9V型肺炎球菌疫苗中多糖含量微孔板蒽酮-硫酸检测方法的建立及验证

Development and validation of microplate anthrone-sulfuric acid assay to quantify carbohydrate in serotype 9V pneumococcal vaccine

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 and validate a microtiter anthrone-sulfuric acid method in determination of polysaccha- rides contained in serotype 9V pneumococcal vaccine, and a preliminary application of the method was performed. Metho- ds Colorimetry in microplate was introduced to the traditional method of anthrone-sulfuric acid in determination of polysac- charides. The concentration of sulfuric acid solution and the heating time were optimized. Absorbance was obtained in an ELISA reader and the polysaccharides content was quantified. The specificity, linearity, precision and accuracy of the method was validated. Results The appropriate concentration of sulfuric acid solution was 6 ： 1 （ sulfuric acid to H20 v/ v） , the heating time was 10 min. A high specificity was obtained in determination of polysaccharides content in serotype 9V pneumococcal polysaccharides and its derivatives and conjugates. A good linear correlation （ r^2 〉0.995 ） was obtained within the concentrational range 20 μg/mL- 120 μg/mL. Intro- and inter-assay CV ranged between 2.92% - 3.32% and 3.10% -4.06% ,respectively. Recovery rates were obtained between 96.17% and 106.63% for three concentration levels in accuracy test. Conclusion This method is effective, accurate and stable in determination of serotype 9V pneumococcal polysaccharides, polysaccharide derivatives and conjugates. Com- pared to the traditional method, it can be operated easily and rapidly, and saved amount for sampling and reagents. It should be suitable in quality control for related vaccine production.