title: A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk.
abstract: 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).
authors.affiliation: Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.
authors.forename: Di
authors.initials: D
authors.lastname: Guan
authors.forename: Peiwu
authors.initials: P
authors.lastname: Li
authors.forename: Yehan
authors.initials: Y
authors.lastname: Cui
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
mesh_terms: D016607:Aflatoxin M1 / Q000032:analysis*; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000032:analysis; D000918:Antibody Specificity; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology; D008892:Milk / Q000737:chemistry*; D009994:Osmolar Concentration; D011042:Poisons / Q000032:analysis*; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011042:Poisons; D016607:Aflatoxin M1