Competitive binding assay for biotin and biotin derivatives, based on avidin and biotin-4-fluorescein

Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay 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 B4F. 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 justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-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 deviations from the ANS assay were ≤5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n≤5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness.

Avidin; Biocytin; Biotin assay; Biotin-4-fluorescein; Fluorescence; Titration.