9V型肺炎球菌多糖蛋白结合物原液中游离多糖含量检测方法的建立

Development of a determination method for free polysaccharide content in bulk of type 9V pneumococcal polysaccharide conjugate

目的建立测定9V型肺炎球菌多糖蛋白结合物原液中游离多糖含量的检测方法。方法对9V型肺炎球菌多糖溶液进行不同浓度的脱氧胆酸钠(NaDOC)酸沉处理,选择仅沉淀结合物而不沉淀多糖的最佳处理条件,筛选出所能沉淀蛋白的最大浓度;对建立的方法进行准确度、精密度、专属性及线性验证。结果 9V型肺炎球菌多糖蛋白结合物原液的最佳NaDOC酸沉处理条件为2%NaDOC-0. 05 mol/L HCl;蛋白浓度低于200μg/m L时,NaDOC酸沉处理条件沉淀蛋白的效果最佳。准确度试验的回收率在90%～99%之间;实验内及实验间均具有良好的精密度(CV均小于10%);在10～100μg/mL范围内采用蒽酮硫酸法测定糖浓度的曲线,线性良好,r^2> 0. 998;NaDOC的终浓度在0. 25%以下,对蒽酮硫酸法检测无干扰。结论 NaDOC酸沉淀法能很好地分离结合物原液中的结合物与游离多糖,具有良好的重复性和准确性,可用于测定9V型肺炎球菌多糖蛋白结合物原液中的游离多糖含量。

Objective To develop a method for determination of free polysaccharide content in bulk of type 9 V pneumococcal polysaccharide conjugate. Methods Type 9 V pneumococcal polysaccharide solution was treated with sodium deoxycholate and hydrochloric acid,at various concentrations. The condition for treatment was optimized,under which the conjugate was completely precipitated while the polysaccharide was not precipitated. On the basis of this,the maximum protein concentration for precipitation was screened. The developed method was verified for accuracy,precision,specificity and linearity. Results The conjugate and protein in type 9 V pneumococcal polysaccharide conjugate was completely precipitated with 2% sodium deoxycholate-0. 05 mol/L bulk of hydrochloric acid at a protein concentration of less than 200 μg/m L. The recovery rate of polysaccharide in accuracy test was 90% ~ 99%. The developed method showed high precision in both intra-and inter-assays,with CVs of less than 10%. The curve for determination of polysaccharide concentration by anthrone-sulfuric method showed good linearity within a concentration range of 10 ~100 μg/mL,with an r^2 value of more than 0. 998. The sodium deoxycholate at a final concentration of less than 0. 25%showed no interference to anthrone-sulfuric acid method. Conclusion The conjugate and free polysaccharide in bulk of type 9 V pneumococcal polysaccharide conjugate was separated effectively by precipitation with sodium deoxycholate and hydrochloric acid. The developed method showed high reproducibility and accuracy,which might be used for quantitative determination of free polysaccharide content in bulk of type 9 V pneumococcal polysaccharide conjugate.