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.