A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk Original Research Article

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M1 (AFM1) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM1 surrogate, was generated by immunizing rabbits with F(ab′)2 fragments from the anti-AFM1 monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM1 mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM1 and the anti-Id antibody (y = 31.91x − 8.47, r = 0.9997). The assay was applied to analyze AFM1 in spiked milk samples. The IC50 value of the surrogate calibrator curve was 2.4 μg mL−1, and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y = 0.81x + 9.82, r = 0.9922).