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Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

Genova, Cecilia, Sampson, Alex, Scott, Simon D., Cantoni, Diego, Neto, Martin, Bentley, Emma, Mattiuzzo, Giada, Wright, Edward, Derveni, Mariliza, Auld, Bethany, and others. (2021) Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes. BIO-PROTOCOL, 11 (21). ISSN 2331-8325. (doi:10.21769/BioProtoc.4236) (KAR id:91352)

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Official URL:
https://doi.org/10.21769/BioProtoc.4236

Abstract

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.

Item Type: Article
DOI/Identification number: 10.21769/BioProtoc.4236
Subjects: Q Science > QR Microbiology > QR355 Virology
Divisions: Divisions > Division of Natural Sciences > Medway School of Pharmacy
Depositing User: Nigel Temperton
Date Deposited: 05 Nov 2021 09:47 UTC
Last Modified: 07 Nov 2022 09:06 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/91352 (The current URI for this page, for reference purposes)
Scott, Simon D.: https://orcid.org/0000-0002-8290-0461
Temperton, Nigel J.: https://orcid.org/0000-0002-7978-3815
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