直接快速免疫组化法检测狂犬病病毒抗原

发布时间：2018-06-12 12:05

本文选题：抗狂犬病病毒单克隆抗体 + 直接快速免疫组化法　；参考：《吉林大学》2017年硕士论文

【摘要】：狂犬病是由狂犬病病毒引起的人兽共患病。人和所有温血动物对狂犬病病毒都易感染。由于该病一旦发病目前尚无法医治,又由于动物传染源广泛存在,控制难度很大,所以对人及动物狂犬病感染的诊断研究十分重要。直接快速免疫组化法(Direct Rapid Immunohistochemical Test,DRIT)是美国CDC狂犬病室新近建立的一种以酶标记抗狂犬病毒单克隆抗体检测狂犬病毒抗原的诊断方法,根据国家用于诊断狂犬病病毒动物的标准操作程序规定,DRIT是一种尚未得到认证的验证d FA方法的标准。美国CDC狂犬病室进行的评价实验显示DRIT与WHO推荐“金标准”DFA检测结果的符合率为100%。本试剂是本单位与中国疾病预防控制中心病毒病预防控制所合作研究的成果。由中国疾病预防控制中心病毒病预防控制所提供抗狂犬病病毒单克隆抗体细胞株、狂犬病病毒阳性脑组织印片和阴性脑组织印片。本单位对单克隆抗体细胞进行培养,对单抗进行纯化和标记,将抗体应用免疫组化方法对狂犬病病毒抗原进行检测,对实验方法进行优化,选择最佳实验方案,配套实验材料、耗材及包装,将所有材料组装成试剂盒。操作过程如下:将抗狂犬病病毒单克隆抗体细胞株(编号:4A12)复苏后进行培养,然后植入到Balb/c小鼠腹腔内产生腹水。回收的腹水用间接ELISA方法检测抗体效价,检测到具有较高的效价。制备的单克隆抗体腹水经过盐析纯化后标记生物素。用间接ELISA方法检测标记后的抗体效价,检测到具有较高的效价。采用接种有狂犬病病毒的细胞板进行小试,确定了该实验方法在检测狂犬病病病毒的可行性。筛选抗体生物素标记物及链霉亲和素HRP酶标记物的最佳工作浓度,抗体生物素标记物的最佳工作浓度为1:100而链霉亲和素HRP酶标记物的最佳工作浓度为1:1000。然后检测狂犬病病毒抗原阳性和阴性脑组织涂片,用该方法可鉴别出狂犬病病毒抗原阳性和阴性。最后,将本试验所需的所有试剂、材料和耗材分装后进行试剂盒的组装。本实验用直接快速免疫组化法(DRIT)确定了4A12单抗株能特异性结合狂犬病病毒及包涵体,且用光学显微镜就可以观察到脑组织内包涵体的存在。因此,该试剂可以应用于直接快速免疫组化法检测狂犬病病毒抗原,且方便了实验操作,节约了实验成本,提高了实验的特异性和可重复性。目前,国内还没有用该方法检测狂犬病病毒的成品试剂,因此,可对该试剂做进一步的研究,使直接快速免疫组化法检测狂犬病病毒抗原的方法在基层实验室得到更加广泛的推广。
[Abstract]:Rabies is a zoonosis caused by rabies virus. People and all warm blooded animals are susceptible to rabies virus. Because once the disease is still untreatable, and because of the widespread existence of animal infectious sources, it is very difficult to control the disease. Therefore, the diagnosis of human and animal rabies infection is very important. Direct rapid immunization group. Direct Rapid Immunohistochemical Test (DRIT) is a newly established diagnostic method for detecting rabies virus antigen by enzyme labeled rabies monoclonal antibody in the CDC rabies room of the United States. According to the national standard operating procedure for the diagnosis of rabies virus animals, DRIT is an uncertified D FA prescription. The standard of the law. The evaluation experiment of the CDC rabies room in the United States showed that the coincidence rate between DRIT and WHO recommended the "gold standard" DFA test results was the result of the cooperation between the unit and the Chinese Center for the prevention and control of the disease control center. The anti rabies provided by the China Center for Disease Control and prevention of viral disease prevention and control. A monoclonal antibody cell line, a rabies virus positive brain tissue seal and a negative brain tissue seal. The unit has cultured the monoclonal antibody cells, purified and marked the monoclonal antibody. The antibody was detected by immunohistochemical method to detect the rabies virus antigen, and the experimental method was optimized, and the best experimental scheme was selected. The experimental materials, materials, and packaging were assembled and all the materials were assembled into a kit. The operation process was as follows: after the recovery of the monoclonal antibody cell line (number: 4A12) of the rabies virus, then implanted into the abdominal cavity of the Balb/c mice to produce ascites. The recovered ascites detected the antibody titer with the indirect ELISA method, and the high titer was detected. The prepared monoclonal antibody ascites was purified by salting out and labeled with biotin. The titer of the labeled antibody was detected by indirect ELISA method and high titer was detected. The feasibility of detecting rabies virus was determined by using the cell plate inoculated with rabies virus, and the antibody biotin marker was screened. The best working concentration of the streptomycin HRP enzyme marker, the best working concentration of the antibody biotin marker was 1:100 and the optimum working concentration of the streptavidin HRP enzyme marker was 1:1000., and then the rabies virus antigen positive and negative brain tissue smear were detected, and the positive and negative of rabies virus antigen was identified by this method. Finally, all the reagents, materials and consumables needed in this experiment were assembled to carry out the kit. The direct rapid immunohistochemical method (DRIT) was used to determine the specific binding of the rabies virus and inclusion body of the 4A12 monoclonal antibody strain, and the presence of inclusion bodies in the brain tissue could be observed by optical microscopy. The direct rapid immunohistochemical method was used to detect rabies virus antigen, and the experimental operation was convenient, the cost of the experiment was saved, the specificity and repeatability of the experiment were improved. At present, it has not been used to detect the finished reagents of rabies virus in China. Therefore, the test agent can be further studied and the direct rapid immunohistochemical method can be used. The method of rabies virus antigen detection has been widely promoted in primary laboratories.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.99;R446.6

【相似文献】