Production of polyclonal antibodies towards the immunodetection of insecticide phosalone

Hapten synthesis for the production of specific insecticide phosalone polyclonal antibodies was carried out starting from an intermediate of the phosalone synthesis, 6-chloro-2-benzoxazolone 1. Two haptens containing different spacers have been prepared: N-5-carboxypentyl-6-chloro-2-benzoxazolone 7 and N-(2-oxo-3-aza-5-carboxypentyl)-6-chloro-2-benzoxazolone 12. Each of these two haptens conjugated to bovine serum albumine (BSA) was used to immunize four rabbits. Immunoassays of phosalone were performed with ELISA using solid-phase bound hapten thyroglobulin conjugate and horseradish peroxidase labelled goat antirabbit IgG. The more sensitive response was observed when the antiserum obtained from the rabbit immunized with the hapten-BSA conjugate containing the N-2-oxo-3-aza-5-carboxypentyl spacer was in competition with the same hapten coupled to thyroglobulin. An identical response was obtained under the same conditions when using benzoxazolone instead of phosalone as competitor, showing a good recognition of the specific aromatic part of the organophosphate insecticide phosalone. Reduction of coating conjugate concentration (from 2 to 0.05 micrograms/well) and also the use of heterologous coating protein instead of homologous did improve the sensitivity, resulting in a concentration of phosalone required to inhibit binding by 50% of 2 mg/l and a detection limit of 0.02 mg/l.