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目的建立一种b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)结合疫苗中游离多糖含量新的检测方法。方法分别对标准蛋白溶液、多糖溶液和标加衍生多糖(AH-PRP)的结合物原液进行脱氧胆酸钠(NaDC)酸沉淀处理,观察该方法对蛋白和多糖的沉淀效果。分别采用NaDC酸沉淀法和乙醇分步沉淀法测定结合疫苗原液中的游离多糖含量。结果不同浓度的标准蛋白溶液经NaDC酸沉淀法处理后,沉淀中蛋白的回收率在96%～99%之间;不同浓度的多糖溶液经NaDC酸沉淀法处理后,上清中的多糖回收率在99%～106%之间;该方法对结合物中的游离多糖能起到很好的分离效果;两种方法测定结合疫苗原液中的游离多糖含量差异有统计学意义(P<0.01)。结论 NaDC酸沉淀法能专一性地沉淀蛋白物质,对游离多糖无沉淀作用,该方法具有良好的重复性和准确性,可用于测定Hib结合疫苗中的游离多糖含量。
Objective To establish a new method for detecting free polysaccharide in Haemophilus influenzae type b (Hib) conjugate vaccine. Methods Sodium dodecyl sulfate (NaDC) was precipitated by the conjugate solution of standard protein solution, polysaccharide solution and AH-PRP, and the precipitation effect of the method on protein and polysaccharide was observed. The content of free polysaccharide in the conjugate vaccine solution was determined by NaDC acid precipitation method and ethanol fractional precipitation method respectively. Results The recoveries of the proteins in the precipitate were between 96% and 99% after NaDC acid precipitation at different concentrations of standard protein solution. The polysaccharide recovery in the supernatant after different concentrations of polysaccharide solution were treated by NaDC acid precipitation (99% ~ 106%). The method can be used for the separation of free polysaccharides in the conjugate. The two methods have significant difference (P <0.01) in the determination of free polysaccharides in the conjugate stock solution. Conclusion NaDC acid precipitation method can precipitate protein material with no precipitation effect on free polysaccharide. The method has good repeatability and accuracy and can be used for determination of free polysaccharide in Hib conjugate vaccine.