Preparation And Identification Of Monoclone Antibody Of Sulfamerazine

Sulfonamides are foremost medicines to control and cure infectious diseases. Owing to their characteristics of cheapness, convenience,stability,effectiveness in some animal diseases, sulfonamides are applied in stock raising industry more and more often. But. sulfonamides will accumulate in animal tissues if they are not used properly ,Normal bacterium will get drug-resistance if people eat contaminated food. Also, it causes intoxication, hypersensitivity. So many countrys and organisations have established the Maximum Residues Limits(MRL) of 0.1mg/kg or even none.The international has established many chemical and physics methods for detecting sulfonamides. Though some of these methods are sensitive and accurate, However, the pretreatment of samples is complicated and expatiatory. Its cost is quite high. All those have restrict its extensive use. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological methods is gradually applied to detecting sulfonamides residues.As one of the important Sulfonamides, Sulfamerazine, (SM1) is larger in proportion to other Sulfonamides.Molecular weight being 250, Sulfamerazine is a half antigen. There is an aromatic amido in molecular of SM1, so diazotization is available. In this study, based on immunological analysis of SM1. Bovine serum albumin (BSA) was used as carrier. Ovalbumin is chosed in the same time for controlled trial and detection. We used the technology of monoclonal antibodies to produce the monoclonal antibodies against SM1, provided capital materials for the immune analysis of SM1.1,SM1 was linked to Bovine Sera Albumin (BSA) and ovum Albumin (OVA) using glutaraldehyde and diazotation as cross-linker respectively in experiment one. Corresponding identification was made by SDS-PAGE and UltraViolet scan.2,A weak immune response or none at all was induced after immunization of mice with those conjugates that used glutaraldehyde . High antibody titers were obtained with conjugates where SM1 was linked to BSA with a diazotation reaction. 3,Using the mature technology of monoclonal antibodies in experiment three, the lymphocytes from Balb/c mouse spleen routinely immunized by BSA-SM1 antigen were fused with NS0 myeloma cells by 50% PEG 4000, and selected cultured with HAT medium. A total of 112 hybridomas producing monoclonal antibodies against SM1 were obtained by screened with SM1-OVA antigen using an indirect enzyme-linked immunosorbent assay (ELISA). and cloned three times by limiting dilution. Three hybridoma cell lines of SM12B1, SM13H5, SM14E11. These hybridomas secret high affinity monoclonal antidodies (McAb) with the titers of 1:512, 1:640, 1:320, determined by ELISA in supernatants respectively. Three cell lines of the hybridoma were intraperitoneally injected into prostane sensitized Balb/c mice for production of ascites antibodies, the titers of which were 1×10-7, 1×10-7, 5×10-6 respectively. These McAbs showed low cross-reactivity to other sulfadiazines and other materia medical(<0.4%).