Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening T-2 toxin in milk

Abstract

To monitor T-2 toxin rapidly in milk, a highly specific against T-2 toxin monoclonal antibody (mAb) was produced and a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed. T-2 toxin was first converted to T-2-adipic anhydride (T-2HA), and then Keyhole limpet hemocyanin (KLH) and virus-like particles (VLP) were synthesized and conjugated to T-2HA as coating and immunogen antigen, respectively. After the inoculation of female Balb/c mice and cell fusions, a high affinity mAb was produced. After optimization, the 50% inhibition concentration (IC50) for the developed ic-ELISA was estimated as 0.137 ng mL−1. Based on this mAb, an optimized ic-ELISA protocol was developed using the dilution method to prepare milk samples. The limits of detection of T-2 toxin in milk was 0.827 ng mL−1, the mean recovery of T-2 toxin in spiked samples (2.5, 5 and 10 ng mL−1) ranged from 85.0% to 98.9%, and the CVs ranged from 6.1% to 8.6%. The ic-ELISA was validated by HPLC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting T-2 toxin in milk.