本文選題：凍干甲型肝炎減毒活疫苗 + 毒種�。� 參考：《浙江大學(xué)》2006年碩士論文

【摘要】：目的 對凍干甲肝減毒活疫苗毒種與成品檢定中的關(guān)鍵性項目的檢定方法進行優(yōu)化。(1)利用單克隆抗體代替常規(guī)的人、猴免疫血清進行毒種外源因子檢查及鑒別試驗。(2)利用Labworks凝膠成像分析系統(tǒng)判斷疫苗RT-PCR滴度；建立病毒滴度檢測的RT-PCR-ELISA法、微量免疫熒光法。(3)確定疫苗牛血清白蛋白殘留量檢測最佳方法。 方法 (1)毒種檢定：將單克隆抗體與甲肝病毒進行中和試驗，觀察單克隆抗體的中和作用；利用單克隆抗體中和毒種，進行外源因子檢查，并應(yīng)用于鑒別試驗。(2)感染性滴度檢測：通過對RT-PCR反應(yīng)液的離子濃度、PH值等條件的優(yōu)化，建立檢測滴度的RT-PCR一步法，利用Labworks凝膠成像分析系統(tǒng)的半定量功能判斷結(jié)果，并與常規(guī)組織培養(yǎng)法比較；將標記有生物素的寡核苷酸引物所擴增的疫苗病毒基因產(chǎn)物，，與微孔反應(yīng)板上的特異性探針進行快速雜交，通過辣根過氧化物酶標記的鏈親和素進行酶聯(lián)顯色，讀取光密度值(OD值)，判斷結(jié)果，并與常規(guī)組織培養(yǎng)法比較；取適宜稀釋度病毒接種細胞己成致密單層的96孔培養(yǎng)板，至增殖高峰時直接在板上固定細胞／病毒系統(tǒng)，用間接免疫熒光法觀察結(jié)果，并與常規(guī)組織培養(yǎng)法比較。(3)牛血清白蛋白殘留量測定：比較疫苗牛血清白蛋白殘留量檢測中反向間接血凝法、酶聯(lián)免疫法的重復(fù)性、精確性。 結(jié)果 (1)此單克隆抗體可中和10~5 CCID_(50)／ml甲肝病毒，將此高效價
[Abstract]:Objective to optimize the method for the determination of live attenuated hepatitis A vaccine strains and products by using monoclonal antibody instead of conventional human. Using Labworks gel imaging analysis system to determine the titer of vaccine RT-PCR, to establish a RT-PCR-ELISA method for virus titer detection. The best method for the detection of bovine serum albumin residue in vaccine was determined by microimmunofluorescence method. Methods the neutralization of monoclonal antibody with hepatitis A virus was studied by neutralizing monoclonal antibody with hepatitis A virus. Using monoclonal antibody to neutralize the virus, the exogenous factors were examined and used in the detection of infectious titer. By optimizing the conditions such as the ionic concentration and PH value of the RT-PCR reaction solution, a one-step RT-PCR method was established to detect the titer. The semi-quantitative functional judgment results of Labworks gel imaging analysis system were used and compared with the conventional tissue culture method. The DNA products of vaccine virus were amplified by oligonucleotide primers labeled with biotin. Rapid hybridization was carried out with specific probes on the microporous reaction plate. Enzyme linked chromogenic reaction was performed with horseradish peroxidase labeled chain avidin. The OD value of optical density was read, and the results were compared with the conventional tissue culture method. The cell / virus system was directly fixed on the plate at the peak of proliferation, and the results were observed by indirect immunofluorescence method. And compared with the routine tissue culture method, the determination of bovine serum albumin residue: the repeatability of reverse indirect hemagglutination and enzyme-linked immunosorbent assay in the detection of bovine serum albumin residue of vaccine was compared. Accuracy. Results 1) this monoclonal antibody can neutralize 10 5 CCIDS 50 / ml hepatitis A virus and has a high titer.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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