肺炎链球菌18C型多糖结合物中游离多糖含量检测方法的建立

目的建立肺炎链球菌18C型多糖结合物中游离多糖含量的测定方法。方法采用不同条件预处理样品,将结合多糖与游离多糖分离,用改良蒽酮法测定结合物中总糖与游离多糖的浓度,计算出结合物中游离多糖的含量,并对检测方法进行验证。结果采用5mol/L氯化钠溶液、85%乙醇溶液及-20℃冻存72h预处理样品,结合多糖与游离多糖的分离率及实验的有效性均最高。采用本方法测定肺炎链球菌18C型多糖结合物中游离多糖含量,结合多糖与游离多糖分离率的变异系数CV值为1.7%,实验有效性变异系数CV值为5.6%;检测3个浓度供试品的平均回收率分别为98.6%、98.5%和97.0%;检测1批多糖结合疫苗中游离多糖含量的CV值为4.2%。结论已建立肺炎链球菌18C型多糖结合物中游离多糖含量的检测方法,该方法可靠性强,准确性高,精密性良好。

Development of A Method for Determination of Free Polysaccharide Content in Streptococcus pneumoniae Type 18C Polysaccharide Conjugate

Objective To develop a method for determination of free polysaccharide content in Streptococcus pneumoniae type 18C polysaccharide conjugate.Methods The samples of conjugate was pre-treated under various conditions to separate conjugated and free polysaccharides,then determined for total and free polysaccharide contents by an improved anthrone colorimetry,based on which the free polysaccharide content was calculated,and the developed method was verified.Results Compared with those pretreated under other conditions,both the separation rates of conjugated and free polysaccharides in the conjugate vaccine pre-treated with 5 mol/L sodium chloride and 85%ethanol and stored in frozen at-20℃for 72 h were high.The variation coefficient(CV)of separation rate of conjugated and free polysaccharides determined by the developed method was 1.7%,and that of effectiveness was 5.6%.The mean recovery rates of test samples at 3 concentrations were 98.6%,98.5%and 97.0%respectively.The CV value of free polysaccharide content in one batch of polysaccharide conjugate vaccine determined by the developed method was 4.2%.Conclusion A method for determination of free polysaccharide content in Streptococcus pneumoniae type 18C polysaccharide conjugate was developed.