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Development and Validation of an Ultrasensitive Chemiluminescent Enzyme Immunoassay for Aflatoxin M 1 in Milk

2005; American Chemical Society; Volume: 53; Issue: 9 Linguagem: Inglês

10.1021/jf0479315

1520-5118

Maria Magliulo, Mara Mirasoli, Patrizia Simoni, R. Lelli, Ottavio Portanti, Aldo Roda,

Analytical chemistry methods development

A fast and ultrasensitive chemiluminescent enzyme immunoassay for aflatoxin M1 in milk samples has been developed and validated. The method is an indirect competitive type format involving the immobilization of an aflatoxin M1−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 M1 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, r2 = 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 M1 in milk samples with a reduction of costs and increased detectability, as compared with previously developed immunoassays. Keywords: Enzyme immunoassay; chemiluminescence; aflatoxin M1; milk