| Tiamulin（TML）is a semi-synthetic diterpene veterinary antibiotics.It is mainly used for the treatment of chronic respiratory diseases in chickens.The standardized use of TML will exert its antibacterial properties well,but if the abuse of TML will residue in animal-derived foods,it will cause potential harm to people’s health,causes food safety and public health problems.The methods of detecting TML include chromatography,and immunological technology.Among them,the test strip detection method in immunological detection is widely used,because it is sensitive,specific,fast,simple,and can realize on-site real-time detection.The preparation of highly specific,high affinity monoclonal antibodies is the basis for the establishment of TML immunoassay methods.In this study,according to the structural of TML,the ketone carbonyl group on the five-membered ring of TML was modificated and the synthesis of artificial complete antigen was synthesized.Mice were immunized;The anti-TML monoclonal antibody with high specificity,high sensitivity and high affinity was developed by cell fusion technology and limiting dilution subcloning technology,established hybridoma cell lines stably secreting anti-TML monoclonal antibody,which lays a foundation for establishing an immunological method for rapid detection of TML.The immunogen TML-BSA was prepared by coupling the tiamulin（TML）with the carrier protein（BSA）by the oxidative method and the active ester method.The same method was used to prepare the coating antigen TML-OVA.The prepared artificial complete antigen was identified by ultraviolet scanning（UV）and polyacrylamide gel electrophoresis（SDS-PAGE）.The results of immunizing Balb/c mice indicated:mouse 2 had the best immune effect,the titer of immune serum was up to 1:1×105,and the half-inhibitory concentration was 4.07ng/mL.Positive hybridoma cell lines were screened by cell fusion technique and limiting dilution subcloning,and 1 cell line was obtained,which was named 4A7.The ascites were prepared by in vivo induction method,and purified from ascites by caprylic acid-ammonium sulfate method to obtain monoclonal antibody.The SDS-PAGE results showed that the antibody of the purified had two bands,one is the heavy chain about 50kDa,another is the light chain about 25kDa,and the purity was about 90%,the indirect ELISA results showed that the titer of the purified antibody could be up to1:5.12×105,IC500 is 0.049 ng/mL,affinity constant Kaff is 1.47×109L/mol。The antibody has no cross-reaction with other antibiotics such as valnemulin and fleroxacin,and there is a cross-reaction rate of 2.7%with rapapalin.In this study,anti-TML monoclonal antibody was prepared,which provides afoundation for the development of TML immunoassay kits and related rapid test products. |
