Monoclonal antibody-based enzyme-linked immunosorbent assay for the insecticide imidacloprid

An enzyme-linked immunosorbent assay (ELISA) was developed for the neonicotinoid insecticide imidacloprid, 1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine using monoclonal antibodies (MAb). Three MAbs, designated as E6A6, E6F3 and H7F7, were raised from mice immunized with an imidacloprid hapten-ovalbumin conjugate. These MAbs performed similarly in indirect competition ELISA (icELISA), so one, E6F3, was selected for detailed study. The equilibrium constants (K_d) and association and dissociation rate constants (k_on, k_off) for five neonicotinoids and one imidacloprid metabolite to E6F3 were determined by kinetic exclusion fluoroimmunoassay (KinExA). Affinities (1/K_d) of E6F3 for acetamiprid and clothianidin were similar, but 50-fold weaker than that of imidacloprid. MAb E6F3 had no measurable affinity for the other neonicotinoids. The icELISA can tolerate up to 15% (v/v) acetone or 20% (v/v) methanol. Assay sensitivity was similar at pH 4-9, 1-10-fold concentration of PBS with or without 0.05% Tween 20, and incubation times of 30-180 min. The half-maximal inhibition and the limit of detection were approximately 0.8 and 0.1 μg/l of imidacloprid in icELISA, and 0.3 and 0.03 μg/l in direct competition ELISA (dcELISA), respectively. Analysis of imidacloprid-fortified water and cucumber samples by the icELISA showed average recoveries from 70 to 120%.