金黄色葡萄球菌α-毒素溶血活性测定方法优化及初步应用

Optimization and preliminary application of a method for determination of hemolytic activity of Staphylococcus aureus α-toxin

目的优化金黄色葡萄球菌α-毒素(α-toxin,AT)溶血活性测定方法,用于无毒突变AT(non-toxic mutant AT,mAT)特异性血清抗体体外功能性评价和中和抗体滴度测定。方法使用AT裂解兔红细胞,释放血红蛋白,通过分光光度法检测溶血活性,并对该方法中的孵育时间、兔红细胞终浓度、Triton X-100浓度进行优化,同时验证方法重复性。对mAT特异性血清抗体进行体外功能性评价,绘制AT溶血曲线,计算AT溶血率50%时的浓度,检测血清半数中和抗体滴度。结果AT溶血活性最适检测条件为:孵育时间90 min,兔红细胞终浓度为2%(V/V),Triton X-100浓度为0.25%。此条件下,分别加入mAT和含mAT的多价抗原血清抗体,对AT溶血活性均起到明显的抑制作用。AT溶血率50%时浓度为1.75μg/ml,变异系数3.75%。mAT和含mAT的多价抗原血清抗体的半数中和抗体滴度初步结果均为1∶64。结论优化的AT溶血活性检测方法重复性良好,且检测时间较短,可用于特异性血清抗体体外功能性评价和中和抗体滴度测定。

Objective To optimize the determination method of Staphylococcus aureusα-toxin(AT)hemolytic activity for in vitro functional evaluation and neutralizing antibody titer determination of non-toxic mutant AT(mAT)specific serum antibody.Methods Rabbit red blood cells were lysed with AT to release hemoglobin.The hemolysis activity was detected by spectrophotometry.The incubation time,rabbit red blood cell final concentration,and Triton X-100 concentration of the method were optimized,and the repeatability of the method was verified.In vitro functional evaluation of mAT-specific serum antibody was carried out,AT hemolysis curve was drawn,the concentration of AT with 50%hemolysis rate was calculated,and the serum 50%neutralization titer(NT50)was detected.Results The optimal conditions for detection of AT hemolytic activity were incubation time 90 min,rabbit red blood cells final concentration 2%(V/V),and Triton X-100 concentration 0.25%.Under these conditions,respectively adding serum antibody of mAT and multi-antigen containing mAT both significantly inhibited the hemolytic activity of AT.When the hemolysis rate was 50%,the AT concentration was 1.75μg/ml and coefficient of varriation was 3.75%.The preliminary results of NT50 of mAT and multi-antigen serum antibody containing mAT were both 1∶64.Conclusion The optimized AT hemolytic activity assay has good repeatability and short detection time,which can be used for functional evaluation of specific serum antibodies and titer determination of neutralizing antibodies in vitro.