Development of a Rapid Focus Reduction Neutralization Test Assay for Measuring SARS-CoV-2 Neutralizing Antibodies

Curr Protoc Immunol. 2020 Dec;131(1):e116. doi: 10.1002/cpim.116.


SARS-CoV-2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS-CoV-2, which causes severe respiratory illness or death. Defining the antibody response to SARS-CoV-2 will be essential for understanding disease progression, long-term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS-CoV-2 antibodies. The basic protocol is a focus reduction neutralization test (FRNT), which involves immunostaining infected cells with a chromogen deposit readout. The alternate protocol is a modification of the FRNT that uses an infectious clone-derived SARS-CoV-2 virus expressing a fluorescent reporter. These protocols are adapted for use in a high-throughput setting, and are compatible with large-scale vaccine studies or clinical testing. © 2020 Wiley Periodicals LLC Basic Protocol: Focus reduction neutralization test Alternate Protocol: mNeonGreen-based focus reduction neutralization test (FRNT-mNG).

Keywords: SARS-Cov-2; antibody neutralization; high-throughput neutralization assay; neutralizing antibodies; serological assay.

Publication types

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

MeSH terms

  • Animals
  • Antibodies, Neutralizing / blood*
  • COVID-19 / epidemiology
  • COVID-19 / immunology*
  • COVID-19 / prevention & control
  • Chlorocebus aethiops
  • Humans
  • Neutralization Tests / methods*
  • Pandemics
  • SARS-CoV-2 / immunology*
  • Vero Cells


  • Antibodies, Neutralizing