CCID_(50)法检测流感病毒滴度方法的建立及验证  

Development and validation of a CCID_(50) method for determination of influenza virus titer

作　　者：孙瑶[1] 申镇维[2,3] 姜春来[1] 孟祥博[4] 徐菲 

机构地区：[1]吉林大学生命科学学院艾滋病疫苗国家工程实验室,吉林长春130012 [2]长春百克生物科技股份公司,吉林长春130012 [3]吉林大学第一医院转化医学研究院免疫学研究所,吉林长春130061 [4]吉林大学中日联谊医院胃肠内科,吉林长春130033

出　　处：《中国生物制品学杂志》2016年第9期978-983,共6页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目"创新生物技术药物的开发"课题(140901GX010013647)

摘　　要：目的建立基于MDCK细胞的三价流感减毒活疫苗病毒滴度检测方法,并进行验证。方法以抗流感病毒血清中和三价流感减毒活疫苗中两种型别病毒,采用CCID_(50)法检测未中和型别病毒感染性滴度,并对方法的专属性、线性、准确性、精密度、耐用性进行验证。结果样品中的辅料成分、其他型别病毒和异源抗病毒血清均不会对病毒滴度测定结果产生干扰;该方法检测H1N1、H3N2和B型流感病毒滴度的线性范围分别为3.81~8.48、3.48~7.65和2.45~6.98 log CCID_(50)/0.2 ml,在线性范围内,线性回归系数(R^2)分别为1、0.994 6和0.994 5;该方法检测3种型别流感病毒滴度重复性和中间精密度的相对标准偏差(relative standard deviation,RSD)均小于15%,平均回收率在85%~115%之间;同一样品不同时间点检出结果差异无统计学意义(P均>0.05)。结论建立的CCID_(50)法专属性、准确度、线性、精密度、耐用性良好,是一种简便、快速、成本低、检测误差小的方法,可用于三价流感减毒活疫苗病毒滴度的检测。Objective To develop and validate a Madin-Darby canine kidney（MDCK） cell line based potency assay to test the titer of trivalent live attenuated influenza vaccine. Methods The infectious titer of non-neutralized virus was determined by CCID50 method. The test was validated for specificity, linearity, accuracy, precision and robustness. Results The virus preservers, other viral strains and heterologous antisera in samples showed no influence on the determination result of virus titer. The linear ranges of titers of influenza H1N1, H3N2 and B viruses were 3. 81 - 8. 48, 3. 48 - 7. 65 and 2. 45 - 6. 98 log CCID50/ 0. 2 ml, with R-2 values of 1, 0. 994 6 and 0. 994 5, respectively. The relative standard deviations（RSDs）of reproducibility and intermediate precision of virus titers of the three types were less than 15%, while the mean recovery rate was 85% - 115%. The determination results of the same sample at various time points showed no significant difference（each P 〉 0. 05）. Conclusion The developed CCID50 method showed high specificity, linearity,accuracy, precision and robustness, which was simple, rapid, low-cost and with small error, and was suitable for determination of virus titer of trivalent live attenuated influenza vaccine.

关 键 词：CCID50 三价流感减毒活疫苗 病毒滴度 MDCK细胞 

分 类 号：R373.13[医药卫生—病原生物学] R392-33[医药卫生—基础医学]