An optimized high-throughput fluorescence plate reader-based RSV neutralization assay

Sun, Yongpeng; Zhang, Wei; Zhao, Qinjian; Cao, Jianli; Zhang, Lu-Jing; Xiang, Jiang-Yan; Si, Jun-Yu; Lin, Xue; Chen, Li; Zheng, Zizheng; Xia, Ningshao

doi:10.1016/j.jviromet.2018.07.004

Cited by 2 publications

(1 citation statement)

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“…Lung homogenates were made using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) on program Lung 02. Pulmonary virus titration was determined via plaque assay, and replication of the RSV A2 genome RNA was detected by measuring the target RSV A2 N gene via qPCR on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) as previously described [15,16]. The left lungs were kept in 10% formaldehyde and paraffin-embedded.…”

Section: Methodsmentioning

confidence: 99%

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein

Zhang

Zhan

et al. 2019

Viruses

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Background: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. Methods: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. Results: Fixation in an optimal concentration of formaldehyde (0.0244%–0.0977%) or paraformaldehyde (0.0625%–1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. Conclusions: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

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Section: Methodsmentioning

confidence: 99%