检测白喉毒素特异性细胞毒性的Vero细胞/MTT法的建立

摘要

目的 建立检测白喉毒素(diphtheria toxin,DT)特异性细胞毒性的Vero细胞/MTT法.方法 用MEM培养液对DT进行不同倍数的预稀释,用目测法确定DT的最适预稀释倍数.用建立的方法检测以最适预稀释倍数稀释的DT,以确定该法的灵敏度和细胞病变半数抑制浓度(IC50),同时绘制剂量-反应曲线以验证该法的重复性.用建立的方法于不同pH、盐浓度或蔗糖浓度条件下检测DT,计算DT回收率以验证该法的耐用性.结果 目测法确定的DT最适预稀释倍数为106倍.建立的方法的平均检测灵敏度为(6.01±1.44)×10-5 lf/ml DT,细胞病变半数抑制率对数值(log IC50)范围为5.02 ～ 5.82,变异系数为5.82％,该法具有较好的重复性.用建立的方法检测不同条件下的DT显示,DT的平均回收率为93.7％ ～ 110.4％,该法具有较好的耐用性.结论 Vero细胞/MTr法可用于检测DT特异性细胞毒性.%Objective To establish Vero cells / MTT method for determination of specific cytotoxicity of diphtheria toxin (DT).Methods DT was diluted multiply with MEM medium.The optimal pre-dilution multiple of DT was detected by microscopic observation.DT diluted in definitive time was detected by the established method to determine the sensitivity and half cytopathic inhibition concentration (IC50) of the method.The dosage-effect curves were drawn to validate repeatability of the method.DTs under different pH,salt concentration or sucrose concentration conditions were detected by the established method,and recovery rates of DT were calculated to validate durability of the method.Results The optimal pre-dilution multiple of DT detected by microscopic observation was 106 time.The sensitivity of the established method was (6.01 ± 1.44) × 10-5 lf/ml DT,logIC50 ranged from 5.02 to 5.82,and its variable coefficient was 5.82％.The average recovery rates of DT under different conditions were 93.7％-110.4％.Conclusion Vero cells/MTT method has good sensitivity,repeatability and durability,and can be applied to determination of the specific cytotoxicity of DT.