Competitive Binding Assay for Biotin and Biotin Derivatives, based on Avidin and Biotin-4-Fluorescein

Biotinylated molecules are extensively employed in bioanalytics and biotechnology; therefore, numerous assays have been developed for quantification of biotin groups. Most assays perform very well in some aspects but suffer either from low sensitivity, or low accuracy, or very variable dose responses for different biotin derivatives. In the present study a competitive binding assay was developed in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the bound B4F fraction. This protocol resulted in a biphasic titration curve, with a linear rise up to saturation of avidin with the biotinylated analyte and a horizontal plateau when the analyte was in excess. The biotin content was calculated from the breakpoint between the two linear segments. As a consequence, the assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages well justified the 10 higher sample consumption (~0.6 nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (10%) was compensated by the 100-fold higher sensitivity and the actual deviations from the reliable ANS-Assay were ≤4%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. (...) The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness, which distinguishes it from other biotin group assays where the advantages and disadvantages are more pronounced.