Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay.
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, opt...
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2022-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0272298 |
| _version_ | 1811214685648715776 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Anders Frische |
| collection | DOAJ |
| description | 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 (SARS-CoV-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. |
| first_indexed | 2024-04-12T06:08:17Z |
| format | Article |
| id | doaj.art-710168d165e6428c9e88698f5035b509 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T06:08:17Z |
| publishDate | 2022-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-710168d165e6428c9e88698f5035b5092022-12-22T03:44:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032022-01-01177e027229810.1371/journal.pone.0272298Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay.Anders FrischePatrick Terrence BrooksMikkel Gybel-BraskSusanne Gjørup SækmoseBitten Aagaard JensenSusan MikkelsenMie Topholm BruunLasse BodingCharlotta Polacek StrandhCharlotte Sværke JørgensenKaren Angeliki KrogfeltAnders FomsgaardRia LassauniereVirus 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 (SARS-CoV-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.https://doi.org/10.1371/journal.pone.0272298 |
| spellingShingle | 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 Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay. PLoS ONE |
| title | Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay. |
| title_full | Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay. |
| title_fullStr | Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay. |
| title_full_unstemmed | Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay. |
| title_short | Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay. |
| title_sort | optimization and evaluation of a live virus sars cov 2 neutralization assay |
| url | https://doi.org/10.1371/journal.pone.0272298 |
| work_keys_str_mv | AT andersfrische optimizationandevaluationofalivevirussarscov2neutralizationassay AT patrickterrencebrooks optimizationandevaluationofalivevirussarscov2neutralizationassay AT mikkelgybelbrask optimizationandevaluationofalivevirussarscov2neutralizationassay AT susannegjørupsækmose optimizationandevaluationofalivevirussarscov2neutralizationassay AT bittenaagaardjensen optimizationandevaluationofalivevirussarscov2neutralizationassay AT susanmikkelsen optimizationandevaluationofalivevirussarscov2neutralizationassay AT mietopholmbruun optimizationandevaluationofalivevirussarscov2neutralizationassay AT lasseboding optimizationandevaluationofalivevirussarscov2neutralizationassay AT charlottapolacekstrandh optimizationandevaluationofalivevirussarscov2neutralizationassay AT charlottesværkejørgensen optimizationandevaluationofalivevirussarscov2neutralizationassay AT karenangelikikrogfelt optimizationandevaluationofalivevirussarscov2neutralizationassay AT andersfomsgaard optimizationandevaluationofalivevirussarscov2neutralizationassay AT rialassauniere optimizationandevaluationofalivevirussarscov2neutralizationassay |