14型肺炎球菌荚膜多糖-破伤风类毒素结合物中游离多糖的测定方法

目的:建立一种简单有效的测定肺炎球菌荚膜多糖-破伤风类毒素结合物中游离多糖的方法。方法:根据脱氧胆酸(DOC)在酸性条件下可快速将蛋白质沉淀的原理,采用不同pH值的1%DOC溶液分别对14型肺炎球菌荚膜多糖(Ps14)、破伤风类毒素(TT)及Ps14-TT结合物进行处理,观察不同pH值1%DOC溶液对它们的影响,来确定最佳反应pH值。随后采用精密度试验和准确度试验对该方法进行验证。结果:Ps14-TT结合物经1%DOC处理后,在酸性条件下(1 mol.L-1HCl),结合物及蛋白质被完全沉淀,而未结合的Ps14存在于上清液中,采用蒽酮法测定结合物当中的游离多糖含量。结论:该方法重复性,稳定性,精密度,准确度均达到检测要求,适用于检测Ps14-TT结合物中游离多糖的测定。

Determination of free polysaccharides in pneumococcal polysaccharide serotype 14-tetanus toxoid conjugates

Objective: To establish a simple and effective assay for determining the concentrations of free polysaccharides in pneumococcal polysaccharide-tetanus toxoid conjugates.Methods: Since deoxycholic acid(DOC) can precipitate protein in acidic conditions,pneumococcal polysaccharide Ps14,tetanus toxoid and Ps14-TT conjugates were treated with deferent pH values 1% DOC solution.The impact of different pH values of 1% DOC solution on them was observed,and the optimal reaction pH was determined.Then this method with precision test and accuracy test were verified.Results: After Ps14-TT conjugates were treated with 1% DOC solution,the conjugates and free protein were completely precipitated in acidic conditions(1 mol·L-1 HCl),with free polysaccharides in the supernatant.The concentration of free polysaccharides then was determined using anthrone assay.Conclusion: The method shows good reproducibility,stability,precision and accuracy,and meets the detection requirements.It is applicable to detect the concentration of free polysaccharides in Ps14-TT.