6B型肺炎球菌荚膜多糖-破伤风类毒素结合物中游离多糖测定方法的建立  

Development of a determination method for free polysaccharide content in serotype 6B pneumococcal polysaccharide conjugate

作　　者：刘爱萍 任珍芸 刘倩 罗树权 陈晓航 李献林 杜娇 谢贵林 

机构地区：[1]兰州生物制品研究所有限责任公司,兰州730046

出　　处：《中国新药杂志》2017年第24期2937-2942,共6页Chinese Journal of New Drugs

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)

摘　　要：目的:建立6B型肺炎球菌荚膜多糖-破伤风类毒素结合物(PS6B-TT)中游离多糖的测定方法,并对方法进行验证。方法:采用脱氧胆酸钠-盐酸沉淀法(NaDC/HCl),取不同浓度的载体蛋白破伤风类毒素(TT),加入1%NaDC溶液混匀,离心后用Lowrry法检测上清中蛋白含量,以确定载体蛋白沉淀范围;用不同离子强度的氯化钠溶液稀释游离多糖对照品与TT的混合物,NaDC沉淀并离心后以蒽酮-硫酸法测定上清中多糖含量、计算回收率,确定游离多糖的最佳分离条件;以NaDC/HCl沉淀6B型多糖测定标准品并绘制标准曲线,与未经NaDC/HCl沉淀的标准品相比较,确定NaDC对蒽酮硫酸法测定结果的影响;由此建立NaDC/HCl沉淀法结合蒽酮硫酸法测定6B型结合物中游离多糖含量的方法,并对方法的专属性、准确度及精密度进行验证。结果:此方法沉淀载体蛋白浓度应控制在500μg·mL^(-1)以下;选择终浓度0.3 mol·L-1氯化钠溶液作为6B型肺炎球菌结合物中游离多糖测定的稀释液;NaDC/HCl沉淀法对蒽酮硫酸法测定多糖含量基本无影响;游离多糖对照品与载体蛋白TT混合物经建立的NaDC/HCl法沉淀后多糖及蛋白回收率分别为98.25%及0;游离糖添加试验回收率均在90%~100%;3批结合物游离糖含量测定值其试验内及试验间CV值均在10%以下。结论:本试验建立的NaDC/HCl沉淀法结合蒽酮硫酸法可有效分离6B型游离多糖与多糖蛋白结合物,并能够准确、稳定地测定结合物中的游离多糖含量。Objective：To develop and validate a method for determination of free polysaccharide in serotype 6B pneumococcal polysaccharide conjugate with TT as carrier protein （PS6B-TT）. Methods：NaDC/HC1 method was adopted. 1% NaDC was mixed with different concentrations of TT, supernatants were collected by centrifugation, and protein content was determined by Lowrry assay to determine the concentration range of carrier protein precipitated with NaDC. The mixtures of free polysaecharide and TT were diluted with different ionic strengths of sodium chloride solution, and the supernatants were collected and tested for polysaccharide content by anthrone-sulfuric acid method to determine the condition for separation of free polysaceharide. The serotype 6B standard for determination of polysaccharide content was precipitated with NaDC/HCl and the standard curve was compared with the normal standard to evaluate the influence of presence of NaDC on determination result of polysaccharide content. The specificity, accuracy and precision were verified. Results：The carrier protein concentration for NaDC precipitation was controlled at less than 500 μg·mL^-1. 0.3 mol·L^-1 sodium chloride served as the diluent for determination of free polysaccharide in serotype 6B pneumococcal conjugate. NaDC/HCl method showed little effect on determination of polysaccharide content by anthrone-sulfuric acid method. After NaDC/HCl precipitation, the recovery rates of mixture of free polysaccharide and TT were 98.25% and 0, respectively. The recovery rates of free polysaccharide in spike test were between 90% - 100%. The intro- and inter-assay CVs of three batches of determination results were less than 10%. Conclusion： The developed NaDC/HCl precipitation combined with anthrone-sulfuric acid method is effective for separation of 6B free polysaccharide from conjugate, accurate and stable for determination of free polysaccharide content.

关 键 词：肺炎多糖蛋白结合物 破伤风类毒素 游离多糖 脱氧胆酸钠 

分 类 号：R392[医药卫生—免疫学] R967[医药卫生—基础医学]