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

Method development for determination of free polysaccharide content in Streptococcus pneumoniae type 19A polysaccharide conjugate

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5g／L脱氧胆酸钠（sodiumdeoxycholate，IX）C）溶液（pH7．3）沉淀不同浓度的破伤风类毒素（tetanustoxoid，1vr），确定不同浓度DOC沉淀载体蛋白1vr的浓度范围。以1g／LDOC分离结合19APS（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3g／LDOC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 determinanon oi lree polysaccnanae 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 he controlled in 〈 150 and 〈200μg/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.