Development and validation of a respiratory syncytial virus neutralizing antibody titer detection method based on pseudoviruses
10.13200/j.cnki.cjb.004683
- VernacularTitle:呼吸道合胞病毒中和抗体效价假病毒检测方法的建立及验证
- Author:
Yu LIU
1
Author Information
1. Beijing Yunling Bio⁃Technology Co., Ltd., Beijing 100023, China
- Publication Type:Journal Article
- Keywords:
Respiratory syncytial virus(RSV);
Neutralizing antibody titer;
Pseudovirus
- From:
Chinese Journal of Biologicals
2026;39(04):468-474
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish and verify a pseudovirus detection method for respiratory syncytial virus(RSV) neutralizing antibodies, in order to provide a rapid, high-throughput and safe method for RSV vaccine development and antiviral drug screening.MethodsThe RSV A2 pseudovirus was prepared, and the pseudovirus detection method for RSV neutralizing antibody was established by optimizing the pseudovirus concentration factor(0, 2, 4, 8 and 16 times), infected cell type(HEK293T, Hep-2, Huh7.5.1, HeLa and Vero cells), cell inoculation density(0. 5 × 10~4, 1. 0 × 10~4, 1. 5 × 10~4, 2. 0 × 10~4 and2. 5 × 10~4 cells/well), cell generation(1, 5, 10, 15, 20 and 25 generations), detection time(6, 12, 24, 36, 48, 60 and 72 h) and virus inoculation amount(5. 0 × 10~3, 2. 5 × 10~3, 1. 25 × 10~3, 6. 25 × 10~2, 3. 125 × 10~2, 1. 562 5 × 10~2 TCID50/mL). In addition, the precision, specificity, accuracy and edge effects of the method were verified. Finally, the established method and plaque-reduction neutralization test(PRNT) were used to detect 30 mouse positive sera simultaneously, and the correlation between the detection results was analyzed.ResultsThe optimal pseudovirus concentration factor was 8, the infected cells were HEK293T cells of 1-10 passages after resuscitation with the inoculation density of 2. 0 × 10~4 cells/well, the detection duration was 48 h, and the virus inoculation amount was 3. 125 × 10~2-2. 5 × 10~3 TCID50/mL. The coefficients of variation(CVs) of repeatability and intermediate precision verification were both less than 15%. No RSV neutralizing activity was detected in normal mouse serum, and high RSV neutralizing activity was detected in positive mouse serum and Nirsevimab monoclonal antibody. The recovery rates of accuracy verification were within the range of 80% to 120%. The ratios of the detection values of edge holes to non-edge holes were 0. 5-0. 7, indicating that there was edge hole effects, and non-edge holes should be used for detection. The results of 30 mouse positive sera detected by the two methods showed a good positive correlation(r = 0. 914 5, P < 0. 000 1).ConclusionThe established RSV neutralizing antibody titer pseudovirus detection method has good precision, specificity, accuracy, with simple and fast operation, which can be used for the evaluation of RSV vaccine immunogenicity and the high-throughput detection of serum neutralizing antibody potency.
