基于补体依赖的水痘-带状疱疹病毒中和抗体效价检测方法的建立及应用

Development and application of a detection method for varicella-zoster virus neutralizing antibody titer based on complement dependence

目的 建立基于补体依赖的水痘-带状疱疹病毒(varicella-zoster virus,VZV)中和抗体效价检测方法并进行应用,以提高传统噬斑减少中和试验检测VZV抗体效价的灵敏度。方法 将抗原(水痘减毒活疫苗)、抗体(人VZV免疫球蛋白)以不同比例、不同时间混合孵育,中和后接种于6孔板培养的人二倍体细胞2BS株培养7~10 d,考马斯亮蓝染色计数噬斑数,通过Karber公式计算50%中和抗体滴度。在最佳中和条件下,通过改变补体(冻干豚鼠血清)添加量来探讨补体对中和试验灵敏度的影响,评估补体的最佳添加剂量。根据确定的中和试验参数,分别采用传统蚀斑法和补体依赖的蚀斑法对12份抗VZV小鼠血清和14份抗VZV人血清进行中和抗体效价检测。结果 确定了检测方法的关键参数:VZV标准抗原使用滴度为500~1 000 PFU/mL,补体在抗原抗体中和体系中添加的比例为1∶10(v/v),中和条件为37℃中和1 h。补体依赖的蚀斑法和传统蚀斑法检测抗VZV小鼠血清抗体均为阳性,但传统蚀斑法检测抗体效价普遍偏低,接种2剂水痘减毒活疫苗的小鼠抗体水平显著高于接种1剂水痘减毒活疫苗的小鼠(t=0.45,P <0.05);两种方法检测抗VZV人血清抗体均为阳性。结论 建立了补体依赖的VZV中和抗体效价检测方法,补体的添加显著提高了中和抗体检测的灵敏度。用该方法评价不同免疫策略的小鼠血清的中和抗体效价,得出2剂疫苗的免疫效果优于1剂。

Objective To develop and apply a method for detecting the titer of varicella-zoster virus(VZV)neutralizing antibodies based on complement dependence,so as to improve the sensitivity of traditional plaque reduction neutralization assay for detection of the titer of VZV antibody.MethodsThe antigen(live attenuated varicella vaccine)and antibody(human VZV immunoglobulin)were mixed in different proportions and different incubation times.After neutralization,the antigen-antibody mixture was inoculated into human diploid cell 2BS strain cultured in a six-well plate.After 7 ~ 10 d of culture,the number of plaques was counted by Coomassie brilliant blue staining,and the 50% neutralizing antibody titer was calculated by Karber′s formula.Under the optimal neutralization conditions obtained,the effect of complement on the sensitivity of neutralization experiment was explored by changing the addition amount of complement(lyophilized guinea pig serum)to evaluate the optimal addition amount of complement.According to the determined neutralization test parameters,the neutralizing antibody titers of 12 anti-VZV mouse sera and 14 anti-VZV human sera were detected by using traditional plaque method and complement-dependent plaque method respectively.ResultsThe key parameters of the detection method were determined:the titer of VZV standard antigen was 500 ~ 1 000 PFU/mL;the proportion of complement added to the antigen-antibody neutralization system was 1∶10(v/v),and the neutralization condition was 37 ℃ for 1 h.Both the complement-dependent plaque method and the traditional plaque method were positive for anti-VZV mouse serum antibody,while the antibody titer detected by the traditional plaque method was generally lower,and the antibody level of mice inoculated with 2 doses of live attenuated varicella vaccine was significantly higher than that of mice inoculated with 1 dose(t = 0.45,P<0.05);Both of the two methods were positive for anti-VZV human serum antibody.ConclusionA complement-dependent detection method for neutralizing antibody titer of VZV was established.The addition of complement significantly improved the sensitivity of neutralization detection.The evaluation of the titers of neutralizing antibodies in mouse serum with different immunization strategies by the method suggested that the immune effect of two doses of vaccine was better than that of one dose.