Influence of the hapten conjugation site on the characteristics of antibodies generated against metabolites of clostebol acetate

4-chloro-androst-4-ene-3,17-dione (CLAD) and 4-chlorotestosterone (clostebo l, beta-CLT or CLT) were made immunogenic by coupling to protein carriers v ia the 3 and 17 positions, respectively. These immunogens were used to elic it polyclonal and monoclonal antibodies to CLAD and to clostebol. The antib odies were characterized in an enzyme immunoassay for sensitivity and speci ficity. Polyclonal antisera generated through position 17 reacted preferent ially with 4-chlorotestosterone-17-acetate (clostebol acetate, CLTA), 4-chl oro-epitestosterone (epi-clostebol, 17 alpha-clostebol, 17 alpha-CLT), and clostebol, whereas polyclonal antisera generated through the 3 position alm ost did not react with these derivatives. Interestingly, the monoclonal ant ibody generated through the 3 position recognized (35%) epi-clostebol. Thes e results suggest that polyclonal antisera generated through the 17 positio n have a broad specificity profile and can be used to analyze by immunoassa y methods urinary metabolites of clostebol acetate and thereby detect the i llegal use of clostebol acetate in livestock farming.