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

A biotinylated enzyme inhibitorsorbent assay (BEISA) for quantitating enzyme and its inhibitor has been developed. The assay is based on the competition between unlabeled enzyme and biotin-labeled enzyme for binding by an immobilized inhibitor or between free inhibitor and the immobilized inhibitor for binding by a biotin-labeled enzyme followed by reaction of the biotin-bound complex with a streptavidin-alkaline phosphatase conjugate. The amount of enzyme or inhibitor can be determined from the intensity of color produced by the alkaline phosphatase acting on its substrate. Trypsin and its inhibitor from egg white (ovomucoid) were used as a model for the BEISA. The results indicated that the BEISA is a simple, sensitive, and specific method that can be used to quantitate the amount of an enzyme or its inhibitor and it is amenable to high-throughput analysis and automation. The BEISA can be also applied to any enzyme that has an appropriate inhibitor.