Mitigation of Biotin Interference in Manual and Automated Immunoassays by Preconjugating Biotinylated Antibodies to the Streptavidin Surface as an Alternative to Biotin Depletion

Background: Streptavidin-to-biotin binding is one of the strongest noncovalent interactions in nature and incorporated into many immunoassays. Biotin-streptavidin coupling assays are susceptible to interference from free biotin in patient specimens, which may falsely decrease or increase results. To prevent biotin interference, we evaluated a method to preconjugate biotinylated antibodies to the assay's streptavidin solid surface before adding patient specimen and compared this technique to a biotin depletion protocol. Methods: Biotin interference in 3 manual ELISAs and 2 automated immunoassays was established. Mitigation of biotin interference by preincubation was evaluated in each assay by adding biotinylated antibody to the streptavidin-coated surface before adding biotin- or PBS-spiked serum. Lastly, the preincubation method was compared to a biotin-depletion protocol to compare the effectiveness of mitigating biotin interference. Results: In the presence of 400 mu g/L biotin, analyte detection was reduced to 10% to 15% of total in the ELISA assays and to 15.2% in the automated sandwich (thyroglobulin) immunoassay. In the automated competitive (free thyroxine) immunoassay, biotin caused an increased detection of 551.6%. Preconjugation of the biotinylated capture antibody to the streptavidin surface in the ELISA assays resulted in 84% to 99% activity recovery, compared to 84% to 97% by a biotin depletion protocol. Similarly, automated sandwich and competitive immunoassays obtained 97.1% and 116.5% recovery by preconjugation, compared to 95.6% and 100.3% by the depletion method, respectively. Conclusion: This study demonstrates how assay redesign to include preconjugation of biotinylated capture antibody to streptavidin is an effective alternative to biotin-depletion methods to mitigate biotin interference.