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目的制备病毒载体疫苗滴度测定用国家标准品。方法以鸡胚成纤维细胞制备痘苗病毒,经密度梯度超速离心纯化后,加入天花疫苗保护剂,分装冻存,分发至我国4个不同实验室,按照统一的结晶紫染色蚀斑法实验方案进行协作标定,每次实验稀释后取2个稀释度,每个稀释度设5个平行孔,计算各实验室检测结果的几何均数,对标准品中痘苗病毒滴度进行赋值。检测病毒标准品于不同温度下保存不同时间的稳定性,并对病毒标准品的各项质量指标进行检定。结果 4个实验室共测定44次,4个实验室间的检测结果差异无统计学意义(P>0.05),各实验室检测结果的几何平均数为3.05×106PFU/ml,95%CI为2.00×106～4.65×106PFU/ml,标准差为0.09,总变异系数1.41%。制备的痘苗病毒标准品于4℃可稳定放置10 d,37℃放置稳定性较差。制备的痘苗病毒标准品装量检查合格(1 ml/支);pH值为6.9;无菌试验合格;分装重量精密性为0.95%。结论制备的痘苗病毒标准品可作为痘病毒载体疫苗滴度测定用标准品。
Objective To prepare a national standard for the determination of viral vector vaccine titers. Methods The vaccinia virus was prepared from chicken embryo fibroblasts. After purified by density gradient ultracentrifugation, smallpox vaccine protectant was added and stored in aliquots and stored in 4 different laboratories in our country. According to the experimental protocol of crystal violet staining Collaborative calibration, each dilution was taken after two dilutions, each dilution set five parallel holes, calculate the geometric mean of each laboratory test results, the standard vaccinia virus titer assignment. The stability of virus standard samples stored at different temperatures for different time was tested, and the quality indexes of virus standard samples were tested. Results Four laboratories were tested 44 times. There was no significant difference between the four laboratories (P> 0.05). The geometric mean of each laboratory was 3.05 × 106 PFU / ml and the 95% CI was 2.00 × 106 ~ 4.65 × 106PFU / ml, the standard deviation of 0.09, the total coefficient of variation of 1.41%. The prepared vaccinia virus standard can be stably stored at 4 ° C for 10 days, and the stability is poor when placed at 37 ° C. Prepared vaccinia virus standard sample amount of qualified inspection (1 ml / support); pH value of 6.9; sterile test pass; packing weight precision of 0.95%. Conclusions The prepared vaccinia virus standard can be used as a standard for the determination of the titer of the poxvirus vector vaccine.