本文選題：抗狂犬病病毒單克隆抗體 + 直接快速免疫組化法　； 參考：《吉林大學》2017年碩士論文

【摘要】：狂犬病是由狂犬病病毒引起的人獸共患病。人和所有溫血動物對狂犬病病毒都易感染。由于該病一旦發(fā)病目前尚無法醫(yī)治,又由于動物傳染源廣泛存在,控制難度很大,所以對人及動物狂犬病感染的診斷研究十分重要。直接快速免疫組化法(Direct Rapid Immunohistochemical Test,DRIT)是美國CDC狂犬病室新近建立的一種以酶標記抗狂犬病毒單克隆抗體檢測狂犬病毒抗原的診斷方法,根據(jù)國家用于診斷狂犬病病毒動物的標準操作程序規(guī)定,DRIT是一種尚未得到認證的驗證d FA方法的標準。美國CDC狂犬病室進行的評價實驗顯示DRIT與WHO推薦“金標準”DFA檢測結(jié)果的符合率為100%。本試劑是本單位與中國疾病預防控制中心病毒病預防控制所合作研究的成果。由中國疾病預防控制中心病毒病預防控制所提供抗狂犬病病毒單克隆抗體細胞株、狂犬病病毒陽性腦組織印片和陰性腦組織印片。本單位對單克隆抗體細胞進行培養(yǎng),對單抗進行純化和標記,將抗體應用免疫組化方法對狂犬病病毒抗原進行檢測,對實驗方法進行優(yōu)化,選擇最佳實驗方案,配套實驗材料、耗材及包裝,將所有材料組裝成試劑盒。操作過程如下:將抗狂犬病病毒單克隆抗體細胞株(編號:4A12)復蘇后進行培養(yǎng),然后植入到Balb/c小鼠腹腔內(nèi)產(chǎn)生腹水。回收的腹水用間接ELISA方法檢測抗體效價,檢測到具有較高的效價。制備的單克隆抗體腹水經(jīng)過鹽析純化后標記生物素。用間接ELISA方法檢測標記后的抗體效價,檢測到具有較高的效價。采用接種有狂犬病病毒的細胞板進行小試,確定了該實驗方法在檢測狂犬病病病毒的可行性。篩選抗體生物素標記物及鏈霉親和素HRP酶標記物的最佳工作濃度,抗體生物素標記物的最佳工作濃度為1:100而鏈霉親和素HRP酶標記物的最佳工作濃度為1:1000。然后檢測狂犬病病毒抗原陽性和陰性腦組織涂片,用該方法可鑒別出狂犬病病毒抗原陽性和陰性。最后,將本試驗所需的所有試劑、材料和耗材分裝后進行試劑盒的組裝。本實驗用直接快速免疫組化法(DRIT)確定了4A12單抗株能特異性結(jié)合狂犬病病毒及包涵體,且用光學顯微鏡就可以觀察到腦組織內(nèi)包涵體的存在。因此,該試劑可以應用于直接快速免疫組化法檢測狂犬病病毒抗原,且方便了實驗操作,節(jié)約了實驗成本,提高了實驗的特異性和可重復性。目前,國內(nèi)還沒有用該方法檢測狂犬病病毒的成品試劑,因此,可對該試劑做進一步的研究,使直接快速免疫組化法檢測狂犬病病毒抗原的方法在基層實驗室得到更加廣泛的推廣。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.99;R446.6

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