Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

Viruses. 2020 May 6;12(5):513. doi: 10.3390/v12050513.


SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.

Keywords: 293T-ACE2; ACE2; ALAYT; COVID-19; SARS-CoV-2; Spike; coronavirus; cytoplasmic tail; lentiviral pseudotype; luciferase; neutralization assay.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2
  • Antibodies, Neutralizing / immunology
  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology*
  • Containment of Biohazards
  • HEK293 Cells
  • Humans
  • Lentivirus
  • Neutralization Tests / methods*
  • Peptidyl-Dipeptidase A / metabolism
  • Plasma / immunology
  • Spike Glycoprotein, Coronavirus / analysis*


  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • Peptidyl-Dipeptidase A
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2