Development of novel monoclonal antibodies-based ultrasensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for aflatoxin B1 detection

Abstract

Monoclonal antibodies (mAbs) that are specific to aflatoxin B1 (AFB1) were produced from hybridoma cell lines 3F6G11 and 9C7C11 by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells that were isolated from a BALB/c mouse that was immunized with AFB1-bovine serum albumin (BSA). Both 3F6G11 and 9C7C11 mAbs are the immunoglobulin G1 isotypes. Competitive direct enzyme-linked immunosorbent assays (cdELISA) were established to characterize these antibodies. In the 3F6G11 mAb based cdELISA, the concentrations causing 50% inhibition of binding of AFB1-horseradish peroxidase to the antibody by AFB1, AFB2, AFG1, and AFG2 were found to be 0.051, 0.050, 1.820, and 1.270 ng/ml, respectively. Using 9C7C11 mAbs, similar IC50 values for AFB1, AFB2, AFG1 and AFG2 were obtained as 0.045, 0.057, 2.530 and 2.120 ng/ml, respectively. A rapid and sensitive gold nanoparticle immunochromatographic strip (immunostrip) was also established for these antibodies. This strip has a detection limit of 1.0 ng/ml for AFB1 and the whole assay can be completed within 10 min. Extensive analysis of 20 samples by 3F6G11 mAb and 9C7C11 mAbs cdELISAs revealed that six samples were slightly contaminated by AFB1 at concentrations from 0.160 to 16.10 ng/g. Results of analyses of 20 samples with an immunostrip assay correlate well with those obtained using cdELISA. The proposed cdELISA and immunostrip methods are highly sensitive for the rapid screening of AFB1 in food samples.