9V型肺炎球菌疫苗中多糖含量微孔板蒽酮-硫酸检测方法的建立及验证

发布时间：2018-01-11 19:25

本文关键词：9V型肺炎球菌疫苗中多糖含量微孔板蒽酮-硫酸检测方法的建立及验证　出处：《微生物学免疫学进展》2016年03期 　论文类型：期刊论文

【摘要】：目的建立微孔板蒽酮-硫酸法检测9V型肺炎球菌疫苗中多糖含量的检测方法,并对其进行验证和初步应用。方法通过对蒽酮-硫酸方法的改进,建立检测9V型肺炎球菌疫苗中多糖含量稳定可靠的微孔板蒽酮-硫酸法。分别对蒽酮-硫酸法中硫酸溶液浓度、加热时间进行优化,并对建立的方法进行特异性、线性、精密度及准确度的验证。结果最佳硫酸溶液比例为6∶1(浓硫酸∶水),最佳加热时间为10 min。在检测9V型肺炎球菌多糖、多糖衍生物及多糖蛋白结合物中多糖含量时,此方法特异性强;在20~120μg/m L范围内,标准曲线线性良好(r20.995);实验内及实验间均具有良好的精密度(CV值分别为2.92%~3.32%和3.10%~4.06%);准确度试验中3个质量浓度的回收率均在96.17%~106.63%之间。结论微孔板蒽酮-硫酸法可被用来有效、准确、稳定地检测9V型肺炎球菌多糖、多糖衍生物及多糖蛋白结合物中多糖的含量,该方法较传统蒽酮-硫酸法操作简便快捷,节约样品、试剂等材料,非常适用于相关的疫苗生产过程中的质量控制。
[Abstract]:Objective to establish a method for the determination of polysaccharides in 9V pneumococcal vaccine by microporous anthrone sulfuric acid method, and to verify and apply it. Methods the method of anthrone-sulfuric acid was improved. A stable and reliable micropore plate anthrone sulfuric acid method was established for the detection of polysaccharide in 9V pneumococcal vaccine. The concentration and heating time of sulfuric acid solution in anthrone-sulfuric acid method were optimized. The specificity, linearity, precision and accuracy of the method were verified. Results the optimum ratio of sulfuric acid solution was 6: 1 (concentrated sulfuric acid: water). The optimum heating time was 10 min. The method was specific in detecting the content of polysaccharide in 9V pneumococcal polysaccharide, polysaccharide derivatives and polysaccharide protein conjugate. In the range of 20 渭 g / mL, the standard curve has a good linearity. The CV values were 2.92% and 3.10% and 4.06%, respectively. The recoveries of the three mass concentrations in the accuracy test were 96.17 and 106.63% respectively. Conclusion the microporous plate anthrone sulfuric acid method can be used effectively and accurately. The content of polysaccharide in 9V pneumococcal polysaccharide, polysaccharide derivative and polysaccharide protein conjugate was determined stably. This method is simpler and faster than the traditional anthrone-sulfuric acid method and saves samples and reagents. It is very suitable for quality control in the process of vaccine production.
【作者单位】：兰州生物制品研究所有限责任公司第一研究室甘肃省疫苗工程技术研究中心;中国生物技术股份有限公司;
【基金】：“十二五”国家高技术发展研究计划(863计划)疫苗研发关键技术及产品研发项目(2012AA02A401)
【分类号】：R392-33
【正文快照】： 肺炎球菌多糖含量是肺炎球菌多糖疫苗及结合疫苗生产过程中重要的质量控制指标之一。目前检测肺炎球菌多糖含量的常用方法为蒽酮-硫酸法[1],此方法中,加热和强酸环境使得多糖糖苷键水解成为单糖,单糖脱水成为糠醛或羟甲基糠醛,再与蒽酮反应产生有色物质,在620 nm处有最大光吸

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