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目的建立用Vero细胞检测柯萨奇病毒A组16型(CA16)滴度的方法,并对其适用性进行初步验证。方法通过对细胞种类的选择、细胞接种浓度和细胞病变判定时间的优化,建立测定CA16滴度的半数细胞感染剂量法(CCID50法),并对其进行初步验证。结果通过对病毒感染后细胞病变情况的观察,确定了以Vero细胞作为CA16病毒滴度检定用细胞。Vero细胞的最佳接种浓度和结果判定时间分别为5×104~1×105细胞/mL(即96孔板内每孔加入的最终细胞量为5×103~1×104细胞)和7 d。由4组人员对6批CA16病毒液进行重复检测,实验结果表明不同实验人员测定结果的变异系数(CV)在1.739%~4.974%之间,说明该方法测定结果重复性好,准确度高。结论该法简便、稳定,可以灵敏、准确地检测CA16病毒的滴度,可用于相关疫苗生产过程中的质量控制。
Objective To establish a method for detecting the titer of Coxsackie A virus type 16 (CA16) in Vero cells and to verify the applicability of the method. Methods Based on the selection of cell types, the concentration of cells inoculated and the determination of cytopathic effect, the half dose of CC16 assay (CCID50 method) was established for the determination of CA16 titer and the preliminary validation was carried out. Results The observation of cytopathic effect after virus infection confirmed that Vero cells were used as CA16 virus titer assay cells. The optimal inoculum concentration and time for determination of Vero cells were 5 × 10 4 to 1 × 10 5 cells / mL (ie, 5 × 10 3 to 1 × 10 4 cells per well in a 96-well plate) and 7 days. Four batches of six batches of CA16 virus solution were tested repeatedly. The experimental results showed that the coefficient of variation (CV) varied from 1.739% to 4.974%. The results showed that the method had good repeatability and high accuracy. Conclusion The method is simple, stable, sensitive and accurate detection of CA16 virus titer, can be used for the quality control of the relevant vaccine production process.