Competitive enzymatic assay of biotin.

Publisher Summary Biotin is a small molecule that has been widely found as a vitamin in living organisms and used as a tag for detection and isolation in biomedical research. A simple and sensitive assay of biotin is often needed in the study of metabolism and in the determination of the degree of conjugation of biotin to biomolecules. Although the bioassay of biotin on the basis of growth of biotin-dependent bacteria is sensitive, it is rarely used in routine laboratory practice because of the long culture time, low reproducibility, and interference caused by many growth-promoting factors other than biotin in a sample. However, with proper design biotin can be assayed with relative ease in a reagent system consisting of its specific protein binder, avidin, or streptavidin. One of the numerous sensitive and nonradioactive methods derived from the avidin–biotin interaction involves coating of biotinylated bovine serum albumin (B-BSA) on a microtiter plate and several subsequent detection steps. This chapter focuses on the simplification of this assay method while enhancing its reproducibility and sensitivity.