Enzyme-linked competitive binding assays based on the biotin/avidin interaction

The strong interaction between biotin and avidin is utilized for the develo pment of enzyme-linked competitive binding assays not only for biotin itsel f, but also for other biomolecules. Alkaline phosphatase, a single substrat e enzyme typically used for heterogeneous types of competitive binding assa ys, is employed also for homogeneous types. For the biotin assay, the heter ogeneous protocol offers a much improved detection capability when compared to the homogeneous type. A simple approach of simulating dose-response cur ves is introduced. An effective analyte concentration attached to an enzyme label is determined by using the same binder, but with a different enzyme label. The analyte system adapted to the biotin/avidin-mediated homogeneous protocol is digoxin and a monoclonal anti-digoxin antibody. The detection capabilities of these assays, particularly the homogeneous types, are shown to be limited by the detectability of the enzyme conjugate employed.