INTERACTION OF IMMOBILIZED AVIDIN WITH AN AEQUORIN-BIOTIN CONJUGATE -AN AEQUORIN-LINKED ASSAY FOR BIOTIN

Biotinylated recombinant aequorin was used in the development of a het erogeneous bioluminescence binding assay for biotin. This assay is bas ed on a competition between a biotinylated aequorin conjugate and biot in for the binding sites of avidin immobilized on solid particles. Dos e-response curves were obtained that relate solid-phase aequorin activ ity to the concentration of biotin. Under certain experimental conditi ons these curves were biphasic; i.e., as the biotin concentration incr eased, the solid-phase aequorin activity first increased reaching a ma ximum and then decreased at higher biotin concentrations. This ''hook' ' effect was observed with four different types of immobilization supp orts. The effect was more pronounced when low concentrations of aequor in-biotin conjugate were used, and diminished at a high conjugate conc entration. This behavior indicates a possible positive cooperativity i n the interaction between the immobilized avidin and biotin. Scatchard plot analysis was also consistent with a positive cooperativity mecha nism. By using the ascending portion of the dose-response curve, the d etection limit of the assay for biotin was 1 x 10(-15) M (100 zmol of biotin in the sample). (C) 1997 Academic Press.