【作者】 董申；

【导师】 金磊；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2005， 硕士

【摘要】 随着现代生物技术的发展,越来越多的重组生物制品投放市场。目前国内大多数重组产品都是采用E.coli 作为宿主菌进行表达。而其中残余宿主菌菌体蛋白是影响制品安全性的主要因素之一,反复使用含一定量菌体蛋白的重组产品有可能引起机体发生过敏反应。为此,必须建立灵敏度高,重复性好的残余宿主菌体蛋白含量测定方法,以保证这类制品的安全有效。在建立药品质量标准时,所建立的分析方法须经验证,其目的是证明采用的方法适合于相应检测要求,验证原则是根据中华人民共和国国家药典附录中《药品质量标准分析方法验证原则》。我们通过实验制定了本室工程菌的残余菌体蛋白的标准品,并且分别建立了三种该残余宿主菌菌体蛋白的检测方法:斑点检测法,直接Elisa 检测法,双抗体夹心Elisa 检测法。通过实验对比,其中双抗体夹心Elisa 检测法测定残余宿主菌菌体蛋白的方法精确度高,检测灵敏度高,相对检测灵敏度均能达到所要求标准。因此选择双抗体夹心Elisa检测法作为残余宿主菌菌体蛋白的监测方法,并且对其实验条件进行了优化,同时对其进行了方法验证,其结果符合国家药典要求。更多还原

【Abstract】 With the development of modern biotechnology, more and morerecombinant bioproducts are being sold in the market, Host Cell Protein isone of the important factors that affect the safety of the products, andrepeated using of recombinant products of host protein of a certain amountmay cause hypersusceptibility to the organisms. High sensitivity and goodrepetition measuring methods to the host cell protein should be set up.During the experiment, fist we prepared a patch of endo-control products asthe standard, and the semi-quantitive-measuring method of blot measuringhost cell protein method is got rid. Through comparison and selection of theexperiments in direct Elisa and double antibody Sandwich Elisa, doubleantibody Sandwich method that is more suitable is determined as the methodto measure Host cell proten in the recombinant Human Growth Hormone,also the methodology validation has been done to this measuring method,and it accords with the standard of Chinese Codex.Through experiments validation, this method has a good linearity.Whenlinearity regression of Y=ax+b is done, the value of r2 , which is bigger than0.994, is high. The measuring limitation is low which is only 1 ng, andquantitive measuring can be processec in the range of 10ng-320ng. Duringthe experiment of precision, it has a high accuracy, and the result is close to更多还原