Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein

EMBO Mol Med. 2021 Mar 5;13(3):e13549. doi: 10.15252/emmm.202013549. Epub 2021 Feb 17.

Abstract

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection.

Keywords: S protein; SARS-CoV-2; flow cytometry; method; seropositivity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adult
  • Antibodies, Neutralizing / blood*
  • Antibodies, Viral / blood*
  • COVID-19 / diagnosis*
  • COVID-19 / immunology*
  • COVID-19 / virology
  • COVID-19 Serological Testing / methods*
  • COVID-19 Serological Testing / statistics & numerical data
  • Enzyme-Linked Immunosorbent Assay
  • ErbB Receptors / genetics
  • Female
  • Flow Cytometry / methods*
  • Flow Cytometry / statistics & numerical data
  • Hep G2 Cells
  • Humans
  • Jurkat Cells
  • Male
  • Middle Aged
  • Neutralization Tests
  • Pandemics
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • SARS-CoV-2 / genetics
  • SARS-CoV-2 / immunology*
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology*

Substances

  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Recombinant Fusion Proteins
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • EGFR protein, human
  • ErbB Receptors