Establishment and preliminary application of neutralizing antibody detection method for human respiratory syncytial virus
10.3760/cma.j.cn112150-20240102-00004
- VernacularTitle:人呼吸道合胞病毒中和抗体检测方法的建立与初步应用
- Author:
Li ZHANG
1
;
Hai LI
;
Lei CAO
;
Hongqiao HU
;
Na WANG
;
Haixin LI
;
Jie JIANG
;
Naiying MAO
;
Xiaomei LI
;
Yan ZHANG
Author Information
1. 山东第一医科大学（山东省医学科学院）公共卫生与健康管理学院，济南 250117
- Keywords:
Human respiratory syncytial virus;
Immunostaining;
Plaque reduction neutralization test;
Neutralizing antibody
- From:
Chinese Journal of Preventive Medicine
2024;58(7):959-966
- CountryChina
- Language:Chinese
-
Abstract:
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 r s 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.
