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目的建立微孔板蒽酮-硫酸法检测9V型肺炎球菌疫苗中多糖含量的检测方法,并对其进行验证和初步应用。方法通过对蒽酮-硫酸方法的改进,建立检测9V型肺炎球菌疫苗中多糖含量稳定可靠的微孔板蒽酮-硫酸法。分别对蒽酮-硫酸法中硫酸溶液浓度、加热时间进行优化,并对建立的方法进行特异性、线性、精密度及准确度的验证。结果最佳硫酸溶液比例为6∶1(浓硫酸∶水),最佳加热时间为10 min。在检测9V型肺炎球菌多糖、多糖衍生物及多糖蛋白结合物中多糖含量时,此方法特异性强;在20~120μg/m L范围内,标准曲线线性良好(r2>0.995);实验内及实验间均具有良好的精密度(CV值分别为2.92%~3.32%和3.10%~4.06%);准确度试验中3个质量浓度的回收率均在96.17%~106.63%之间。结论微孔板蒽酮-硫酸法可被用来有效、准确、稳定地检测9V型肺炎球菌多糖、多糖衍生物及多糖蛋白结合物中多糖的含量,该方法较传统蒽酮-硫酸法操作简便快捷,节约样品、试剂等材料,非常适用于相关的疫苗生产过程中的质量控制。
Objective To establish a method for the determination of polysaccharide in 9V pneumococcal vaccine by anthrone-sulfuric acid method with microplate and verify the preliminary application. Methods The anthrone-sulfuric acid method was established to establish a stable and reliable microplate anthrone-sulfuric acid method for detecting polysaccharide content in 9V pneumococcal vaccine. The concentration and heating time of sulfuric acid solution in anthrone-sulfuric acid method were optimized, and the specificity, linearity, precision and accuracy of the established method were verified. The optimum ratio of sulfuric acid solution was 6: 1 (concentrated sulfuric acid: water), the optimal heating time was 10 min. The specificity of this method was very good when detecting the polysaccharide content of polysaccharide, polysaccharide derivatives and polysaccharide conjugates of 9V pneumococcal polysaccharide, polysaccharide derivatives and polysaccharide conjugates. The calibration curve was linear (r2> 0.995) in the range of 20 ~ 120μg / All the experiments had good precision (CV values were 2.92% ~ 3.32% and 3.10% ~ 4.06% respectively). The recoveries of three mass concentrations in the accuracy test ranged from 96.17% to 106.63%. Conclusion Microplate anthrone-sulfuric acid method can be used to effectively, accurately and stably detect the content of polysaccharides in polysaccharide, polysaccharide derivatives and polysaccharide conjugates of 9V pneumococcal polysaccharide. The method is simpler than the traditional anthrone-sulfuric acid method Fast, save samples, reagents and other materials, is very suitable for the relevant quality control in the vaccine production process.