柯萨奇病毒A组16型感染性滴度检测用国家参考品的制备及标定

Establishment and calibration of national reference for infective titer detection of coxsackievirus A16

目的 制备柯萨奇病毒A组16型（coxsackievirus A16,CA16）感染性滴度检测用国家参考品,为CA16疫苗生产过程中的病毒滴度控制和CA16疫苗的免疫效果评价提供国家参考品.方法 将验证合格的CA16（C1亚型）培养液分装（0.5 ml/支）制成候选参考品.3个独立实验室分别对候选参考品进行协作标定,用细胞培养法检测CA16感染性滴度,用Behrens-K（a）rber法计算半数细胞感染量（50％ cell culture infective does,CCID50）.同时,将CA16参考品分别置于-20、4、22～25（室温）和37℃条件下或反复冻融,进行热稳定性、长期稳定性和冻存稳定性研究.结果 制备的CA16参考品的病毒滴度为（6.80±0.95） lgCCID50/ml.CA16参考品于-60℃保存12个月、-20℃保存6个月和4℃保存28 d后的病毒滴度无显著下降,表明稳定性较好;在22～25℃（室温）和37℃条件下,CA16参考品每天的病毒滴度损失率分别为3.4％和4.4％,而且分别保存27和21 d后病毒滴度无明显下降;对CA16参考品反复冻融的病毒滴度损失率为4.8％/次.结论 成功制备了CA16感染性滴度检测用候选国家参考品,将CA16参考品赋值确定为（6.80±0.95） lgCCID50/ml.

Objective To establish the national reference for detecting infective titer of coxsackievirus A16 （CA16）,in order to provide the national reference for viral titer control during CA16 vaccine production and immune effect evaluation of CA16 vaccine.Methods The qualified CA16 culture was divided into 0.5 ml aliquots as candidate reference.Candidate reference was collaboratively calibrated by 3 independent laboratories.The infective titer of CA16 was detected by cell culture method,and 50% cell culture infective does （CCID50） of CA16 was calculated by Behrens-K（a）rber method.Meanwhile,CA16 candidate reference was stored at-60 ℃ for 12 months,at-20 ℃ for 6 months,at 4 ℃ for 28 days,at 22-25 ℃ （room temperature） for 7 days and at 37 ℃ for 7 days or frozen and thawed repeatedly,and the stability under different circumstances was evaluated.Results The viral titer of CA16 candidate reference was （6.80 ± 0.95） lgCCID50/ml.The viral titers of CA16 candidate reference had no significant reduction after keeping in storage at-60 ℃ for 12 months,-20 ℃ for 6 months,and 4 ℃ for 28 days,indicating high stability at these conditions.The viral titer loss rate of CA16 candidate reference at 22-25 ℃ （room temperature） and 37 ℃ were 3.4% and 4.4% per day respectively,and respective titer loss after 27 and 21 d storage was not significant.The viral titer loss rate of CA16 candidate reference was 4.8% per time under repeated freeze-thaw condition.Conclusion CA16 candidate reference meets the requirement as a national reference,and its valuation was （6.80 ± 0.95） lgCCID50/ml.