An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M1 (AFM1), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM1 of 1.74 × 109 L/mol and no cross-reactivity to aflatoxin B1, B2, G1 and G2. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM1 in milk and infant milk products. Assays were performed in the AFM1-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.