CA16滴度微量检测方法的建立及其验证

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。

Development and verification of the microtitration assay for the infectious titer of CA16

Objective To develop a microtitration assay for the infectious titer of coxsackievirus A group 16 strain （CA16） on Vero cell and verify its adaptivity. Methods CCIDs0 （ Cell culture infection dose-50） assay for the infectious titer of CA16 was developed and verified by optimization of experimental condition. Results Vero cell was selected in detection of titer of CAI6 by observing the CPE induced by the virus infection. The optimal Vero cell concentration for inoculation and detection timing are 5 ×10^4 - 1 ×10^5 cells/mL （5 ×10^3 - 1 ×10^4cells/well in 96-well plates） and 7 days, respectively. The determinated coefficients of variation （CV） are in a range 1. 739% -4. 974%, which suggest that the established method is accurate and reliable. Conclusion The developed CCID50 assay used on Vero cell is a simple, sensitive and reliable technique for detection titer of CA16, and would be useful for quality control in the process of vaccine production.