人血清中寨卡病毒微量中和抗体检测方法的建立和应用  

Establishment and application of micro-neutralization test to determine neutralizing antibody against ZIKA virus in sera

作　　者：纪洵敏 苏娟[2] 易丽娜 彭拓华 谈琦琪 张欢[2] 孙九峰 吴德[2] 张贤昌 柯昌文[2] Ji Xunmin Su Juan Yi lina Peng Tuohua Tan Qiqi Zhang Huan Sun Jiufeng Wu De Zhang Xianchang Ke Changwen(Guangdong Provincial Institute of Biological Products and Materia Medica, Guangzhou 510440, China （Ji XM, Peng TH, Zhang XC Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 511430, China （ Su J, Tan QQ, Zhang H, Wu D, Ke CW Guangdong Provincial Institute of Public Health, Guangzhou 511430, China （Yi LN, Sun JF)

机构地区：[1]广东省生物制品与药物研究所,广州510440 [2]广东省疾病预防控制中心病原微生物检验所,广州511430 [3]广东省公共卫生研究院,广州511430

出　　处：《中华实验和临床病毒学杂志》2017年第4期367-371,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：广东省科技计划项目（20160250）;中美新发传染病合作项目（EID）子项目12（NU2GGH000961-05-00）

摘　　要：目的 建立人血清寨卡病毒（ZIKV）微量中和抗体检测方法,并用10例经核酸检测和/或病毒分离确诊的寨卡输入病例的急性期和恢复期血清样本进行分析验证.方法 采用分离自输入寨卡病例的寨卡病毒株开展组织培养微量中和抗体测定,在BHK21、VERO和VERO-E6 3种细胞系中选择感染寨卡病毒后能产生典型CPE的细胞系制备病毒储备液,滴定病毒滴度;使用100TCID50的病毒液与连续4倍稀释的经56℃ 30 min灭活的血清于37℃中和2h,然后加入细胞悬液.5％二氧化碳培养箱孵育,逐日观察CPE.结果 BHK21、VERO和VERO-E63种细胞系对寨卡病毒感染的敏感度不同,其中VERO细胞最为敏感,可出现典型的CPE;应用VERO细胞系建立寨卡病毒微量中和抗体检测方法能准确反映寨卡确诊病例急性期和恢复期血清中和抗体水平,并能作为一种特异性诊断方法.结论 寨卡病毒微量中和抗体检测方法不仅可用于人感染寨卡病毒的实验室诊断,还可用于人群血清流行病学调查.Objective To develop a micro-neutralization test for determination of neutralizing antibody against ZIKA virus （ZIKV） in human sera and to verify the acute and convalescent serum samples of 10 ZIKA virus-infected cases diagnosed by nucleic acid detection and/or virus isolation.Methods ZIKV isolated from ZIKA cases was used to determine micro-neutralization antibody.The virus solution was prepared by infecting BHK21,VERO and VERO-E6 cell lines and viral titer was tested;100 TCID50 viral solution and 4 times diluted sera which were inactivated at 56 ℃ for 30 min were neutralized,then added the cell suspension and incubated in 5% CO2 incubator at 37 ℃ for 7 d.The CPE was observed every day.Results The sensitivity of BHK21,VERO and VERO-E6 was different after infection with ZIKA virus.VERO cell line was the most sensitive and showed typical CPE.VERO cell line was used to establish a micro-neutralization test for determination of neutralizing antibody against ZIKA virus in sera.Conclusions The neutralizing antibody test for zika virus in sera is a special and usefulmethod to diagnose human infection of ZIKV and to conduct population based epidemiological investigation.

关 键 词：微量中和抗体 寨卡病毒 VERO细胞系 

分 类 号：R446.6[医药卫生—诊断学] R511[医药卫生—临床医学]