Production and characterization of monoclonal antibody for class-specific determination of O,O-dimethyl organophosphorus pesticides and effect of heterologous coating antigens on immunoassay sensitivity

A generic hapten (H1), 3-(4-Dimethoxyphosphorothioyloxy phenyl)propanoic acid, was synthesized to produce monoclonal antibodies (mAbs) for the determination of organophosphorus pesticides (OPs) in a class-specific manner. Six heterologous haptens were designed to study the effect of hapten heterology on immunoassay sensitivity. Several mice were immunized with this H1-BSA immunogen. Spleen cells of two immunized mice were fused with myeloma cells, and the resulting hybridomas were screened using H1-OVA. In a competitive indirect enzyme-linked immunosorbent assay (ELISA) format, three hybridoma cell lines (B4-C6, D12-B5, E5-H2) that produced mAbs with high selectivity and broad specificity were selected and expanded. The monoclonal antibody D12-B5 with higher titer was chosen for further study. In heterologous assay, the combination of D12-B5 and coating antigen H7-OVA constituted a particularly sensitive assay for competitive indirect ELISA and showed broad specificity for the determination of OPs, including parathion-methyl, chlorpyrifos-methyl, tolclofos-methyl, fenthion, malathion, fenitrothion. This combination resulted in the IC50 of 0.58–10.47 µg/ml.