Production Of Tiamulin Complete Immunogen And Development Of Anti-tiamulin Ployclone Antibody

Tiamulin (Tiamulin, TML) is a dedicated pleuromutilins animals antibiotics. Itsstrong activity against Mycoplasma, Streptococcus and Staphylococcus aureus,together with the ability to encourage growth make it widely used in veterinary drugand feed additives. However, TML residues in animal foods may cause a seriouspotential threat to human health. Currently, the methods for detecting residualtiamulin are mainly physical and chemical detection methods. Despite the delicacyand accuracy by using these instruments analytical methods, the sample process iscumbersome, time-consuming and requires expensive equipment. In this study weproduced complete antigen according to TML’s molecular structure characteristics,and accomplished the identification of the complete immungen, laying foundation forfuture immunological detection methods.1, The preparation and identification of complete immungenThe TML hapten was combined to bovine serum albumin(BSA) and ovalbumin(OVA)by mixed acid anhydride method(MA), and N,N-carbonyldiimidazole method(CDI)respectively, obtaining the immunogen TML-BSA and coating antigenTML-OVA.UV scan and SDS-PAGE gel electrophoresis were used to identify the immunogenResults indicated the successful coupling and the coupling ratios were1:23.6,according to the results of SDS-PAGE.Complete immungen TML-BSA were used toimmunize3BALB/c mice. we set50μg doses with immune interval of two weeks,and a total of4times immunization; mouse serum antibody titers were determined byindirect ELISA, while the sensitivities of serum antibodies were determined byindirect competitive ELISA. The results showed that antibody titers of NO.2andNO.3mouse were1:6400. In sensitivity experiments, the antibody titer of all3micewere suppressed by TML standard.The sensitivity of NO.3mouse was the best,whose IC50value was559ng/mL. 2, Preparation of polyclonal antibody and establishment of immunological detectionmethodComplete antigen TML-BSA was used to immunize female New Zealand whiterabbits with a dose of300ug and the interval of two weeks for total4times.Polyclonal antibodies were obtained after the purification of antiserum and then wereindentified for its immunologic characteristics. Results showed that the polyclonalantibodies have great specificity with the titer of1:1.28×105, the IC50of200.04ng/mL and the less than1%cross-reactivity with normal antibiotics such as tylosinand roxithromycin. We established the indirect complete ELISA to detect TML byusing the prepared polyclonal antibody. Linearity of the standard curve is fine withthe R2of0.9906and sensitivity of181.28ng/mL.