Production of polyclonal antibodies towards the immunodetection of insecticide phosalone

Hapten synthesis for the production of specific insecticide phosalone polyc lonal antibodies was carried out starting from an intermediate of the phosa lone synthesis, 6-chloro-2-benzoxazolone 1. Two haptens containing differen t 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 t hese two haptens conjugated to bovine serum albumine (BSA) was used to immu nize four rabbits. Immunoassays of phosalone were performed with ELISA usin g solid-phase bound hapten thyroglobulin conjugate and horseradish peroxida se labelled goat antirabbit IgG, The more sensitive response was observed w hen the antiserum obtained from the rabbit immunized with the hapten-BSA co njugate containing the N-2-oxo-3-aza-5-carboxypentyl spacer was in competit ion with the same hapten coupled to thyroglobulin. An identical response wa s obtained under the same conditions when using benzoxazolone instead of ph osalone as competitor, showing a good recognition of the specific aromatic part of the organophosphate insecticide phosalone. Reduction of coating con jugate concentration (from 2 to 0.05 mu g/well) and also the use of heterol ogous coating protein instead of homologous did improve the sensitivity, re sulting in a concentration of phosalone required to inhibit binding by 50% of 2 mg/l and a detection limit of 0.02 mg/l.