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

Abstract

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.