人呼吸道合胞病毒中和抗体检测方法的建立与初步应用

Establishment and preliminary application of neutralizing antibody detection method for human respiratory syncytial virus

目的建立用于人呼吸道合胞病毒(HRSV)中和抗体滴度检测的噬斑减少中和实验(PRNT),并进行条件优化与初步应用。方法应用CHO表达系统制备帕利珠单克隆抗体(Palivizumab),同时对细胞种类、细胞培养时间、固定通透方法、封闭方法等影响因素进行优化。验证该方法的重复性,并与传统PRNT验证其相关性。利用优化的PRNT方法检测BALB/c小鼠血清(融合蛋白肌肉注射免疫)对HRSV A和B亚型的中和抗体滴度。结果Palivizumab的表达量约为50 mg/L。PRNT的最佳工作条件为:培养细胞为HEp-2细胞,细胞培养时间为2 d,最佳固定通透方式为4%(V/V)多聚甲醛室温固定15 min后0.2%(V/V)Triton X-100通透15 min,去掉封闭步骤。优化后该方法的重复性验证总体变异系数均<15%,与传统PRNT呈现良好的线性关系,Spearman相关系数rs高达0.983。在检测小鼠血清对HRSV A亚型long株和B亚型9320株的中和抗体滴度时,融合蛋白联合AlOH和CpG佐剂诱导小鼠产生的中和抗体滴度最高。结论本研究建立的HRSV中和抗体检测方法快速、可重复、高通量,能够适用于A、B亚型HRSV的中和抗体检测。

Objective To establish a Plaque-reduction Neutralization Test(PRNT)for the detection of neutralizing antibody titers of Human Respiratory Syncytial Virus(HRSV)and optimize the conditions for preliminary application.Methods The CHO expression system was used to produce palivizumab monoclonal antibody(palivizumab)and the influencing factors such as cell type,cell culture duration,fixation and permeabilization protocols,and blocking agents.The reproducibility of the method was verified and its correlation was verified with conventional PRNT.Finally,the optimized PRNT assay was further used to determine neutralizing antibody titers against HRSV subtypes A and B in BALB/c mouse serum(immunized by intramuscular injection of HRSV fusion proteins).Results Palivizumab was expressed at approximately 50 mg/L.The optimal working conditions for PRNT were as follows:culturing HEp-2 cells for 2 days,fixing with 4%(V/V)paraformaldehyde at room temperature for 15 min followed by 0.2%(V/V)Triton X-100 permeabilization for 15 minutes as the optimal fixation-permeabilization and removing the blocking step.The overall coefficient of variation(CV)for the reproducibility validation of this method was<15%,showing a good linear relationship with the conventional PRNT.The Spearman correlation coefficient rs was 0.983.This method was used to detect neutralizing antibody titers in mouse sera against HRSV subtype A strain long and subtype B strain 9320,and the fusion proteins combined with AlOH and CpG adjuvant induced the highest neutralizing antibody titers in mice.Conclusion The HRSV neutralizing antibody assay established in this study is rapid,reproducible,high-throughput,and can be used to detect neutralizing antibodies to HRSV subtypes A and B.