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A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. In this work, we improve the hitherto existing neutralization assay system to assess SARS-CoV-2 inhibitors using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913246/ https://www.ncbi.nlm.nih.gov/pubmed/33540924 http://dx.doi.org/10.3390/pathogens10020153 |
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author | Pısıl, Yalçın Shida, Hisatoshi Miura, Tomoyuki |
author_facet | Pısıl, Yalçın Shida, Hisatoshi Miura, Tomoyuki |
author_sort | Pısıl, Yalçın |
collection | PubMed |
description | Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. In this work, we improve the hitherto existing neutralization assay system to assess SARS-CoV-2 inhibitors using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein (LpVspike +) and angiotensin-converting enzyme 2 (ACE2)-transfected cat Crandell–Rees feline kidney (CRFK) cells as the host cell line. Our method was 10-fold more sensitive compared to the typical human embryonic kidney 293T (HEK293T) cell system, and it was successfully applied to quantify the titers of convalescent antisera and monoclonal anti-spike antibodies required for pseudo virus neutralization. The 50% inhibition dilution (ID50) of two human convalescent sera, SARS-CoV-2 immunoglobulin G (IgG) and SARS-CoV-2 immunoglobulin M (IgM), which were 1:350 (±1:20) and 1:1250 (±1:350), respectively. The 50% inhibitory concentration (IC50) of the IgG, IgM and immunoglobulin A (IgA) anti-SARS-CoV-2 monoclonal antibodies (mAbs) against LpVspike(+) were 0.45 (±0.1), 0.002 (±0.001) and 0.004 (±0.001) µg mL(−1), respectively. We also found that reagents typically used to enhance infection were not effective in the CFRK system. This methodology is both efficient and safe; it can be employed by researchers to evaluate neutralizing monoclonal antibodies and contribute to the discovery of new antiviral inhibitors against SARS-CoV-2. |
format | Online Article Text |
id | pubmed-7913246 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-79132462021-02-28 A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells Pısıl, Yalçın Shida, Hisatoshi Miura, Tomoyuki Pathogens Article Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. In this work, we improve the hitherto existing neutralization assay system to assess SARS-CoV-2 inhibitors using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein (LpVspike +) and angiotensin-converting enzyme 2 (ACE2)-transfected cat Crandell–Rees feline kidney (CRFK) cells as the host cell line. Our method was 10-fold more sensitive compared to the typical human embryonic kidney 293T (HEK293T) cell system, and it was successfully applied to quantify the titers of convalescent antisera and monoclonal anti-spike antibodies required for pseudo virus neutralization. The 50% inhibition dilution (ID50) of two human convalescent sera, SARS-CoV-2 immunoglobulin G (IgG) and SARS-CoV-2 immunoglobulin M (IgM), which were 1:350 (±1:20) and 1:1250 (±1:350), respectively. The 50% inhibitory concentration (IC50) of the IgG, IgM and immunoglobulin A (IgA) anti-SARS-CoV-2 monoclonal antibodies (mAbs) against LpVspike(+) were 0.45 (±0.1), 0.002 (±0.001) and 0.004 (±0.001) µg mL(−1), respectively. We also found that reagents typically used to enhance infection were not effective in the CFRK system. This methodology is both efficient and safe; it can be employed by researchers to evaluate neutralizing monoclonal antibodies and contribute to the discovery of new antiviral inhibitors against SARS-CoV-2. MDPI 2021-02-02 /pmc/articles/PMC7913246/ /pubmed/33540924 http://dx.doi.org/10.3390/pathogens10020153 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Pısıl, Yalçın Shida, Hisatoshi Miura, Tomoyuki A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells |
title | A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells |
title_full | A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells |
title_fullStr | A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells |
title_full_unstemmed | A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells |
title_short | A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells |
title_sort | neutralization assay based on pseudo-typed lentivirus with sars cov-2 spike protein in ace2-expressing crfk cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913246/ https://www.ncbi.nlm.nih.gov/pubmed/33540924 http://dx.doi.org/10.3390/pathogens10020153 |
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