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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.

The properties of binding proteins that control the nature and magnitude of inhibition of the enzyme-ligand conjugates in homogeneous enzyme-linked competitive binding assays were investigated. An assay for biotin that employed adenosine deaminase as the enzyme-label was used as a model system because of the availability of several biotin-specific binders with different characteristics. It was found that the association constant between the ligand and the binding protein, as well as the depth of the binding pocket, affect the response characteristics of the assay. In addition, the reported data suggest that steric-hindrance effects also contribute toward the sensitivity and detection-limit capabilities of the assay.

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