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

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M(1) (AFM(1)) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM(1) surrogate, was generated by immunizing rabbits with F(ab')(2) fragments from the anti-AFM(1) monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM(1) 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 AFM(1) and the anti-Id antibody (y=31.91x-8.47, r=0.9997). The assay was applied to analyze AFM(1) in spiked milk samples. The IC(50) 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).