5型肺炎球菌结合物原液中游离多糖分离方法的建立及验证

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...

Establishment and verification of a separation method for free polysaccharide in type 5 pneumococcal polysaccharide conjugate#br#

 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 added PS of 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 determination 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 interfered 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 vartation 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.