19A型肺炎链球菌多糖结合物中游离多糖含量测定方法的建立

摘要

目的 建立19A型肺炎链球菌多糖(Streptococcus pneumoniae type 19A polysaccharide,19APS)结合物中游离PS含量的测定方法.方法 在一定条件下分别用1、3和5 g/L脱氧胆酸钠(sodium deoxycholate,DOC)溶液(pH7.3)沉淀不同浓度的破伤风类毒素(tetanus toxoid,TT),确定不同浓度DOC沉淀载体蛋白TT的浓度范围.以1 g/L DOC分离结合19 APS(19APS-TT)和游离19APS,测定上清液中的游离PS含量,并验证该DOC沉淀法的准确度和精密度.结果 分别以1和3 g/L DOC沉淀19APS-TT时,TT浓度最好分别控制在＜150和＜200μg/ml.该DOC沉淀法的准确度和精密度均良好,加标PS回收率为94.0％～107.2％,相对标准偏差均＜4％.结论 成功建立了19APS-TT中游离PS含量的测定方法.%Objective To develop a method for determination of free polysaccharide content in Streptococcus pneumoniae type 19A polysaccharide (19APS) conjugate.Methods Tetanus toxoid (TT) solutions of different concentrations were precipitated using 1,3 and 5 g/L sodium deoxycholate (DOC) (pH7.3),respectively,under certain conditions.The concentration ranges of carrier protein TT precipitated with different concentrations of DOC were determined.The conjugated 19APS (19APS-TT) and free 19APS were separated using 1 g/L DOC,and free PS contents in supernatant were detected.The accuracy and precision of the DOC precipitation method were validated.Results When 19APS-TT was precipitated using 1 and 3 g/L DOC,the TT concentrations should be controlled in ＜ 150 and ＜200 μg/ml,respectively.The DOC precipitation method had good accuracy and precision.The adding recovery rates of PS were 94.0％-107.2％,and the relative standard deviations of the DOC precipitation method were all ＜4％.Conclusion The method for determination of free polysaccharide content in 19APS-TT is developed successfully.