Development of a Monoclonal Antibody-Based ELISA for the Detection of Alternaria Mycotoxin Tenuazonic Acid in Food Samples

Abstract

In this study, a specific monoclonal antibody (mAb) against Alternaria mycotoxin tenuazonic acid (TeA) was prepared and a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of TeA was developed. Tenuazonic acid coupled with carboxymethoxylamine hemihydrochloride (TeA-CMO) was conjugated to keyhole limpet hemocyanin (KLH) as an immunogen for Balb/c mice injection. The hybridoma cell line 3F10 secreting the mAb specific to TeA was obtained, and then by intraperitoneal injection and the caprylic acid-ammonium sulfate precipitation, the purified mAb was prepared and identified as the immunoglobulin G1 isotypes. The affinity constant (kD) of the mAb was 3.045 E−3 M by SPR analysis. Based on this mAb, an ic-ELISA was established after optimization of assay condition, namely the concentration of coating antigen and antibody were 250 ng/mL and 125 ng/mL respectively, competition time of antigen-antibody was 40 min, and incubation time of secondary antibody was 40 min in 0.01 M PBS buffer (pH 7.4). Under the optimized condition, the IC50 value and the detection limit (LOD) were 18.50 ng/mL and 1.00 ng/mL respectively. The average recovery rate from spiked beer, apple juice, and grape juice was from 85.0 to 120.0%. A squared coefficient of correlation (R2) between ic-ELISA and HPLC method was 0.9732. The established ic-ELISA provides an acceptable technique for the detection of TeA residue in food samples.