Sulphonamide antibodies: From specific polyclonals to generic monoclonals

Polyclonal antibodies (PAbs) against eight different sulphonamides were rai sed in rabbits. The aromatic amino group, common to all sulphonamides, was used for linking the different sulphonamides to the carrier proteins (bovin e serum albumin (BSA) and keyhole limper haemocyanin (KLH)) and enzyme (hor seradish peroxidase (HRP)), using different coupling procedures. The compet itive direct ELISAs (cdELISAs) developed with these antisera and HRP-conjug ates showed high sensitivity (0.2-8.0 ng ml(-1) at 50% inhibition) and high specificity. The performances of these antibodies were compared with PAbs raised in mice against two sulphonamide derivatives (N-1-[4-(carboxymethyl) -2-thiazolyl]sulphanilamide (TS) and N-1-[4-methyl-5-[2-(4-carboxyethyl-1-h ydroxyphenyl)]-azo-2-pyridyl]sulphanilamide (PS)) linked to proteins (BSA a nd KLH) in such a way that the common aromatic amino group was distal to th e protein. In competitive indirect ELISAs (ciELISAs), these PAbs recognized several structurally different sulphonamides. The PAbs from mice immunized with TS-BSA reacted with sulphonamides containing thiazolyl, thiadiazolyl, pyridazinyl and isoxazolyl groups. The PAbs from mice immunized with PS-KL H reacted with sulphonamides containing pyrimidinyl, pyridazinyl, quinoxali nyl and pyridinyl groups. The spleen cells of the mice were fused with myel oma cells to obtain monoclonal antibodies (MAbs) producing hybridomas. So f ar, with only one of the mice (immunized with TS-BSA), this resulted in fou r different MAbs which recognized several sulphonamides. By use of the best MAbs (27G3A9B10 and 4E10B12B6E12) and an optimized ciELISA protocol, eight structurally different sulphonamides showed 50% inhibition at concentratio ns less than 100 ng ml(-1) or ng/well. However, other relevant sulphonamide s (such as sulphadimidine, sulphatroxazole and sulphachloropyrazine) were d etected at a high level only.