Synthesis of derivatives and production of antiserum for class specific detection of pyrethroids by indirect ELISA

Two series of haptens including 3-phenoxybenzoic acid (PBA) and 3-(2-chloro-3, 3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclo-propanecarboxylic acid (CF3MPA) were used to prepare immunogens through attachment of 4-C or 6-C handles. Class selective antibodies were produced by immunising rabbits. Ab502 showed the highest reactivity towards tau-fluvalinate (IC50 1.3 ng mL-1), -cyhalothrin (IC50 2.3 ng mL-1), cyfluthrin (IC50 2.2 ng mL-1) and fenpropathrin (IC50 18.5 ng mL-1) among the antibodies in a competitive ELISA. The effects of methanol, pH and salt concentration were optimised for maximum efficiency of the ELISA (Enzyme-Linked ImmunoSorbent Assay). Ab502 (1:80000)/2-OVA-1(0.2 g mL-1) was chosen for ELISA optimisation. Finally, 0.05 M phosphate buffered saline (PBS) at pH 6.5 containing 30% methanol (v/v) was used to dilute the standards. Target analytes in honey samples were extracted with ethyl acetate by sonication. The samples were spiked with three different concentrations of each compound (tau-fluvalinate, 0.5 ng g-1, 3 ng g-1, 12 ng g-1; -cyhalothrin and cyfluthrin 1 ng g-1, 5 ng g-1, 65 ng g-1). The recoveries were 36-59% at the lowest spiking concentration and 61-81% at the higher concentration. This assay might be useful to screen pyrethroid residues in honey or other matrix.