Development and Validation of a Multiplex Microsphere Immunoassay Using Dried Blood Spots for SARS-CoV-2 Seroprevalence: Application in First Responders in Colorado, USA

J Clin Microbiol. 2021 May 19;59(6):e00290-21. doi: 10.1128/JCM.00290-21. Print 2021 May 19.


Serological testing of large representative populations for antibodies to SARS-CoV-2 is needed to estimate seroprevalence, transmission dynamics, and the duration of antibody responses from natural infection and vaccination. In this study, a high-throughput SARS-CoV-2 multiplex microsphere immunoassay (MMIA) was developed for the receptor binding domain (RBD) and nucleocapsid (N) that was more sensitive than enzyme-linked immunosorbent assay (ELISA) (98% versus 87%). The MMIA was then applied and validated in 264 first responders in Colorado using serum and dried blood spot (DBS) eluates, compared to ELISA, and evaluated for neutralizing antibodies. Four percent (11/264) of first responders were seropositive in July to August 2020. Serum and DBS were highly correlated for anti-RBD and anti-N antibodies (R = 0.83, P < 0.0001 and R = 0.87, P < 0.0001, respectively) by MMIA. The MMIA accurately predicted SARS-CoV-2 neutralizing antibodies using DBS (R = 0.76, P = 0.037). On repeat antibody testing 3 months later, anti-RBD IgG decreased less rapidly than anti-N IgG measured by MMIA, with a median change in geometric median fluorescence intensity of 62% versus 79% (P < 0.01) for anti-RBD and anti-N IgG, respectively. This novel MMIA using DBS could be scalable for rapid and affordable SARS-CoV-2 serosurveillance in the United States and globally.

Keywords: COVID-19 antibody testing; SARS-CoV-2 infection serological testing; dried blood spot testing; enzyme immunoassay; immunological surveillance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Viral
  • COVID-19 Serological Testing
  • COVID-19*
  • Colorado
  • Emergency Responders*
  • Humans
  • Immunoassay
  • Microspheres
  • SARS-CoV-2
  • Seroepidemiologic Studies


  • Antibodies, Viral