Abstract
This chapter describes competitive binding assays for biotin that use the bioluminescent protein aequorin as the label. Heterogeneous and homogeneous competitive binding assays using aequorin bioluminescence have been developed for detection of biotin. In the heterogeneous system, an aequorin–biotin conjugate competes with unlabeled biotin for binding to immobilized avidin, followed by a separation step to remove the unbound conjugate. Bioluminescence assays are best performed on luminometers operated in a photon-counting mode. A variety of such instruments is commercially available. In addition, modern scintillation counters may be used to perform bioluminescence assays. The relative amounts of avidin and biotinylated aequorin (conjugate) influence the response characteristics of the assay. Therefore, the amounts of avidin and biotinylated aequorin must be optimized in order to enhance assay performance. The development of a homogeneous assay for biotin using aequorin is based on the ability of avidin in solution to inhibit the bioluminescence of biotinylated aequorin. To establish the optimal amount of avidin to use in the assay, a binder dilution curve is constructed by varying the amount of avidin in the presence of a fixed amount of biotinylated aequorin, and measuring the degree of signal inhibition.
