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.

Research highlights
► In our experiments, a novel semi-solid complete medium was applied to select single clone. Hybridoma colonies obtained from this medium are monoclonal from the start. It avoids the step of recloning and saves much time.
► The monoclonal antibodies described in this paper are the most specific for AFM1 so far. 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 monoclonal antibodies described in this paper, an ultra-sensitive competitive enzyme immunoassay for aflatoxin M1 was developed. This method showed a detection limit of 3 ng/L, rather below the EU maximum level. Several physicochemical factors (pH, ionic strength and blocking solution) that affect assay performance were studied and optimized.
► The ultra-sensitive method was validated, by recovery test and comparing with reference HPLC method. The recovery and accuracy results demonstrated that the method was feasible and reliable for the analysis of real samples.