【作者】 魏丽丽；

【导师】 彭远义；

【作者基本信息】 西南大学 ， 预防兽医学， 2007， 硕士

【摘要】 四环素类药物（tetracyclines，TCs）抗菌谱广、疗效好，已经广泛应用于各种感染疾病的治疗。但近年来其在动物组织中的残留危害已经引起了人们的高度关注，同时其检测方法也在不断的改进，目前国内还没有见到利用四环素单克隆抗体建立四环素残留检测方法的正式报道。因此，本研究在查阅大量资料的基础上制备了盐酸四环素单克隆抗体，建立了四环素残留的免疫学检测方法，为试剂盒的开发应用奠定了基础。试验结果如下：1．采用重氮法合成了TC-BSA、TC-OVA，通过紫外扫描对人工抗原进行鉴定。紫外扫描图谱显示TC-BSA和TC-OVA分别在276.6nm和278nm波长处有最大吸收峰，此外还有TC特征峰，进一步说明半抗原TC已分别与载体BSA和OVA交联。2．以TC-BSA作为免疫抗原，免疫8周龄BALB／C系小鼠。三免后，将小鼠断尾采血分离血清，以TC-OVA为包被抗原，用间接ELISA法和间接竞争ELISA法筛选血清效价高且特异性强的阳性鼠。取阳性小鼠的脾细胞与SP2／0细胞用50％PEG-1500进行融合。通过检测、克隆、筛选等步骤，最终筛选出两株产抗TC单抗的细胞株，并对细胞的培养上清液进行单抗亚型鉴定，表明两株细胞所产单抗为IgG2b型抗体。将一株杂交瘤细胞注射小鼠腹腔产生腹水，并将腹水用辛酸-硫酸胺法纯化，纯化后腹水的蛋白含量为1.326mg／mL，然后用SDS-PAGE进行纯度鉴定，电泳结果为两条清晰的条带，一条为重链，一条为轻链，纯度较高。3．应用纯化后的单抗初步建立间接竞争ELISA法，用方阵滴定法确定了抗原最佳包被浓度（5μg／mL）、单抗纯化后的工作浓度为1∶8000，并对其整个反应体系的反应条件进行了优化，以此建立了TC残留的ELISA检测方法；绘制了TC对E₁₀细胞株所产单克隆抗体的抑制曲线，标准曲线为y=-20.439x+81.03（R²=0.9498），检测限（LOD）为0.68ng／mL，IC₅₀为31.62ng／mL；TC单克隆抗体与环丙沙星、氧氟沙星、庆大霉素、青霉素钾、磺胺嘧啶、链霉素六种药物均无交叉反应性。结果表明，试验建立的TC残留检测的ELISA方法灵敏度高、特异性好。4．将盐酸四环素系列浓度药物添加到牛奶中，然后作其标准曲线，建立其回归方程；再分别取高、中、低3个盐酸四环素药物浓度作回收试验，采取与标准曲线相同的样品处理方法，得到盐酸四环素在牛奶中的添加回收率在61.3％-76.5％之间。结果表明：应用TC单抗建立的间接竞争ELISA方法能用于检测牛奶中四环素的残留。更多还原

【Abstract】 Tetracycline （TC） is widely used in treatment and prevention of animal disease since it has the advantages of broad-spetrum and curative effect.Recently, however, due to its acute side-effect to people’s healthy resulting from the residue is animal tissue, it has brought people’s great attention to the residue in animal food. In order to establish a sensitive method to detect TC residue, monoclonal antibody against TC has been prepared and the Ci-ELISA method has been developed in this study.1. The immunogen（TC-BSA）and coating antigen（TC-OVA） were prepared, and both antigens were identified by UV scan. The results were as follows: The maximum absorbance peaks of TC-BSA and TC-OVA were at 276.6nm and 278nm wavelength respectively. And there were a characteristic absorbance peak for the two antigens. The results showed that TC and carrier proteins condensate successfully.2. TC-BSA was used as immunogen to immunize 8-week-old BALB/C mice for obtaining the antiserum. TC-OVA was used as coating antigen. The positive mice of high antiserum titre against TC were selected by the Indirect Enzyme linked immunosorbent assay （ELISA）. Spleen cells from the positive mice were fused with sp2/0 cells by 50%PEG-1500.Through detecting、cloning and selecting, two kinds of hybridization tumor cells that can secrete the monoclonal antibodies against TC was selected. The identification of subclasses indicated that the monoclonal antibodies secreted by E（10） and D₅ were antibodies of IgG2b group. Mice were injected with by hybrid cells to produce ascites which has to be purified, and the purified protein content was 1.326 mg/mL. SDS-PAGE figure appeared two clear strips, among which one was heavy chain and the other was light chain.3. Ci-ELISA method was developed preliminarily with the purified monoclonal antibodies. The working contents of coating antigen and monoclonal antibody were determined, which is 5μg/mL and 1:8000 respectively, and the reaction conditions was optimized. Through analysis of Ci-ELISA, the standard curve was drawed, and its standard equation was y=-20.439x+81.03 （R²=0.9498）;LOD was 0.68ng/mL;IC₅₀ was 31.62ng/mL. The crossing reaction results indicated the method of Ci-ELISA has high sensitivity and good specificity.4. A series of concentrations of TC was added into milk, then the standard curve was drawed by milk mediator. Three kinds of TC concentration（high concentration, medium concentration, low concentration）was added to milk to measure TC recovery rate, and the recovery rate was between 61.3% and 76.5 %.The final results showed that the TC residue in milk can be detected by the established Ci-ELISA with the prepared monoclonal antibody against TC.更多还原