Development of a biotinylated nanobody for sensitive detection of aflatoxin B1 in cereal via ELISA

Aflatoxin B1 (AFB1) contamination is a severe threat to food safety and human health, and requires continuous monitoring. In this study, we developed a biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) by using biotinylated nanobody Nb26 and streptavidin-conjugated polymerized horseradish peroxide (SA-PolyHRP) for sensitive and rapid detection of AFB1 in cereal. Under the optimal condition, the IC50 value of the BA-ELISA was improved to 0.21 ng mL-1 for AFB1, satisfying the requirement of detection limit in practical applications. The total assay time of our strategy is reduced to 50 min from 2 h in conventional competitive ELISA. Additionally, the BA-ELISA saves as much as 98% of the antibody in comparison to the previous classic ELISA. Our work also demonstrated an interesting phenomenon that the biotinylated Nb26 achieved better selectivity to AFB1, which could possibly result from the steric hindrance that interferes reaction between the Nb26 and the AFB1 analogs. Furthermore, the assay was used to detect AFB1 in two cereal samples, and the results were in good agreement with that obtained by high performance liquid chromatography. The developed BA-ELISA can be used for routine screening analysis of AFB1, and offers a promising strategy for measuring low concentrations of food contaminants.