应用中国仓鼠卵巢细胞法检测百日咳毒素毒性方法的建立及初步验证

Establishment and primary validation of Chinese hamster ovary cell-based assay for determination of residual toxicity of pertussis toxin

目的 建立用中国仓鼠卵巢（Chinese hamster ovary,CHO）细胞检测百日咳毒素（pertussis toxin,PT）毒性的方法,并对其适用性进行初步验证.方法 根据PT能特异地使CHO细胞簇集的特性,以国家PT标准品为参考品,取多批脱毒后PT进行检测,摸索适宜的孵育时间、细胞悬液浓度等实验条件,建立PT毒性的体外检测方法.对PT参考品进行倍比系列稀释,确定本法的检测限.对5批脱毒后PT进行重复检测,验证本法的重复性.结果 利用不同浓度CHO细胞对脱毒PT进行测定,确定最佳细胞浓度为5×104/ml,并通过最佳浓度细胞确定簇集结果的最佳判定时间为PT接种后48 h.本法对PT标准品的检测限定为125.0～ 500.0 pg/ml.用本法重复测定5批脱毒PT的残余毒性,变异系数为0.0％～34.6％,说明该法重复性良好.结论 建立的CHO细胞检测法快捷、简便,能灵敏、准确地检测脱毒PT的残余毒性,可用于相关疫苗产品的质量控制.

Objective To establish Chinese hamster ovary cell clustering method for determination of residual toxicity of pertussis toxin （PT） and validate its applicability.Methods The PT national standard was used as a reference and several lots of detoxified PTs as samples.The optimal incubation time and cell concentration were explored to establish the method for determination of PT toxicity in vitro.Meanwhile,the detection limit was determined by doubling dilution of the PT reference.The repeatability of the method was validated by repeat detection of 5 lots of detoxified PTs.Results The optimal CHO cell concentration and incubation time were 5 × 104/ml and 48 h after inoculation of PT,respectively.The detection limit ranged from 125.0 to 500.0 pg/ml.The coefficient of variation was 0.0％-34.6％,suggesting that the method had a good repeatability.Conclusion The method developed is rapid,sensitive and reliable,and could be used for quality control of residual toxicity in vaccine products.