柯萨奇病毒A组10型免疫荧光检测方法的建立及其在灭活验证中的应用  

Establishing an immunofluorescence assay for the detection of CVA10 and its application in inactivation verification

作　　者：张宇昊 杨婷[1] 李华[1] 岳磊[1] 向虹 谢天宏[1] 张也[1] 谢忠平[1] ZHANG Yu-hao;YANG Ting;LI Hua;YUE Lei;XIANG Hong;XIE Tian-hong;ZHANG Ye;XIE Zhong-ping(Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Kunming,Yunnan 650118,China)

机构地区：[1]中国医学科学院、北京协和医学院、医学生物学研究所,云南昆明650118

出　　处：《中国病毒病杂志》2023年第1期48-54,共7页Chinese Journal of Viral Diseases

基　　金：中国医学科学院医学与健康科技创新工程(重大协同创新项目)(2021-I2M-1-043)。

摘　　要：目的建立检测柯萨奇病毒A组10型(coxsackievirus A10,CVA10)病毒颗粒的免疫荧光检测方法,使用该方法对病毒的灭活进行辅助验证。方法在非洲绿猴肾细胞(African green monkey kidney cell,Vero)、人恶性胚胎横纹肌瘤细胞(human malignant embryonic rhabdomyoma cells,RD)、非洲绿猴胚胎肾细胞(African green monkey embryonic kidney cells,MA104)三种细胞上检测病毒滴度,绘制滴度曲线确定最敏感细胞;以兔抗CVA10多克隆抗体为一抗、Alexa Fluor®594偶联的山羊抗兔IgG为荧光二抗,对实验条件进行优化,建立检测CVA10的免疫荧光方法,并与灭活验证中细胞盲传实验结果进行比较。结果RD细胞为CVA10最敏感细胞,一抗最佳稀释度为1∶100,二抗最佳稀释度为1∶200。病毒感染RD细胞后24 h可检测到的病毒量下限为50 CCID_(50)/ml,感染4 d时的检测下限为0.5 CCID_(50)/ml。Vero细胞为CVA10的较敏感细胞,相同条件下,病毒感染Vero细胞后24 h可检测到的病毒下限为80 CCID_(50)/ml,5 d时的检测下限为1 CCID_(50)/ml。灭活验证中,免疫荧光结果与细胞盲传实验结果具有一致性。结论成功建立了CVA10的免疫荧光检测方法,该方法可辅助细胞盲传实验进行灭活验证。Objective To develop an immunofluorescence assay for the detection of coxsackievirus A10(CVA10),and to discuss its application in the verification of CVA10 virus inactivation.Methods Virus titer curve was mapped based on the virus titers detected on three types of cells:African green monkey kidney cells(Vero),human malignant embryonic rhabdomyoma cells(RD),and African green monkey embryonic kidney cells(MA104)to de⁃termine the most sensitive cell line.The rabbit anti-CVA10 polyclonal antibody was used as the primary antibody,and Alexa Fluor®594-conjugated goat anti-rabbit IgG was used as the fluorescent secondary antibody.The experi⁃mental conditions were optimized,an immunofluorescence method was developed for the detection of CVA10,and the method was applied to the inactivation verification.Results RD was found to be the most sensitive cell line for CVA10 culture.The optimal dilution was 1∶100 for primary antibody and was 1∶200 for secondary antibody.The lower limit of the detectable virus was 50 CCID_(50)/ml at 24 h after virus infection in RD cells,and was 0.5 CCID_(50)/ml at 4 days of infection.Vero cell was relatively sensitive cell line for CVA10 culture.Under the same conditions,the lower limit of virus detection in Vero cells after infection was 80 CCID_(50)/ml at 24 h and 1 CCID_(50) at 5 d.In the verifi⁃cation of virus inactivation,the immunofluorescence results were consistent with the results of blind cell passaging experiments.Conclusions The immunofluorescence detection method of CVA10 is established successfully,which can assist the blind cell transmission test for inactivation verification.

关 键 词：柯萨奇病毒A组10型 灭活验证 免疫荧光检测 敏感性 

分 类 号：R-331[医药卫生]