Preparation And Preliminary Application Of The Monoclonal Antibodies Against Sulfamethoxazole

Sulfamethoxazole(SMZ), is mainly used for respiratory and urinary tract infection in the veterinary clinic. Cotrimoxazole(TMP-SMZ),which is SMZ combined with Antibacterial synergist trimethoprim (TMP) , has wide antibiotic spectrum, formal curative effect, stable property, easy to use, inexpensive and can be domesticly mass-produced etc. advantages, making it become one of the most widely used Sulfonamides antibiotics in veterinary clinic. At the same time, abuse, overcharging add in the animal and animal feed become common occurrence events, which leading to too high residual in animal products. It not only has great poison side effects for the animals themselves, but also has tremendous damage on the human health . Many countries and organizations have prescription for the maximum residue limit (MRL) of Sulfonamides. South Korea, the United States, the European Community (EEC), Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) of Codex Alimentarius Commission (CAC), etc. have the prescription presents total Sulfonamides and/or specific individual Sulfonamides must be no more than 0.1 mg/kg MRL, and even Japan ordains that certain individual Sulfonamide should not be detected. Therefore, it has important practical significance to develop efficient, accurate and inexpensive Sulfonamides inspection methods.At present, the main methods used for Sulfonamides detection are the traditional methods, including microbiology method, titration, spectrophotometry etc. ; chromatography technology, including gas chromatography (GC), high-performance liquid chromatography (HPLC), gas / liquid chromatography-mass spectrometry (GC / LC-MS), capillary electrophoresis (CE), supercritical fluid chromatography (SFC); immunological analysis, including the ELISA, Fluoro-immunoassay (FIA), colloidal gold immunization method (CGIA), immune biosensor (IB), and so on. Traditional methods are low sensitive and specific, easy to be interfered with other substances, and long time needed; although the chromatography has high sensitivity, but it also needs high-level detection equipment, expensive equipment, high technical requirements of operation, and preprocessing is complicated; Immunological methods are high specific, high sensitive, simple preprocessing, easy to operate and to promote the use. And the ELISA can detect a number of samples simultaneously, with high specificity, high sensitivity, high speed. It is an accurate, safe and practical detection technology. In this research, the chemical synthesis methods were used for preparation of SMZ complete antigens. Monoclonal antibodies with high sensitivity, specificity and valence were prepared through monoclonal antibody technology. The ELISA detection method was founded, and was preliminary applied in the detection of SMZ in pork, eggs, honey and milk.As SMZ molecular weight is very low, only 253.28, is a semi-antigen. Diazo method was used to make the SMZ combined with the carrier protein HSA and BSA by aryl-primary amino group structure of SMZ. Coupling ratio of the two artificial antigen is 23:1 and 21:1 respectively.SMZ-BSA was used to immunize 7-week BALB/ c female mouse. The serum titer had reached more than 1:400000 before fusion. Through the monoclonal antibody technology and modern cell fusion technique, two monoclonal hybridoma cell line called 3G11 and 2H10 were obtained. Stability experiments showed that these two cell lines could secrete monoclonal antibody against SMZ stably.Through the foundation of indirect competition ELISA method, the best work concentration of ascites was got, which is 1:1×106 above. SMZ standard curve was prepared by 3G11 ascites, with the linear equation: y = 0.206x -0.0317 (R2 = 0.9885), LOD 3.29 ng/ml, LOQ 23.06 ng/ml. The cross reaction experiment of congeneric Sulfonamides drugs and two non-Sulfonamides drugs showed that the SMZ monoclonal antibody has good specificity, with less than 10 percent inhibition rate anti other drugs.In addition, additive reclamation test had been done for animal foods which with relatively more SMZ used. The preprocessing approach of samples was optimized. The measured drug additive reclamation test rates were: milk 98.9% to129.1%, egg yolk 78.7% to 102.3%, egg white 71.9% to 85.3%, the whole eggs79.3% to 93.6%, pork 89.7% to 116.2%, honey 74% to 111.7%. Experiment results showed that the SMZ monoclonal antibody prepared by this research can be used for animal food SMZ residue detection. It provided a high quality antibody for the development of SMZ residual ELISA rapid detection kit or immune colloidal gold detection paper.