目的:本文以对生物制品生物效价检测进行规范的《中国药典》2020年版新增9401指导原则为标准进行EV71抗原检测方法的验证,进一步浅析生物制品生物活性/效价检测的方法学验证.方法:依据《中国药典》2020年版9401指导原则,结合EV71抗原检测方法特点及验证目的对其进行专属性、相对准确度、精密度、线性和范围5个方面进行方法学验证.结果:EV71抗原检测方法专属性强,6个效价水平几何变异系数为4.7％-13.0％,均小于15％,相对偏倚为-6.05％～9.03％,均在±15％范围内,定量范围为225％～31％,线性回归相关系数为0.999 5,5方面验证均符合要求,且适用于该检测方法的使用.结论:本原则可具体指导生物学检测方法验证,在对不同检测方法进行验证时,根据检测目的、方法的原理、方法的技术特点、被检物的成分等设计具体方案.

目的 比较干燥保存及湿保存肠道病毒71型(enterovirus 71,EV71)抗原检测板的稳定性.方法 用羊抗EV71多克隆抗体包被96孔酶标板,制备20块EV71抗原检测板,10块板置空气干燥后真空包装,-25℃保存;另外10块板用洗涤液浸泡,封口后置4℃保存.分别于保存0、20、40、60、90、120、180d时,检测5份EV71样品.结果 干燥保存EV71抗原检测板检测5份样品7个时间点的均值分别为315.0、299.4、7 795.3、827.6、261.4 U/mL,湿保存的EV71抗原检测板检测结果均值分别为354.3、347.1、6 766.6、749.7、277.3 U/mL,各时间点两种方法检测结果差异无统计学意义(P＞0.05).两种方法保存180 d时,EV71抗原检测板均可保持较好的活性(P＞0.05).结论 两种方法保存EV71抗原检测板6个月内,均具有良好的稳定性.

Objective:To study the effect of different immune doses in rabbit anti-enterovirus 71 (EV71) serum preparation on the antibody titer.Methods:Three doses of 5, 50 and 100 μg were selected in this experiment. There were 3 groups of 3 rabbits in each group.After the mixture of EV71 inactivated vaccine and Freund’s adjuvant, the rabbits in the three groups were immunized by intramuscular injection. The rabbits were immunized once at 0, 7, 14 and 28 d, respectively. Blood samples were collected every 7 d, serum was separated and neutralizing antibody titer was detected.Results:The antibody titer of 100 μg was significantly higher than that of 5 μg ( t=-3.282) and 50 μg ( t=-2.486), on day 42 ( P<0.05). There was a linear relationship between the titer of rabbit serum antibody and the immune dose (within the range of 5-100 μg). The linear model was established and the regression equation y=478.229 x-2 041.820 was obtained. Conclusion:Among the 3 immune doses selected in this experiment, the titer of serum antibody increases with the increase of immune dose, and shows a linear relationship.