{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T19:40:59Z","timestamp":1778010059050,"version":"3.51.4"},"reference-count":37,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T00:00:00Z","timestamp":1658966400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008389","name":"Uddannelses- og Forskningsministeriet","doi-asserted-by":"publisher","award":["0237-00006B"],"award-info":[{"award-number":["0237-00006B"]}],"id":[{"id":"10.13039\/100008389","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>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 &lt;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.<\/jats:p>","DOI":"10.1371\/journal.pone.0272298","type":"journal-article","created":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T18:07:42Z","timestamp":1659031662000},"page":"e0272298","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":24,"title":["Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2841-7655","authenticated-orcid":true,"given":"Anders","family":"Frische","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick Terrence","family":"Brooks","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5716-5899","authenticated-orcid":true,"given":"Mikkel","family":"Gybel-Brask","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1346-0052","authenticated-orcid":true,"given":"Susanne Gj\u00f8rup","family":"S\u00e6kmose","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bitten Aagaard","family":"Jensen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Susan","family":"Mikkelsen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mie Topholm","family":"Bruun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lasse","family":"Boding","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Charlotta Polacek","family":"Strandh","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Charlotte Sv\u00e6rke","family":"J\u00f8rgensen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karen Angeliki","family":"Krogfelt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anders","family":"Fomsgaard","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3910-7185","authenticated-orcid":true,"given":"Ria","family":"Lassauniere","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2022,7,28]]},"reference":[{"issue":"6561","key":"pone.0272298.ref001","doi-asserted-by":"crossref","DOI":"10.1126\/science.abj0299","article-title":"Immune correlates of protection by mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates","volume":"373","author":"KS Corbett","year":"2021","journal-title":"Science"},{"issue":"1","key":"pone.0272298.ref002","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-020-17892-0","article-title":"A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation","volume":"11","author":"AE Muruato","year":"2020","journal-title":"Nat Commun"},{"issue":"10","key":"pone.0272298.ref003","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1371\/journal.pone.0075677","article-title":"High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses","volume":"8","author":"P Sehr","year":"2013","journal-title":"PLoS One"},{"issue":"19","key":"pone.0272298.ref004","doi-asserted-by":"crossref","first-page":"12148","DOI":"10.1128\/JVI.79.19.12148-12163.2005","article-title":"Comparing Antigenicity and Immunogenicity of Engineered gp120","volume":"79","author":"S Selvarajah","year":"2005","journal-title":"J Virol"},{"issue":"6","key":"pone.0272298.ref005","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1111\/irv.12333","article-title":"Optimisation of a micro-neutralisation assay and its application in antigenic characterisation of influenza viruses","volume":"9","author":"Y Lin","year":"2015","journal-title":"Influenza Other Respi Viruses"},{"issue":"1","key":"pone.0272298.ref006","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/S0042-6822(03)00192-2","article-title":"Genetic and antigenic characterization of novel pestivirus genotypes: Implications for classification","volume":"311","author":"P Becher","year":"2003","journal-title":"Virology"},{"issue":"1","key":"pone.0272298.ref007","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.vaccine.2016.11.060","article-title":"ViroSpot microneutralization assay for antigenic characterization of human influenza viruses","volume":"35","author":"CA van Baalen","year":"2017","journal-title":"Vaccine"},{"issue":"1","key":"pone.0272298.ref008","doi-asserted-by":"crossref","first-page":"2011","DOI":"10.1038\/s41598-019-39276-1","article-title":"Insights into the antigenic advancement of influenza A(H3N2) viruses, 2011\u20132018","volume":"9","author":"PA Jorquera","year":"2019","journal-title":"Sci Rep"},{"issue":"6555","key":"pone.0272298.ref009","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1126\/science.abi7994","article-title":"SARS-CoV-2 immune evasion by the B.1.427\/B.1.429 variant of concern","volume":"373","author":"M McCallum","year":"2021","journal-title":"Science"},{"issue":"5956","key":"pone.0272298.ref010","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1126\/science.1175868","article-title":"Structural Basis of Immune Evasion at the Site of CD4 Attachment on HIV-1 gp120","volume":"326","author":"L Chen","year":"2009","journal-title":"Science"},{"issue":"1\u20132","key":"pone.0272298.ref011","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/j.jim.2011.08.018","article-title":"Development and validation of a standardized ELISA for the detection of soluble Fc-epsilon-RI in human serum","volume":"373","author":"W Lexmond","year":"2011","journal-title":"J Immunol Methods"},{"issue":"2","key":"pone.0272298.ref012","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1007\/s11095-005-9045-3","article-title":"Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement","volume":"23","author":"JW Lee","year":"2006","journal-title":"Pharm Res"},{"key":"pone.0272298.ref013","doi-asserted-by":"crossref","first-page":"1259","DOI":"10.1016\/j.jchromb.2008.11.022","article-title":"Method validation of protein biomarkers in support of drug development or clinical diagnosis\/prognosis","volume":"877","author":"JW Lee","year":"2009","journal-title":"J Chromatogr B Analyt Technol Biomed Life Sci"},{"issue":"2","key":"pone.0272298.ref014","doi-asserted-by":"crossref","first-page":"E156","DOI":"10.1208\/aapsj0902017","article-title":"Key elements of bioanalytical method validation for macromolecules","volume":"9","author":"M Kelley","year":"2007","journal-title":"AAPS J"},{"issue":"6","key":"pone.0272298.ref015","doi-asserted-by":"crossref","first-page":"1430","DOI":"10.4269\/ajtmh.17-0948","article-title":"Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue","volume":"99","author":"MC Whiteman","year":"2018","journal-title":"Am J Trop Med Hyg"},{"issue":"1","key":"pone.0272298.ref016","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/j.biologicals.2003.09.003","article-title":"Validation of the anthrax lethal toxin neutralization assay","volume":"32","author":"D Hering","year":"2004","journal-title":"Biologicals"},{"issue":"5","key":"pone.0272298.ref017","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1208\/s12248-020-00491-8","article-title":"Fit-for-Purpose Validation and Establishment of Assay Acceptance and Reporting Criteria of Dendritic Cell Activation Assay Contributing to the Assessment of Immunogenicity Risk","volume":"22","author":"D Wickramarachchi","year":"2020","journal-title":"AAPS J"},{"issue":"3","key":"pone.0272298.ref018","doi-asserted-by":"crossref","first-page":"790","DOI":"10.1016\/j.vaccine.2009.10.066","article-title":"A practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses","volume":"28","author":"A Hassantoufighi","year":"2010","journal-title":"Vaccine"},{"issue":"3","key":"pone.0272298.ref019","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1093\/oxfordjournals.aje.a118408","article-title":"A simple method of estimating fifty per cent endpoints","volume":"27","author":"LJ Reed","year":"1938","journal-title":"Am J Epidemiol"},{"issue":"6","key":"pone.0272298.ref020","first-page":"179","article-title":"A practical guide to immunoassay method validation","volume":"19","author":"U Andreasson","year":"2015","journal-title":"Front Neurol"},{"issue":"5","key":"pone.0272298.ref021","doi-asserted-by":"crossref","first-page":"e02596","DOI":"10.1128\/JCM.02596-20","article-title":"Comparison of 16 serological SARS-CoV-2 immunoassays in 16 clinical laboratories","volume":"59","author":"LH Harritsh\u00f8j","year":"2021","journal-title":"J Clin Microbiol"},{"issue":"2","key":"pone.0272298.ref022","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1080\/02786826.2020.1829536","article-title":"The influence of temperature, humidity, and simulated sunlight on the infectivity of SARS-CoV-2 in aerosols","volume":"55","author":"P Dabisch","year":"2021","journal-title":"Aerosol Sci Technol"},{"issue":"5","key":"pone.0272298.ref023","doi-asserted-by":"crossref","first-page":"962","DOI":"10.4269\/ajtmh.12-0461","article-title":"Optimization and validation of a plaque reduction neutralization test for the detection of neutralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development","volume":"88","author":"TM Timiryasova","year":"2013","journal-title":"Am J Trop Med Hyg"},{"key":"pone.0272298.ref024","doi-asserted-by":"crossref","first-page":"112626","DOI":"10.1016\/j.jim.2019.06.017","article-title":"Rapid fluorescent focus inhibition test optimization and validation: Improved detection of neutralizing antibodies to rabies virus","volume":"474","author":"TM Timiryasova","year":"2019","journal-title":"J Immunol Methods"},{"issue":"27","key":"pone.0272298.ref025","doi-asserted-by":"crossref","DOI":"10.2807\/1560-7917.ES.2021.26.27.2100568","article-title":"SARS-CoV-2 neutralising antibody testing in Europe: Towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data","volume":"27","author":"D Nguyen","year":"2021","journal-title":"Eurosurveillance"},{"key":"pone.0272298.ref026","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/j.jim.2014.02.013","article-title":"Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells","volume":"409","author":"M Sarzotti-Kelsoe","year":"2014","journal-title":"J Immunol Methods"},{"issue":"10","key":"pone.0272298.ref027","doi-asserted-by":"crossref","first-page":"2096","DOI":"10.1002\/jmv.25986","article-title":"Evaluation of SARS-CoV-2 neutralizing antibodies using a CPE-based colorimetric live virus micro-neutralization assay in human serum samples","volume":"92","author":"A Manenti","year":"2020","journal-title":"J Med Virol"},{"issue":"3","key":"pone.0272298.ref028","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1111\/tme.12746","article-title":"Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels","volume":"31","author":"H Harvala","year":"2021","journal-title":"Transfus Med"},{"issue":"2","key":"pone.0272298.ref029","doi-asserted-by":"crossref","first-page":"e2170","DOI":"10.1002\/rmv.2170","article-title":"Viral infection neutralization tests: A focus on severe acute respiratory syndrome-coronavirus-2 with implications for convalescent plasma therapy","volume":"31","author":"D Focosi","year":"2021","journal-title":"Rev Med Virol"},{"issue":"1","key":"pone.0272298.ref030","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1002\/jmv.27287","article-title":"Correlation between a quantitative anti-SARS-CoV-2 IgG ELISA and neutralization activity","volume":"94","author":"R Dolscheid-Pommerich","year":"2022","journal-title":"J Med Virol"},{"issue":"12","key":"pone.0272298.ref031","doi-asserted-by":"crossref","first-page":"6696","DOI":"10.1002\/jmv.27251","article-title":"Performance assessment of seven SARS-CoV-2 IgG enzyme-linked immunosorbent assays","volume":"93","author":"K Deshpande","year":"2021","journal-title":"J Med Virol"},{"issue":"6561","key":"pone.0272298.ref032","doi-asserted-by":"crossref","first-page":"1372","DOI":"10.1126\/science.abj4176","article-title":"Durability of mRNA-1273 vaccine-induced antibodies against SARS-CoV-2 variants","volume":"373","author":"A Pegu","year":"2021","journal-title":"Science"},{"issue":"3","key":"pone.0272298.ref033","doi-asserted-by":"crossref","first-page":"e0017021","DOI":"10.1128\/mSphere.00170-21","article-title":"Throughput Clinical Assay for Measuring SARS-CoV-2- Neutralizing Antibody Titers","volume":"6","author":"R Vandergaast","year":"2021","journal-title":"mSphere"},{"key":"pone.0272298.ref034","doi-asserted-by":"crossref","first-page":"114031","DOI":"10.1016\/j.jviromet.2020.114031","article-title":"Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma","volume":"288","author":"C von Rhein","year":"2021","journal-title":"J Virol Methods"},{"issue":"1","key":"pone.0272298.ref035","doi-asserted-by":"crossref","first-page":"13","DOI":"10.3390\/vaccines9010013","article-title":"Comparison of Four SARS-CoV-2 Neutralization Assays","volume":"9","author":"L Riepler","year":"2020","journal-title":"Vaccines"},{"issue":"June","key":"pone.0272298.ref036","first-page":"1","article-title":"In vitro Characterization of Fitness and Convalescent Antibody Neutralization of SARS-CoV-2 Cluster 5 Variant Emerging in Mink at Danish Farms","volume":"12","author":"R Lassauni\u00e8re","year":"2021","journal-title":"Front Microbiol"},{"issue":"5","key":"pone.0272298.ref037","doi-asserted-by":"crossref","first-page":"E2212073","DOI":"10.1001\/jamanetworkopen.2022.12073","article-title":"Neutralizing Antibodies Against the SARS-CoV-2 Omicron Variant (BA.1) 1 to 18 Weeks after the Second and Third Doses of the BNT162b2 mRNA Vaccine","volume":"5","author":"R Lassauni\u00e8re","year":"2022","journal-title":"JAMA Netw Open"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0272298","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,7,28]],"date-time":"2022-07-28T18:08:36Z","timestamp":1659031716000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0272298"}},"subtitle":[],"editor":[{"given":"Etsuro","family":"Ito","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2022,7,28]]},"references-count":37,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2022,7,28]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0272298","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,7,28]]}}}