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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 standard virus candidate strains for detecting Enterovirus 71 (EV71) neutralizing antibodies, and to standardize the detection method of EV71 neutralizing antibodies. Methods By cross-neutralization test of the parental and non-parental strains, corresponding geometric mean titers (GMT) were compared to select the 10 strains of EV71 to have the ideal ability of neutralizing the parents and having a more extensive cross-neutralization effect Of the strains. The selected strains were prepared according to the relevant requirements of the Chinese Pharmacopoeia (2010 edition), and after freeze-drying, a batch of standard virus candidate strains that met the epidemic characteristics of our country were prepared. The virus titers of candidate strains were collaborated with three laboratories Calibration, and initially applied to the detection of human serum samples. Results The strains EV71 V03 (523-07T) with strong cross-neutralizing ability, clear separation and passage were screened out as candidate strains. The prepared candidate strains were sub-packed with accuracy, moisture content, sterility test, mycoplasma test and others The results showed that the average virus titer of the candidate strains was 4.83 lg TCID50 / ml (95% CI: 4.53-5.12 lgTCID50 / ml) after the joint calibration. The coefficient of variation (CV) was 3.16%. By using candidate strains and neutralizing antibody standards to detect natural infection of human serum samples, EV71 could reduce the difference between laboratory tests from 4.4 to 1.6. CONCLUSION: The candidate strain EV71 / 523-07T, which has been initially screened, has the ideal neutralization effect of the parent strain and has a wide range of cross-neutralization properties. It is expected to be a candidate candidate strain for the detection of neutralizing antibodies against EV71.