Development and Validation of an Ultrasensitive Chemiluminescent Enzyme Immunoassay for Aflatoxin Mi in Milk

摘要

A fast and ultrasensitive Chemiluminescent enzyme immunoassay for aflatoxin M_1 in milk samples has been developed and validated.The method is an indirect competitive type format involving the immobilization of an aflatoxin M_1-bovine serum albumin conjugate on 384 well black polystyrene microtiter plates and the use of a secondary antibody labeled with horseradish peroxidase detected with a luminol-based substrate.Aflatoxin M_1 standard solutions were prepared in milk-based buffer,and milk samples were analyzed without any cleanup procedure.The limit of quantification was 1 ppt,the coefficient of variation was below 9% for both intra-and interassay precision,and the recovery ranged from 96 to 122%.The method is specific,and other aflatoxins do not significantly cross-react with the antibody.Twenty-four milk samples were analyzed,and a good correlation was observed(y = 0.98x + 1.71,r~2 = 0.98,n = 24)when the data were compared with a reference high-performance liquid chromatography method with a fluorescent detector.The developed method is suitable for an accurate,sensitive,and high-throughput screening of aflatoxin M_1,in milk samples with a reduction of costs and increased detectability,as compared with previously developed immunoassays.