Development and validation of a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the determination of the aflatoxin M1 levels in milk

• The obtained antibody exhibit excellent sensitivity and specificity to aflatoxin M1.
• The developed ic-ELISA can determination of aflatoxin M1 in milk.
• The ic-ELISA can serve as an effective screening tool in any routine laboratory.

A sensitive monoclonal antibody (mAb) against aflatoxin M1 (AFM1) was generated to quickly monitor the AFM1 residues in milk. Then, a mAb-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established that utilizes simple sample preparation and clean-up methods. The obtained 3D8 mAb, which is an IgG1 isotype mAb, displayed an IC50 value of 64.75 ng L−1 for AFM1 and did not exhibit measurable cross-reactivity with other aflatoxins and antibiotics. The decision limit (CCα, α = 1%), detection capability (CCβ, β = 5%), and LOQ value for the AFM1 matrix calibration method were 24 ng L−1, 27.5 ng L−1, and 35 ng L−1 in the milk matrices, respectively. The AFM1 recovery ranged from 85.3% to 107.6%. The CVs were less than 13.8%. A positive correlation (r > 0.99) was observed between the ic-ELISA and HPLC-MS/MS results. This ic-ELISA would be a useful tool for screening the AFM1 residues in milk.

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