冻干甲型肝炎减毒活疫苗检定方法的研究

发布时间：2018-06-12 04:26

本文选题：冻干甲型肝炎减毒活疫苗 + 毒种　；参考：《浙江大学》2006年硕士论文

【摘要】：目的对冻干甲肝减毒活疫苗毒种与成品检定中的关键性项目的检定方法进行优化。(1)利用单克隆抗体代替常规的人、猴免疫血清进行毒种外源因子检查及鉴别试验。(2)利用Labworks凝胶成像分析系统判断疫苗RT-PCR滴度；建立病毒滴度检测的RT-PCR-ELISA法、微量免疫荧光法。(3)确定疫苗牛血清白蛋白残留量检测最佳方法。方法 (1)毒种检定：将单克隆抗体与甲肝病毒进行中和试验，观察单克隆抗体的中和作用；利用单克隆抗体中和毒种，进行外源因子检查，并应用于鉴别试验。(2)感染性滴度检测：通过对RT-PCR反应液的离子浓度、PH值等条件的优化，建立检测滴度的RT-PCR一步法，利用Labworks凝胶成像分析系统的半定量功能判断结果，并与常规组织培养法比较；将标记有生物素的寡核苷酸引物所扩增的疫苗病毒基因产物，，与微孔反应板上的特异性探针进行快速杂交，通过辣根过氧化物酶标记的链亲和素进行酶联显色，读取光密度值(OD值)，判断结果，并与常规组织培养法比较；取适宜稀释度病毒接种细胞己成致密单层的96孔培养板，至增殖高峰时直接在板上固定细胞／病毒系统，用间接免疫荧光法观察结果，并与常规组织培养法比较。(3)牛血清白蛋白残留量测定：比较疫苗牛血清白蛋白残留量检测中反向间接血凝法、酶联免疫法的重复性、精确性。结果 (1)此单克隆抗体可中和10~5 CCID_(50)／ml甲肝病毒，将此高效价
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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