口服轮状病毒活疫苗病毒滴度评价国家参考品的制备

目的 制备口服轮状病毒(rotavirus,RV)活疫苗病毒滴度评价的国家参考品.方法 选取检定合格的口服RV活疫苗原液,加入保护剂,1.0 ml/安瓿分装,冷冻干燥,制备病毒滴度参考品.用微量细胞病变法检测参考品的病毒滴度,用Karber法计算半数细胞培养感染量(50％ cell culture infective dose,CCID50).由3家实验室对参考品的病毒滴度进行协同标定,采用单因素方差分析和最小显著差数法对3家实验室的标定结果进行统计学分析.另外,将参考品于-20℃放置1年、4℃放置1年、37℃放置14d、-60℃放置2年,测定病毒滴度,分析参考品的热稳定性和长期稳定性.结果 经单因素方差分析比较,3家实验室检测结果的差异无统计学意义(F=0.379,P=0.686).采用最小显著差数法对3家实验室检测结果的均值进行两两比较,差异亦无统计学意义(s(x)值均为0.078 2,P值均＞0.05).参考品的平均病毒滴度为6.5 lgCCID50/ml,于不同温度放置一段时间后病毒滴度下降均未超过1.0 lgCCID50/ml.长期稳定性试验结果的变异系数为4.9％.结论 制备了可用于口服RV活疫苗病毒滴度检测的国家参考品.

Preparation of a national determination reference of virus titer for live oral rotavirus vaccines

Objective To prepare a national evaluation reference of virus titer for live oral rotavirus (RV) vaccines.Methods A bulk of qualified live attenuated RV vaccine was mixed with preservative,packed into ampoules (1.0 ml/vial) and lyophilized.The virus titer of the reference preparation was determined by a micro-cytopathic method and 50％ cell culture infective dose (CCID50) was calculated with Karber method.The reference was collaboratively evaluated in 3 different laboratories and results were analyzed by one way ANOVA and least-significant difference test.In addition,the virus titers were determined after the reference was stored at-20 ℃ for 1 year,4 ℃ for 1 year,37 ℃ for 14 d and -60 ℃ for 2 years,respectively.Results There was no significant difference among results from the 3 laboratories by one way ANOVA (F=0.379,P=0.686) or by pairwise comparison with least-significant difference test (s(x) =0.078 2,all P values ＞0.05).The average titer of the reference was 6.5 lgCCID50/ml.The reduction of titer was no more than 1.0 lgCCID50/ml after the preparation was stored at different temperatures for different periods.The coefficient of variation in long-time stability test was 4.9％.Conclusion The reference prepared can be used for virus titer evaluation of live oral RV vaccines.