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目的:建立和验证有效分离5型肺炎球菌结合物原液(结合物原液)中游离多糖的方法，以供测定游离多糖含量。方法:通过游离多糖添加回收率试验以及氯化钠浓度对咔唑-硫酸法吸光度值影响试验，选择结合物原液中游离多糖和多糖-蛋白结合物分离时最佳的氯化钠浓度；通过蛋白回收率试验筛选出该条件下所能沉降蛋白质的浓度范围；比较标准曲线经脱氧胆酸钠(sodium deoxycholate，NaDC)/HCl法处理前后吸光度值的变化，验证NaDC/HCl法对多糖含量检测的影响；对所建立方法的专属性、精密度及准确度进行验证。结果:NaDC/HCl法处理结合物原液时的最佳氯化钠浓度为0.15 mol/L；结合物原液中蛋白浓度在100 μg/ml以内，NaDC/HCl法能实现蛋白质的完全沉淀；NaDC对多糖含量检测无干扰；该方法专一沉淀蛋白质，而对游离多糖无影响；准确度验证试验的回收率在93.17%～102.59%之间；日间以及日内的精密度变异系数均小于10%。结论:NaDC/HCl法能充分分离结合物原液中的多糖-蛋白结合物和游离多糖，具有良好的准确度和重复性，适用于5型肺炎球菌结合物原液中游离多糖含量的测定。“,”Objective:To develop and verify a separation method for free polysaccharide(PS) content detection in type 5 pneumococcal polysaccharide conjugate.Methods:Based on the results of added PS recovery test and influence of NaCl concentration on the absorbance value, the optimal concentration of NaCl in the samples was determined. Then the concentration range of carrier protein precipitated was screened by protein recovery test. Afterwards, glucuronic acid standard was precipitated with sodium deoxycholate (NaDC)/HCl and compared with the normal standard to evaluate effect of NaDC on determination result. The specificity, accuracy and precision of this method were verified.Results:The optimal concentration of NaCl is 0.15 mol/L. Protein was completely precipitated at concentration of less than 100 μg/ml. NaDC did not interfer with the detection results.This method had good specificity and the recovery rate was between 93.17% and 102.59%. Besides, this method showed high precision both in intra- and inter-assays, with coefficients of variation of less than 10%.Conclusion:Free PS can be separated completely from the conjugate precipitated with NaDC/HCl, indicating this method has superior accuracy and repeatability, suitable for detection of free PS content in type 5 pneumococcal conjugate.