tetano
Editor, Senior Moderator
J Biol Chem
. 2021 Oct 19;101290.
doi: 10.1016/j.jbc.2021.101290. Online ahead of print.
Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein
Marion Gransagne[SUP] 1 [/SUP], Gabriel Aymé[SUP] 2 [/SUP], Sébastien Brier[SUP] 3 [/SUP], Gaëlle Chauveau-Le Friec[SUP] 2 [/SUP], Véronique Meriaux[SUP] 2 [/SUP], Mireille Nowakowski[SUP] 4 [/SUP], François Dejardin[SUP] 4 [/SUP], Sylvain Levallois[SUP] 5 [/SUP], Guilherme Dias de Melo[SUP] 6 [/SUP], Flora Donati[SUP] 7 [/SUP], Matthieu Prot[SUP] 8 [/SUP], Sébastien Brûlé[SUP] 9 [/SUP], Bertrand Raynal[SUP] 9 [/SUP], Jacques Bellalou[SUP] 4 [/SUP], Pedro Goncalves[SUP] 10 [/SUP], Xavier Montagutelli[SUP] 11 [/SUP], James P Di Santo[SUP] 10 [/SUP], Françoise Lazarini[SUP] 12 [/SUP], Patrick England[SUP] 9 [/SUP], Stéphane Petres[SUP] 4 [/SUP], Nicolas Escriou[SUP] 1 [/SUP], Pierre Lafaye[SUP] 13 [/SUP]
Affiliations
Abstract
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 are 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, 7 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.
Keywords: Antibody engineering; Covid19; Nanobodies; Single domain antibodies; diagnostic; hydrogen‐deuterium exchange; immunochemistry; nucleoprotein; phage display.
. 2021 Oct 19;101290.
doi: 10.1016/j.jbc.2021.101290. Online ahead of print.
Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein
Marion Gransagne[SUP] 1 [/SUP], Gabriel Aymé[SUP] 2 [/SUP], Sébastien Brier[SUP] 3 [/SUP], Gaëlle Chauveau-Le Friec[SUP] 2 [/SUP], Véronique Meriaux[SUP] 2 [/SUP], Mireille Nowakowski[SUP] 4 [/SUP], François Dejardin[SUP] 4 [/SUP], Sylvain Levallois[SUP] 5 [/SUP], Guilherme Dias de Melo[SUP] 6 [/SUP], Flora Donati[SUP] 7 [/SUP], Matthieu Prot[SUP] 8 [/SUP], Sébastien Brûlé[SUP] 9 [/SUP], Bertrand Raynal[SUP] 9 [/SUP], Jacques Bellalou[SUP] 4 [/SUP], Pedro Goncalves[SUP] 10 [/SUP], Xavier Montagutelli[SUP] 11 [/SUP], James P Di Santo[SUP] 10 [/SUP], Françoise Lazarini[SUP] 12 [/SUP], Patrick England[SUP] 9 [/SUP], Stéphane Petres[SUP] 4 [/SUP], Nicolas Escriou[SUP] 1 [/SUP], Pierre Lafaye[SUP] 13 [/SUP]
Affiliations
- PMID: 34678315
- PMCID: PMC8526496
- DOI: 10.1016/j.jbc.2021.101290
Abstract
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 are 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, 7 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.
Keywords: Antibody engineering; Covid19; Nanobodies; Single domain antibodies; diagnostic; hydrogen‐deuterium exchange; immunochemistry; nucleoprotein; phage display.