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

•An ultra-sensitive anti-AFM1 monoclonal antibody was produced.•The moderate hapten-to-protein coupling ratio (˜2) in the coating antigen could improve the immunoassay sensitivity.•The sensitivity of the FM-ICTS assay was comparable to the ciELISA and superior to the CG-ICTS assay.

A rapid lateral flow fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MAb 1D3 for AFM1 was 23 ng/L, and the cross-reactivity (CR) of the MAb with aflatoxin M2, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 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 IC50 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 AFM1 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 AFM1 residues in milk.