AviTag-nanobody based enzyme immunoassays for sensitive determination of aflatoxin B1 in cereal

Aflatoxin B1 (AFB1) is a well-known carcinogen for human health, and requires sensitive and rapid methods for monitoring. Here, we developed an Avi-tagged nanobody (AviTag-Nb) and incorporated it into the biotin-streptavidin-amplified ELISA (BA-ELISA) and magnetic bead-based ELISA (MB-ELISA) to detect AFB1. After optimization, BA-ELISA and MB-ELISA achieved 50% inhibitory concentrations (IC50) of 0.28 and 0.53 ng/mL, respectively, and provided results within 50 and 26 min, making them more sensitive and time-saving compared to the classic ELISA. The detection limits (LODs) of BA-ELISA and MB-ELISA were down to 0.07 and 0.12 ng/mL, respectively, ensuring their practical application in AFB1 analysis. Additionally, both assays provided satisfactory recoveries (90.0%–97.2%) and favourable relative standard deviation (4.00–10.5%) when tested with spiked cereal samples, aligning well with the results obtained from HPLC. Therefore, the AviTag-Nb shows promise as a valuable reagent in immunoassays, and the proposed BA-ELISA and MB-ELISA methods can serve as practical analytical tools for determining AFB1 in real samples.