【作者】 赵东；

【导师】 宁长申； 张改平；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 泰乐菌素是最常见的饲料添加剂成分之一,同时该药也作为畜禽疫病和蜜蜂美洲幼虫腐臭病的防治药物,无论作为饲料添加剂或是治病的药物,效果良好。所以该药研发不久,迅速在全球广泛应用,但其耐约菌株急速增加,欧盟首先禁止了泰乐菌素作为饲料添加剂,各国也相继制定了一系列的残留控制体系。在其残留检测方法中,免疫分析法因其敏感、特异、简单易操作,作为一种泰乐菌素残留的筛选方法,有独特优势。本研究在分析泰乐菌素免疫特性的基础上,运用单克隆抗体技术生产其单抗,并组装了快速检测试剂盒和试纸条。1、试验一把半抗原TL和载体蛋白BSA、OVA偶连制备了全抗原,并利用SDS-PAGE和紫外扫描做了相应的鉴定:用TL-BSA抗原免疫Balb/c小鼠,将免疫小鼠脾细胞与NS0瘤细胞用50%的PEG4000进行细胞融合,用HAT培养基筛选培养,以TL-OVA抗原检测杂交瘤细胞培养上清,获得了15株产生泰乐菌素抗体的杂交瘤细胞株。对强阳性克隆以有限稀释法连续克隆三次,建立1株分泌高亲和力抗TL单克隆抗体杂交瘤细胞株TL6E9,培养上清的ELISA效价分别为1:300。以TL6E9杂交瘤细胞,腹腔注射Balb/c小鼠诱生腹水,其ELISA效价为2.5x10^-6,免疫球蛋白亚类为IgG2a。单抗与其他药品的交叉反应率非常低。3、试验二利用基于试验一制备的抗泰乐菌素的单克隆抗体,依据酶联免疫吸附实验和胶体金技术原理,分别组装了间接抑制ELISA检测试剂盒和免疫检测试纸条,并用于检测蜂蜜、牛奶、畜禽的饲料、组织或是代谢物中泰乐菌素的残留。试剂检测盒泰乐菌素残留可以在一个小时内完成;试纸条则可以在8到10分钟内完成。试剂盒和试纸条的最低检测限在10ng/mL,半量抑制浓度（IC50）在30ng/mL;试剂盒和试纸条可以广泛的用于对泰乐菌素残留的定性、半定量和定量检测。更多还原

【Abstract】 Tylosin（TL） is one of the most additives, the same as it is the good medicine for livestock,poultry and bees.Tylosin was widely used through the world as soon as it was developed. But resistance of bacteria against tylosin elevated quickly,so Europe Alliance prohibited that man used TL as one of additives firstly and then other country formulated a series of systems to control the residues of TL. Among the current analysis methods for TL, Immunoassays which provide the advantages of sensitivity, specificity and ease-of-use have more advantages.In this study, Based on immunological analysis of TL, We used the technology of monoclonal antibodies to produce the monoclonal antibodies against TL, and assembled rapid ELISA kit and strip.TL was linked to Bovine Sera Albumin （BSA） and ovum Albumin （OVA）,Corresponding identification was made by SDS-PAGE and UltraViolet scan. A weak immune response or none at all was induced after immunization of mice with those conjugates. High antibody titers were obtained with conjugates where TL was linked to BSA .Using the mature technology of monoclonal antibodies, the lymphocytes from Balb/c mouse spleen routinely immunized by BSA-TL antigen were fused with NSO myeloma cells by 50% PEG 4000, and selected cultured with HAT medium. A total of 15 hybridomas producing monoclonal antibodies against TL were obtained by screened with TL-OVA antigen using an indirect enzyme-linked immunosorbent assay （ELISA）,and cloned three times by limiting dilution. One hybridoma cell line of TL6E9, the hybridoma secret high affinity monoclonal antidodies （McAb） with the titer of 1:300, determined by ELISA in supernatants. The cell line of the hybridoma 6E9 were intraperitoneally injected into prostane sensitized Balb/c mice for production of ascites antibodies, the titer of which was 2.5×10^-6. The immunoglobulin isotype of the McAb was identified with the Immunoglobulin Isotype Kit as IgG2a. These McAbs showed low cross-reactivity to materia medical.Based on the anti-TL McAb and the enzyme-linked immunosorbent assay and Colloidal gold principle, an indirect enzyme-linked immunosorbent assay （ELISA） kit and a lateral flow immunoassay （LFIA） strip was developed and applied to the screening for TL residues. By TL-kit, the test of TL residues could be accomplished within 60 minutes. By TL- strip, the test could be accomplished within 8 to 10 minutes. It was shown that detection limit of the test strip and kit was as low as 10 ng/mL of TL. The half maximal inhibition concentration （IC50） as determined visually was 30 ng/mL. Therefore, the kit and test strip is very useful for the quantitative, semi-quantitative or qualitative detection of TL residues.更多还原