肠道病毒71型中和抗体检测用标准病毒候选株的筛选及制备

Screening and preparation of candidate standard strain for determination of neutralizing antibody against enterovirus 71

目的筛选并制备肠道病毒71型(Enterovirus 71,EV71)中和抗体检测用标准病毒候选株,规范EV71中和抗体检测方法。方法通过对亲本株与非亲本株交叉中和试验,进行相应的几何平均滴度(GMT)比较,从10株EV71毒株中筛选出具有理想亲本中和能力、又具有较广泛交叉中和作用的毒株。筛选出的毒株按《中国药典》三部(2010版)相关要求,冷冻干燥后制备1批符合我国疾病流行特征的标准病毒候选株,联合3家实验室对候选株的病毒滴度进行协作标定,并初步应用于人血清样品的检测。结果筛选出交叉中和能力强、分离、传代背景清晰的EV71 V03株(523-07T)作为候选病毒株;制备的候选病毒株分装精确度、水分含量、无菌检查、支原体检查、其他外源因子检查等项目均符合《中国药典》三部(2010版)相关要求;经联合标定,候选毒株的平均病毒滴度为4.83 lgTCID50/ml(95%CI:4.53～5.12 lgTCID50/ml),变异系数为3.16%;应用候选毒株和中和抗体定值标准品检测EV71自然感染人血清样品,可将实验室间检测差异由4.4倍降至1.6倍。结论初步筛选出的候选株EV71/523-07T株具有理想的亲本株中和作用,又具有较广泛的交叉中和特性,有望成为EV71中和抗体检测的标准毒株候选株。

Objective To screen and prepare the candidate standard strain to standardize the method for determination of neutralizing antibody against enterovirus71（EV71）.Methods One virus strain with strong neutralizing capability to parent strain and cross-neutralizing capability to non-parent strains was screened from ten EV71 stains by cross-neutralization test in which the GMTs of parent and non-parent strains were compared.According to requirements in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）,one batch of candidate standard EV71 strain with the epidemic characteristics in China was prepared by lyophilization,cooperatively calibrated for titer by three independent laboratories,and preliminarily applied to determination of human serum samples.Results EV71 / V03（523-07T） strain with homogeneous cross-neutralizing capability and clear background of isolation and subculture was screened,of which the precision of filling,moisture content,as well as the results of sterility test,mycoplasma test and examination on adventitious agents met the requirements in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）.The titer of the candidate strain was 4.83 lgTCID50 / ml（95%CI： 4.53-5.12 lgTCID50 / ml）,with a coefficient of variation of 3.16%.By combined application of the candidate strain and quantitation standard for EV71 neutralizing antibody,the difference between determination results of human serum samples naturally infected with EV71 in various laboratories decreased from 4.4 folds to 1.6 folds.Conclusion The screened candidate strain EV71 / V03（523-07T）,with satisfactory neutralizing capability to parent strain and cross-neutralizing capability to various non-parent strains,might be used as a candidate standard strain for determination of neutralizing antibody against EV71.