Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases

Front Immunol. 2021 Nov 19:12:748291. doi: 10.3389/fimmu.2021.748291. eCollection 2021.


Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Keywords: Antibodies; COVID-19; COVID-19 immune response; Correlates of Protection; SARS-CoV-2; Serological biomarkers; WHO International Standard.

Publication types

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

MeSH terms

  • Antibodies, Neutralizing / blood
  • Antibodies, Neutralizing / immunology
  • Antibodies, Viral / blood
  • Antibodies, Viral / immunology
  • Antigens, Viral / immunology
  • Biomarkers / blood
  • COVID-19 / blood
  • COVID-19 / diagnosis
  • COVID-19 / immunology*
  • COVID-19 Serological Testing / standards
  • Calibration
  • Convalescence*
  • Humans
  • Immunity, Humoral*
  • Immunoglobulin Isotypes / blood
  • Immunoglobulin Isotypes / immunology
  • Reference Standards
  • SARS-CoV-2 / immunology*
  • Severity of Illness Index


  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Antigens, Viral
  • Biomarkers
  • Immunoglobulin Isotypes