Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk

• A hybridoma cell secreting monoclonal antibody specific to AFM1 was obtained.
• A sensitive ELISA with IC50 of 0.022 ng/mL for AFM1 was established.
• An immunostrip with a detection limit of 0.1 ng/mL for AFM1 was developed.
• Four of test samples were contaminated with AFM1 ranging from 0.002 to 0.054 ng/mL.

Monoclonal antibody (mAb) that is specific to AFM1 was generated from the hybridoma cell line, 10F3C10, which was obtained by the fusion of mouse NS1 myeloma cells with the spleen cells of mouse that had been immunized with AFM1-bovine serum albumin (BSA). The 10F3C10 mAb is belong to the immunoglobulin G1 isotype. Both competitive direct and indirect enzyme-linked immunosorbent assay (ELISA) was utilized to characterize the mAb for AFM1. The concentrations of AFM1, AFB1 and AFG1 that caused 50% inhibition (IC50) of the binding of AFM1-horseradish peroxidase (AFM1-HRP) to the antibody were found to be 0.022, 0.310 and 2.12 ng/mL, respectively. The immunochromatographic strip (immunostrip) assay with mAb-gold nanoparticle conjugates as a detection marker exhibited a visual limit of detection of 0.1 ng/mL for AFM1 and the analysis took a total of 10 min. Closely examining 17 milk-based samples using cdELISA revealed that four were slightly contaminated with AFM1 at concentrations from 0.002 to 0.054 ng/mL. All milk samples were negative in the immunostrip test because the levels of contaminant were below the detection limit of the strip. Notably, the presented cdELISA and immunostrip methods are highly sensitive methods for detecting AFM1 in milk.