Automated flow fluorescent immunoassay for part per trillion detection of the neonicotinoid insecticide thiamethoxam

An ultra sensitive automated flow fluorescent immunoassay was developed using the KinExA™ 3000 system for quantitative analysis of the neonicotinoid insecticide thiamethoxam. Five new monoclonal antibodies were obtained and screened with a competitive ELISA. One monoclonal antibody designated as E6VI was evaluated for sensitivity, selectivity and solvent tolerance with the KinExA. Sensitivity determined from the concentration of half-maximal inhibition (IC50) was obtained by plotting KinExA signals to a four-parameter sigmoidal curve as a function of analyte concentrations. For the most sensitive clone, the IC50 and the limit of detection were approximately 30 pg ml−1 and 16 pg ml−1, respectively. Cross-reactivity was estimated by measuring the equilibrium constants (Kd) for four other neonicotinoid insecticides (clothianidin, imidacloprid, dinotefuran, and acetamiprid). E6VI was very specific to thiamethoxam with <0.11% cross-reactivity for tested neonicotinoids. An excellent correlation (r2 = 0.99) was obtained between spiked and measured concentrations of thiamethoxam in stream and tap water, potato, cucumber, and apple samples.