柯萨奇病毒A16型中和抗体检测毒株的制备及应用  

Preparation and application of the virus strains for detection of neutralizing antibody against Coxsackievirus A16

作　　者：谢学超 胡家垒 胡月梅 赵丽丽 陈磊[1] 肖海峰 徐颖之 李国顺[1] 顾美荣[1] 张改梅[1] XIE Xue-chao;HU Jia-lei;HU Yue-mei;ZHAO Li-li;CHEN Lei;XIAO Hai-feng;XU Ying-zhi;LI Guo-shun;GU Mei-rong;ZHANG Gai-mei(Beijing Minhai Biotechnology Co.,Ltd.R&D Center Daxing District,Beijing;Beijing New Joint Vaccine Engineering Technology Research Center,Beijing 102600,China)

机构地区：[1]北京民海生物科技有限公司研发中心北京市新型联合疫苗工程技术研究中心,北京102600 [2]江苏省南京市疾病预防控制中心(江苏省公共卫生研究院),江苏南京210000

出　　处：《微生物学免疫学进展》2022年第3期8-14,共7页Progress In Microbiology and Immunology

基　　金：北京市科技计划课题(Z201100005420004)。

摘　　要：目的制备柯萨奇病毒A16型(coxsackievirus A16,CV-A16)中和抗体检测毒株,并对其在中和抗体检测中的应用进行研究。方法对CV-A16检测毒株建立3级种子批并进行相关检定。由3名实验人员对工作种子批分别进行3次病毒标定,同时分别与其他3个型别(EV-A71、CV-A10、CV-A6)的免疫血清进行交叉中和。将检测毒株分别与对人自然感染血清和小鼠免疫血清样品进行6次中和抗体效价测定,计算中和抗体效价最大值与最小值的倍数差(Max-Min)和变异系数(coefficient of variation,CV)。用对比检测毒株对人自然感染血清进行3次中和抗体效价测定,与检测毒株进行比较。结果建立的CV-A16检测毒株的3级种子批无菌检查及支原体检查等均符合《中华人民共和国药典》2020版(三部)要求;经标定,平均病毒滴度为7.444 lgCCID_(50)/mL(95%CI:7.231~7.645 lgCCID_(50)/mL),CV为2.85%;与EV-A71、CV-A10、CV-A6这3种型别免疫血清均无交叉反应,特异性良好;人自然感染血清和小鼠免疫血清的中和抗体效价最大值与最小值的倍数差(Max-Min)均<4,CV为0.00%~12.79%。检测毒株与对比检测毒株检测的中和抗体效价结果差异无统计学意义(P<0.05)。结论本实验室筛选的CV-A16中和抗体检测用毒株具有较好的特异性,可以很好地应用于后续CV-A16中和抗体的相关检测。Objective To prepare coxsackievirus A16(coxsackievirus A16,CV-A16)strains for detection of neutralizing antibody titers.Methods The research,master and working CV-A16 virus seed banks were established and characterized.The viral titer of the working virus seed bank was determined three times by three experimenters,and cross-neutralization of CV-A16 by immune sera against three virus strains EV-A71,CV-A10 and CV-A6 was performed.Determination of neutralizing antibodies against CV-A16 in serum from naturally infected human and immunized mouse was conducted six times.The difference between the maximum and the minimum values(Max-Min)and the coefficient of variation of serum neutralizing antibody assay were calculated.While neutralizing antibody titers in serum from naturally infected human were also measured three times by other virus strains.Results The sterility testing and mycoplasma detection results of virus bank meet the relevant requirements of the Chinese Pharmacopoeia(VolumeⅢ,2020 edition).The average titer of the working virus seed bank was 7.444 lgCCID_(50)/mL(95%CI:7.231~7.645 lgCCID_(50)/mL),and the coefficient of variation(CV)was 2.85%.There was no cross-reaction between CV-A16 and the immune serum against EV-A71,CV-A10 and CV-A6,and the specificity was good;the Max-Min values of the neutralizing antibody titers against CV-A16 in serum from naturally infected human and immunized mouse were both less than 4,and the CV of neutralizing antibody titers was 0.00%-12.79%.There was no significant difference(P<0.05)in the results of neutralizing antibody titers against between the virus for detection and other viruses as control.Conclusion The CV-A16 strain has good specificity,and can be well used for the titer determination of neutralizing antibody against CV-A16.

关 键 词：手足口病 柯萨奇病毒 中和抗体 检测毒株 

分 类 号：R373.23[医药卫生—病原生物学]