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Optimization and evaluation of a live virus SARS-CoV-2 neutralization assay.
Frische, Anders; Brooks, Patrick Terrence; Gybel-Brask, Mikkel; Sækmose, Susanne Gjørup; Jensen, Bitten Aagaard; Mikkelsen, Susan; Bruun, Mie Topholm; Boding, Lasse; Strandh, Charlotta Polacek; Jørgensen, Charlotte Sværke; Krogfelt, Karen Angeliki; Fomsgaard, Anders; Lassauniere, Ria.
  • Frische A; Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark.
  • Brooks PT; Department of Clinical Immunology, Copenhagen University Hospital, Copenhagen, Denmark.
  • Gybel-Brask M; Department of Clinical Immunology, Copenhagen University Hospital, Copenhagen, Denmark.
  • Sækmose SG; Department of Clinical Immunology, Zealand University Hospital, Naestved, Denmark.
  • Jensen BA; Department of Clinical Immunology, Aalborg University Hospital, Aalborg, Denmark.
  • Mikkelsen S; Department of Clinical Immunology, Aarhus University Hospital, Aarhus, Denmark.
  • Bruun MT; Department of Clinical Immunology, Odense University Hospital, Odense, Denmark.
  • Boding L; Danish National Biobank, Statens Serum Institut, Copenhagen, Denmark.
  • Strandh CP; Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark.
  • Jørgensen CS; Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark.
  • Krogfelt KA; Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark.
  • Fomsgaard A; Department of Molecular and Medicinal Biology, Institute for Science and Environment, Roskilde University, Roskilde, Denmark.
  • Lassauniere R; Department of Virus & Microbiological Special Diagnostics, Statens Serum Institut, Copenhagen, Denmark.
PLoS One ; 17(7): e0272298, 2022.
Article in English | MEDLINE | ID: covidwho-1963049
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 (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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Experimental Studies / Prognostic study Topics: Vaccines / Variants Limits: Humans Language: English Journal: PLoS One Journal subject: Science / Medicine Year: 2022 Document Type: Article Affiliation country: Journal.pone.0272298

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Full text: Available Collection: International databases Database: MEDLINE Main subject: SARS-CoV-2 / COVID-19 Type of study: Experimental Studies / Prognostic study Topics: Vaccines / Variants Limits: Humans Language: English Journal: PLoS One Journal subject: Science / Medicine Year: 2022 Document Type: Article Affiliation country: Journal.pone.0272298