Observation of “Hook effects” in the Inhibition and Dose-Response Curves of Biotin Assays Based on the Interaction of Biotinylated Glucose Oxidase with (Strept)avidin

Abstract

The Inhibition of highly biotinylated enzymes by avidin and streptavidin has been used in the development of homogeneous assays for biotin and other analytes. Usually, this inhibition occurs in a similar fashion for both avidin and streptavidin. Specifically, the curves that relate the Inhibition of the enzymatic activity with the concentration of avidin or streptavidin have a sigmoidal shape; i.e., the inhibition of the enzyme-biotin conjugates increases gradually with increasing amounts of avidin or streptavidin and arrives at a plateau at high binding protein concentrations. However, when these two biotin-specific binding proteins interact with biotinylated glucose oxidase a significant difference in their inhibitory action is observed. In particular, the inhibition curves have a sigmoidal shape for streptavidin, while those for avidin exhibit a maximum (“hook”) at low avidin concentrations. This difference in the reactivity of the two proteins with biotinylated enzymes influences both the shape of the dose-response curve and the detection limits of homogeneous enzyme-linked competitive binding assays for biotin. © 1993, American Chemical Society. All rights reserved.