Validation of a combined ELISA to detect IgG, IgA and IgM antibody responses to SARS-CoV-2 in mild or moderate non-hospitalised patients

Research output: Contribution to journalArticlepeer-review


  • AM Cook
  • L.J. Williams
  • D. Kay
  • T. Plant
  • G. Wallis
  • S. Beck
  • SE Jossi
  • M. Perez-Toledo
  • M.L. Newby
  • J.D. Allen
  • M. Crispin
  • S. Harding
  • Alex Richter


Background: Frequently SARS-CoV-2 results in mild or moderate disease with potentially lower concentrations of antibodies compared to those that are hospitalised. Here, we validated an ELISA using SARS-CoV-2 trimeric spike glycoprotein, with targeted detection of IgG, IgA and IgM (IgGAM) using serum and dried blood spots (DBS) from adults with mild or moderate disease.

Methods: Targeting the SARS-CoV-2 trimeric spike, a combined anti-IgG, IgA and IgM serology ELISA assay was developed using 62 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 624 COVID-19 negative samples. The assay was validated using 73 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 359 COVID-19 negative serum samples with an additional 81 DBSs. The assay was further validated in 226 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, ≥14 days post symptom onset and 426 COVID-19 negative clinical samples.

Results: A sensitivity and specificity of 98.6% (95% CI, 92.6–100.0), 98.3% (95% CI, 96.4–99.4), respectively, was observed following validation of the SARS-CoV-2 ELISA. No cross-reactivities with endemic coronaviruses or other human viruses were observed, and no change in results were recorded for interfering substances. The assay was stable at temperature extremes and components were stable for 15 days once opened. A matrix comparison showed DBS to correlate with serum results. Clinical validation of the assay reported a sensitivity of 94.7% (95% CI, 90.9–97.2%) and a specificity of 98.4% (95% CI, 96.6–99.3%).

Conclusions: The human anti-IgGAM SARS-CoV-2 ELISA provides accurate and sensitive detection of SARS-CoV-2 antibodies in non-hospitalised adults with mild or moderate disease. The use of dried blood spots makes the assay accessible to the wider community.

Bibliographic note

Funding Information: We thank Lia van der Hoek for seasonal coronaviruses samples used in the cross reactivity study from the Amsterdam Cohort Studies (ACS) on HIV infection and AIDS. The ACS is a collaboration among the Public Health Service of Amsterdam, the Amsterdam University Medical Center of the University of Amsterdam, the Sanquin Blood Supply Foundation, the Medical Center Jan van Goyen and the HIV Focus Center of the DC-Clinics. It is part of the Netherlands HIV Monitoring Foundation and financially supported by the Center for Infectious Disease Control of the Netherlands National Institute for Public Health and the Environment. The authors thank all ACS participants for their contribution, as well as the ACS study nurses, data managers and lab technicians.


Original languageEnglish
Article number113046
Number of pages7
JournalJournal of Immunological Methods
Early online date26 Mar 2021
Publication statusPublished - Jul 2021


  • COVID-19, Dried blood spot, Low prevalence, Mild disease, Serological, Trimeric spike protein

ASJC Scopus subject areas

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