Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay

Anders Frische, Patrick Terrence Brooks, Mikkel Gybel-Brask, Susanne Gjørup Sækmose, Bitten Aagaard Jensen, Susan Mikkelsen, Mie Topholm Bruun, Lasse Boding, Charlotta Polacek Strandh, Charlotte Sværke Jørgensen, Karen Angeliki Krogfelt, Anders Fomsgaard, Ria Lassauniere*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

2 Downloads (Pure)

Abstract

Virus neutralization assays provide a means to quantitate functional antibody responses that block virus infection. These assays are instrumental in defining vaccine and therapeutic antibody potency, immune evasion by viral variants, and post-infection immunity. Here we describe the development, optimization and evaluation of a live virus microneutralization assay specific for severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2). In this assay, SARS-CoV-2 clinical isolates are pre-incubated with serial diluted antibody and added to Vero E6 cells. Replicating virus is quantitated by enzyme-linked immunosorbent assay (ELISA) targeting the SARS-CoV-2 nucleocapsid protein and the standardized 50% virus inhibition titer calculated. We evaluated critical test parameters that include virus titration, assay linearity, number of cells, viral dose, incubation period post-inoculation, and normalization methods. Virus titration at 96 hours was determined optimal to account for different growth kinetics of clinical isolates. Nucleocapsid protein levels directly correlated with virus inoculum, with the strongest correlation at 24 hours post-inoculation. Variance was minimized by infecting a cell monolayer, rather than a cell suspension. Neutralization titers modestly decreased with increasing numbers of Vero E6 cells and virus amount. Application of two different normalization models effectively reduced the intermediate precision coefficient of variance to <16.5%. The SARS-CoV-2 microneutralization assay described and evaluated here is based on the influenza virus microneutralization assay described by WHO, and are proposed as a standard assay for comparing neutralization investigations.

Original languageEnglish
Article numbere0272298
JournalPLOS ONE
Volume17
Issue number7
Number of pages22
ISSN1932-6203
DOIs
Publication statusPublished - 28. Jul 2022

Bibliographical note

Publisher Copyright:
© 2022 Frische et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Fingerprint

Dive into the research topics of 'Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay'. Together they form a unique fingerprint.

Cite this