SARS-CoV-2-Specific Antibody Detection for Seroepidemiology: A Multiplex Analysis Approach Accounting for Accurate Seroprevalence

J Infect Dis. 2020 Oct 1;222(9):1452-1461. doi: 10.1093/infdis/jiaa479.


Background: The COVID-19 pandemic necessitates better understanding of the kinetics of antibody production induced by infection with SARS-CoV-2. We aimed to develop a high-throughput multiplex assay to detect antibodies to SARS-CoV-2 to assess immunity to the virus in the general population.

Methods: Spike protein subunits S1 and receptor binding domain, and nucleoprotein were coupled to microspheres. Sera collected before emergence of SARS-CoV-2 (n = 224) and of non-SARS-CoV-2 influenza-like illness (n = 184), and laboratory-confirmed cases of SARS-CoV-2 infection (n = 115) with various severities of COVID-19 were tested for SARS-CoV-2-specific IgG concentrations.

Results: Our assay discriminated SARS-CoV-2-induced antibodies and those induced by other viruses. The assay specificity was 95.1%-99.0% with sensitivity 83.6%-95.7%. By merging the test results for all 3 antigens a specificity of 100% was achieved with a sensitivity of at least 90%. Hospitalized COVID-19 patients developed higher IgG concentrations and the rate of IgG production increased faster compared to nonhospitalized cases.

Conclusions: The bead-based serological assay for quantitation of SARS-CoV-2-specific antibodies proved to be robust and can be conducted in many laboratories. We demonstrated that testing of antibodies against multiple antigens increases sensitivity and specificity compared to single-antigen-specific IgG determination.

Keywords: COVID-19; IgG; RBD; endemic coronavirus; influenza-like Illness (ILI); multiplex bead-based immune assay; nucleoprotein; sensitivity; specificity; spike S1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptive Immunity
  • Adult
  • Aged
  • Aged, 80 and over
  • Antibodies, Viral / blood*
  • Area Under Curve
  • Betacoronavirus / immunology*
  • COVID-19
  • Case-Control Studies
  • Coronavirus Infections / blood*
  • Coronavirus Infections / epidemiology*
  • Female
  • Humans
  • Immunoassay
  • Immunoglobulin G / blood*
  • Male
  • Middle Aged
  • Netherlands / epidemiology
  • Nuclear Proteins / immunology
  • Pandemics*
  • Patient Acuity
  • Pneumonia, Viral / blood*
  • Pneumonia, Viral / epidemiology*
  • ROC Curve
  • SARS-CoV-2
  • Seroconversion
  • Seroepidemiologic Studies
  • Spike Glycoprotein, Coronavirus / immunology


  • Antibodies, Viral
  • Immunoglobulin G
  • Nuclear Proteins
  • Spike Glycoprotein, Coronavirus