Sulphonamide Antibodies: From Specific Polyclonals to Generic Monoclonals

Polyclonal antibodies (PAbs) against eight different sulphonamides were raised in rabbits. The aromatic amino group, common to all sulphonamides, was used for linking the different sulphonamides to the carrier proteins (bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)) and enzyme (horseradish peroxidase (HRP)), using different coupling procedures. The competitive direct ELISAs (cdELISAs) developed with these antisera and HRP-conjugates 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 (N¹-[4-(carboxymethyl)-2-thiazolyl]sulphanilamide (TS) and N¹-[4-methyl-5-\[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulphanilamide (PS)) linked to proteins (BSA and KLH) in such a way that the common aromatic amino group was distal to the 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-KLH reacted with sulphonamides containing pyrimidinyl, pyridazinyl, quinoxalinyl and pyridinyl groups. The spleen cells of the mice were fused with myeloma cells to obtain monoclonal antibodies (MAbs) producing hybridomas. So far, with only one of the mice (immunized with TS-BSA), this resulted in four 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 concentrations less than 100 ng ml^-1 or 5 ng/well. However, other relevant sulphonamides (such as sulphadimidine, sulphatroxazole and sulphachloropyrazine) were detected at a high level only.