Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved..
The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:298 |
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| Enthalten in: |
The Journal of biological chemistry - 298(2022), 1 vom: 01. Jan., Seite 101290 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Gransagne, Marion [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 07.02.2022 Date Revised 07.02.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jbc.2021.101290 |
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| Förderinstitution / Projekttitel: |
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| 100 | 1 | |a Gransagne, Marion |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein |
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| 500 | |a Date Revised 07.02.2022 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a COVID-19 | |
| 650 | 4 | |a antibody engineering | |
| 650 | 4 | |a diagnostic | |
| 650 | 4 | |a hydrogen-deuterium exchange | |
| 650 | 4 | |a immunochemistry | |
| 650 | 4 | |a nanobodies | |
| 650 | 4 | |a nucleoprotein | |
| 650 | 4 | |a phage display | |
| 650 | 4 | |a single domain antibodies | |
| 650 | 7 | |a Nucleocapsid Proteins |2 NLM | |
| 650 | 7 | |a Single-Domain Antibodies |2 NLM | |
| 700 | 1 | |a Aymé, Gabriel |e verfasserin |4 aut | |
| 700 | 1 | |a Brier, Sébastien |e verfasserin |4 aut | |
| 700 | 1 | |a Chauveau-Le Friec, Gaëlle |e verfasserin |4 aut | |
| 700 | 1 | |a Meriaux, Véronique |e verfasserin |4 aut | |
| 700 | 1 | |a Nowakowski, Mireille |e verfasserin |4 aut | |
| 700 | 1 | |a Dejardin, François |e verfasserin |4 aut | |
| 700 | 1 | |a Levallois, Sylvain |e verfasserin |4 aut | |
| 700 | 1 | |a Dias de Melo, Guilherme |e verfasserin |4 aut | |
| 700 | 1 | |a Donati, Flora |e verfasserin |4 aut | |
| 700 | 1 | |a Prot, Matthieu |e verfasserin |4 aut | |
| 700 | 1 | |a Brûlé, Sébastien |e verfasserin |4 aut | |
| 700 | 1 | |a Raynal, Bertrand |e verfasserin |4 aut | |
| 700 | 1 | |a Bellalou, Jacques |e verfasserin |4 aut | |
| 700 | 1 | |a Goncalves, Pedro |e verfasserin |4 aut | |
| 700 | 1 | |a Montagutelli, Xavier |e verfasserin |4 aut | |
| 700 | 1 | |a Di Santo, James P |e verfasserin |4 aut | |
| 700 | 1 | |a Lazarini, Françoise |e verfasserin |4 aut | |
| 700 | 1 | |a England, Patrick |e verfasserin |4 aut | |
| 700 | 1 | |a Petres, Stéphane |e verfasserin |4 aut | |
| 700 | 1 | |a Escriou, Nicolas |e verfasserin |4 aut | |
| 700 | 1 | |a Lafaye, Pierre |e verfasserin |4 aut | |
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