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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。
Objective: To establish a simple and effective method for the determination of free polysaccharides in the pneumococcal capsular polysaccharide-tetanus toxoid conjugate. Methods: According to the principle that deoxycholic acid (DOC) can quickly precipitate protein under acidic conditions, different concentrations of 1% DOC solutions were used to detect the levels of Ps14, TT, And Ps14-TT conjugate were treated to observe the effects of different pH 1% DOC solution to determine the optimal reaction pH. This method is then validated using precision tests and accuracy tests. Results: After treated with 1% DOC, Ps14-TT conjugates were completely precipitated in acidic condition (1 mol·L-1 HCl) and unbound Ps14 was present in the supernatant. Anthrone Method to determine the content of free polysaccharide in the conjugate. Conclusion: The repeatability, stability, precision and accuracy of the method meet the test requirements and are suitable for the determination of free polysaccharide in the Ps14-TT conjugate.