期刊
BIO-PROTOCOL
卷 11, 期 21, 页码 -出版社
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.4236
关键词
SARS-CoV-2 coronavirus; Lentiviral pseudotype; Virus neutralisation; Spike variants; Pseudotype lyophilisation; COVID-19
类别
资金
- MRC [MC_PC_19060, MC_PC_20016]
- MRC [MC_PC_19060, MC_PC_20016] Funding Source: UKRI
This protocol outlines a method for producing and testing high-titre viral pseudotype particles with the SARS-CoV-2 spike protein for neutralisation assays. Results show that these particles can be neutralised by convalescent plasma and monoclonal antibodies, and can be stored at various temperatures for distribution and application.
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.
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