substrate. Trypsin and its in hibitor from egg white (ovomucoid) were used as a model for the BEISA. The results indicated that the BEISA is a simple, sensitive, and specific metho d that can be used to quantitate the amount of an enzyme or its inhibitor a nd it is amenable to high-throughput analysis and automation. The BEISA can be also applied to any enzyme that has an appropriate inhibitor. (C) 1998 Academic Press.