Multi-analyte enzyme-linked immunosorbent assay for organophosphorus pesticides and neonicotinoid insecticides using a bispecific monoclonal antibody

A bispecific monoclonal antibody (BsMcAb) with antigen-binding sites for two classes of insecticides was used to develop a multi-analyte immunoassay for determination of organophosphorus pesticides and neonicotinoid insecticides. A trioma cell line ON-D6 was produced by fusion of HAT sensitive hybridomas secreting antibody to organophosphorus pesticides with spleen lymphocytes from the BALB/c mice which were immunized by artificial antigens of neonicotinoid insecticides. The broadly selective BsMcAb was raised to recognize eight organophosphorus pesticides (parathion-methyl, parathion, fenitrothion, EPN, paraoxon-ethyl, paraoxon-methyl, fenitrooxon, and cyanophos) and two neonicotinoid insecticides (imidacloprid, and imidaclothiz). A multi-analyte ELISA was established by using the BsMcAb, and several key parameters were optimized to enhance the sensitivity of the immunoassay. The developed immunoassay showed average IC50 values of 114.6 and 129.8 ng mL(-1) for eight organophosphorus pesticides and two neonicotinoid insecticides, respectively. The recoveries from the spiked samples ranged between 86.8 and 125.0% for the detection of single pesticide residues of parathion-methyl and imidacloprid in tap water, pond water, grape, cucumber, and soil. ELISA results were validated by comparison with gas chromatography (GC) analyses. The results showed that this multi-analyte ELISA is useful for the determination of neonicotinoid insecticides and organophosphorus pesticides in real samples.