An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

A rapid lateral flow fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay has been developed for the detection of aflatoxin M 1 (AFM 1 ) residues in milk. For this purpose, an ultra-sensitive anti-AFM 1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC 50 value of the MAb 1D3 for AFM 1 was 23 ng/L, and the cross-reactivity (CR) of the MAb with aflatoxin M 2 , aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 and aflatoxin G 2 was 18%, 9%, <0.15%, 43% and <0.3%, respectively. The MAb was covalently conjugated with carboxylate-modified FMs, and the FMs were used as the label in a competitive immunochromatography assay. Using a moderate hapten-to-protein coupling ratio (˜2) in the coating antigen resulted in improved immunoassay sensitivity. Under optimal conditions, the IC 50 value of the assay was 36.3 ng/L with the limit of detection (LOD) of 4.4 ng/L in milk samples using a fluorescence reader, and the recoveries of AFM 1 in spiked milk samples ranged from 76.6 to 110.8% with coefficient of variation (CV) of 4–14.7%. The whole procedure could be completed within 30 min. The results demonstrated that the FM-ICTS assay is easy to conduct, rapid, highly sensitive and specific for the detection of AFM 1 residues in milk.