Development And Identification Of Monoclonal Antibodies To Sulfadiazine And Sulfonamides

In order to establish methods to detect multiple residues of sulfonamides (SAs) and residues of sulfadiazine (SD) by the method of immunoassay, two strains of unique hybridomas that produced monoclonal antibodies (MAbs) reacted with several sulfonamides and SD were isolated respectively. The results are as following:1. A hapten N-sulfanilyl-4-aminobenzoic acid, referred to as SDL, was synthesized that including the structure of sulfanilamide common to all sulfonamides and with the spacer arm of benzene ring. Immunizing antigen (SDL—HSA) and coating antigen (SDL—OVA) were synthesized by conjugating SDL to human serum albumin (HSA) and ovalbumin (OVA) with the same method of mixed—anhydride. Among 7 hybridoma cell lines, hybridoma that named SDL—4 with the highest ascite productivity and antibody titer(> 1:1.28 × 10⁶) was isolated after fusing and screening of hybridoma supernatants by indirect ELISA and competitive indirect ELISA(CiELISA). The purified IgG of SDL—4 with the isotype of IgG2b was used in CiELISA .The sensitivities (expressed as IC50 values) of SDL — 4 for sulfamonomethoxine(SMM), sulfadimethoxine(SDM), SD, sulfaquinoxaline(SQX), and sulfamethazine(SM₂) were 251.3, 374.9, 117.5, 19.5, and 233.1 ng/mL respetively. The results showed that MAb produced by SDL—4 can be used in immunoassay of multiple residues of SAs.2. An immunizing antigen SD—HSA was synthesized by conjugating SD to HSA with the method of diazotization and coupling. By the same method, a coating antigen SD-OVA was synthesized by conjugating SD to OVA. At the same time, an enzyme— labeled conjugate of SD—OVA and horseradish peroxidase (HRP) was synthesized by the method of periodate oxidation. Among 4 hybridoma cell lines, hybridoma that named SD—4 with the highest ascite productivity and antibody titer(>1:1.28× 10⁶) was isolated after fusing and screening of hybridoma supernatants by indirect ELISA and competitive indirect ELISA(CiELISA). The purified IgG of SD—4 with the isotype of IgG2b was used in competitive direct ELISA with IC50 of 25.6 ng/mL and stable detection range of 1 ng/mL - 1000 ng/mL. The cross—reactivities for SMM, sulfametoxydiazine(SMD), SDM, SM₂, SQX and sulfamethoxazole(SMZ) were 1.3%, 1.0%, 0.6%, <0.1%, < 0.01%, and <0.01% respectively.The results showed that MAb produced by SD—4 can be used in immunoassay of residues of SD.