Preparation And Identification Of Monoclonal Antibodies Against Sulfamethazine

Sulfamethazine(Sulfamethazine,SM2)is a relatively broad type of sulfa drug.It has a unique structure of amino-benzene sulfonamide.It is a broad-spectrum antibacterial drug,because of its quick effect,low cost,and easy acquired,it is widely used for the prevention and treatment of animal protozoal diseases and bacterial diseases.SM2 can promote animal growth,so it is often used as a feed additive in animal husbandry for long-term use.Irrational use of SM2 for prolonged low doses or high doses during treatment can easily lead to residues in livestock products,potentially threatening human health.Countries around the world have already made corresponding provisions for the residues of sulfa drugs in related foods.EU countries require that the residual content of sulfa drugs should not exceed 100 ?g/kg,and the residual content of individual drugs must not exceed 25 ?g/kg;in Japan,relevant regulations shall not detect residues containing sulfa drugs in foods;he Ministry of Agriculture of the People's Republic of China stated in Document No.235 in December 2002 that the residue limit of sulfa drugs in fat,muscle,liver and kidney of all animals must not exceed 100 ?g/kg,and SM2 is included in the veterinary drug residue monitoring important content.Currently,SM2 is detected using microbiology,instrumentation,and enzyme-linked immunosorbent assays.The microbiological method has a high detection rate,but the sensitivity is low,and the selectivity is poor.The instrument detection method is time-consuming,and the procedure sare complicated.and the instrument is too expensive and can only be completed by professional operators.The enzyme-linked immunosorbent assay has high sensitivity,high speed,and batch screening,and does not require large-scale equipment and is more suitable for primary screening of large numbers of samples.In this experiment,diazotization was used to couple SM2 with limpet hemocyanin(SM2,KLH)as immunizing antigens and SM2 and bovine serum albumin(SM2,BSA)were synthesized as coating antigens,BCA method,UV scanning and SDS-PAGE was used to verify the successful coupling of SM2 to the carrier protein;BALB/c mice were immunized with the synthetic SM2-KLH artificial full antigen,and four hybrids were obtained by hybridoma technique and limiting dilution method to obtain a stable strain.Hybridoma cell lines secreting anti-SM2 specific antibodies.Then,the cell line was expanded,and mice were injected intraperitoneally to obtain ascites by means of induction and then purified to obtain the desired antibody.As identified by immunoglobulin subtype,the heavy chain of 1E10 was IgG1 type and the light chain was Kappa.The optimal coating concentration for the antigen to achieve 50% inhibition was 1:6000 and the optimal working concentration of the antibody was 1:5000.The innovation of the method provides new ideas for future research.Based on the enzyme-linked immunosorbent assay(ELISA)method,this subject optimizes the detection conditions and can detect it more effectively and sensitively,laying the foundation for subsequent research.