دورية أكاديمية
Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein
| العنوان: | Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein |
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| المؤلفون: | Gransagne, Marion, Aymé, Gabriel, Brier, Sébastien, Chauveau-Le Friec, Gaëlle, Meriaux, Véronique, Nowakowski, Mireille, Déjardin, François, Levallois, Sylvain, Dias de Melo, Guilherme, Donati, Flora, Prot, Matthieu, Brûlé, Sébastien, Raynal, Bertrand, Bellalou, Jacques, Goncalves, Pedro, Montagutelli, Xavier, Di Santo, James, P, Lazarini, Françoise, England, Patrick, Petres, Stéphane, Escriou, Nicolas, Lafaye, Pierre |
| المساهمون: | Département de Santé Globale - Department Global Health, Institut Pasteur Paris (IP)-Université Paris Cité (UPCité), Ingénierie des Anticorps (plate-forme) - Antibody Engineering (Platform), Institut Pasteur Paris (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme Technologique de RMN Biologique et HDX-MS - Biological NMR and HDX-MS Technological Platform, Plateforme technologique Production et purification de protéines recombinantes – Production and Purification of Recombinant Proteins Technological Platform (PPR), Biologie des Infections - Biology of Infection, Institut Pasteur Paris (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Lyssavirus, épidémiologie et neuropathologie - Lyssavirus Epidemiology and Neuropathology, Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae Paris (CNR - laboratoire coordonnateur), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Immunité Innée - Innate Immunity, Génétique de la souris - Mouse Genetics, Perception et Mémoire / Perception and Memory, Institut Pasteur Paris (IP)-Centre National de la Recherche Scientifique (CNRS), P. G. and J. P. D. received support from the ANR grant “COVARIMM.”We want to thank Nicolas Wolff and Baptiste Colcombet-Cazenave for their technical help for the production of the different VHHs. The strain BetaCoV/France/IDF0372/2020 was supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Pr. Sylvie van der Werf. The human sample from which strain BetaCoV/France/IDF0372/2020 was isolated was provided by Dr X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, France. Moreover, the strain BetaCoV/France/IDF0372/2020 was supplied through the European Virus Archive goes Global (Evag) platform, a project that has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 653316. We are grateful to the National Reference Center for Respiratory Viruses for providing the variants of concern used in mouse experiments, to Sylvie van der Werf and Etienne Simon-Lorière for support and scientific advice, and to Grégory Jouvion for histological evaluation. We thank the Institut Pasteur Histology platform for histological slides preparation. Mouse experiments on variants of concern were supported by ANR grants HUMOCID (ANR-20-COVI-0028-01) and IBEID (ANR-10-LABX-62-IBEID) and by the EU Horizon 2020 RECOVER project (No. 101003589). The authors would like to thank the DIM 1HEALTH région Ile-de-France scheme for funding the Centrifection project that allowed to purchase the Optima ultracentrifuge. The CACSICE Equipex ANR-11-EQPX-0008 is acknowledged., ANR-20-COVI-0053,CoVarImm,Variation de la réponse immune systémique et muqueuse pendant l'infection par le SRAS-CoV-2 et la convalescence(2020), ANR-20-COVI-0028,HuMoCID,Développement de modèles murins de COVID-19(2020), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), European Project: 653316,H2020,H2020-INFRAIA-2014-2015,EVAg(2015) |
| المصدر: | ISSN: 0021-9258. |
| بيانات النشر: | HAL CCSD American Society for Biochemistry and Molecular Biology |
| سنة النشر: | 2022 |
| مصطلحات موضوعية: | COVID-19, antibody engineering, diagnostic, hydrogen-deuterium exchange, immunochemistry, nanobodies, nucleoprotein, phage display, single domain antibodies, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM] |
| الوصف: | International audience ; 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. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| العلاقة: | info:eu-repo/semantics/altIdentifier/pmid/34678315; info:eu-repo/grantAgreement//653316/EU/European Virus Archive goes global/EVAg; pasteur-03524979; https://pasteur.hal.science/pasteur-03524979Test; https://pasteur.hal.science/pasteur-03524979/documentTest; https://pasteur.hal.science/pasteur-03524979/file/VHH%20anti%20SARS-CoV2%20nucleoprotein%20Lafaye%20JBC%20%281%29.pdfTest; PUBMED: 34678315; PUBMEDCENTRAL: PMC8526496 |
| DOI: | 10.1016/j.jbc.2021.101290 |
| الإتاحة: | https://doi.org/10.1016/j.jbc.2021.101290Test https://pasteur.hal.science/pasteur-03524979Test https://pasteur.hal.science/pasteur-03524979/documentTest https://pasteur.hal.science/pasteur-03524979/file/VHH%20anti%20SARS-CoV2%20nucleoprotein%20Lafaye%20JBC%20%281%29.pdfTest |
| حقوق: | http://creativecommons.org/licenses/byTest/ ; info:eu-repo/semantics/OpenAccess |
| رقم الانضمام: | edsbas.29F8D662 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1016/j.jbc.2021.101290 |
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