Preparation And Application Of The Monoclonal Antibody For Tylosin

Macrolides is a class of antibiotics that are usually used as veterinary drugs. Forexample, tylosin, tilmicosin and acetylisovaleryl tylosion are the commonly usedrepresentatives for the treatment of various diseases in livestock and poultry. As thesedrugs are widely used, their residues in the foods of animal origin become a publicconcern. The European Union banned tylosin as feed additive in1999, and later othercountries also formulated a series of residual control system for macrolides. Among thedetection methods for macrolides residues, immunoassay is widely used due to itsmerits of high specificity, simple, sensitive and fast.In this study, tylosin was derivatized with6-aminohexanoic acid andaminophenylacetic acid respectively to synthesized two novel haptens（STYL、PTYL）.The two haptens were coupled with bovine serum albumin and ovalbumin respectivelyto produce two immunogens and two coating antigens（STYL-BSA、STYL-OVA、PTYL-BSA、PTYL-OVA）by using of mixed anhydride method. The hapten/carriercoupling ratios in the conjugates were in the range of13-16:1. The two immunogenswere used to immunize Balb/c mice. During multiple immunizations, serum titers ofthese mice were determined by indirect ELISA method to evaluate different coatingantigen/antibody combinations. The results showed that the immunogen of STYL-BSAhad better immunogenicity. Then the spleen cells from the mice with the higheset titerwere fused with SP2/0myeloma cells to produce hybridomas. The positive hybridomacells were screened by using indirect competitive ELISA and then subcloned by usingthe limited dilution method. Finally, two hybridoma cell lines secreting the monoclonalantibody for tylosin were obtained. The ascites from the mice were purified by acrylicacid-ammonium sulfate precipipitation method to produce the monoclonal antibody.The obtained antibody simultaneously recognized tylosin, tilmicosin,acetylisovaleryltylosion, and the metabolite of tylosin （desmycosin）. Then theantibody was used to develop a heterologous indirect competitive ELISA to determinethe four antibiotics. The crossreactivities to the four antibiotics were in the range of46%-100%, the IC50values were in the range of15.8-34.0ng/mL, and the limits ofdetection were in the range of1.6-2.0ng/mL. The four analytes were fortified into blank milk at levels of20-100ng/mL for analysis. The recoveries were in the range of78%-96%with coefficients of variation in the range of4.2%-11.4%.Therefore, this method could be used as a rapid screen tool for routine monitoringthe residues of these macrolides antibiotics in animal derived foods to ensure the safetyof animal original food.