34] Competitive enzyme-linked immunosorbent assay for biotin

Abstract

This chapter proposes a competitive enzyme-linked immunosorbent assay (ELISA) method for the rapid and sensitive determination of biotin concentrations. This method is based on the measurement of residual horseradish peroxidase [conjugated to streptavidin, streptavidin–horseradish peroxidase (HRP)] activities after streptavidin–HRP has reacted with biotin in sample solutions. The detection limit is 1 pg/ml in simple aqueous media and 5 pg/ml in bacterial growth media. The strategy of the competitive enzyme-linked immunosorbent assay (ELISA) method for the determination of biotin concentration is based on the competition between streptavidin-conjugated horseradish peroxidase (streptavidin–HRP, in excess amount) in solutions of variable biotin concentrations. The residual free form of streptavidin–HRP is immobilized with biotinylated goat anti-rabbit immunoglobulin (IgG) precoated on the wells of microtiter plates, and finally, the peroxidase activities are assayed and correlated with the biotin concentrations. When more biotin molecules are present initially in the competition reactions, less free streptavidin–HRP would be available to react with the biotinylated IgG. Biotin concentrations could be estimated from the calibration curves relating standard biotin concentrations and the horseradish peroxidase activities.