Simoa Serology Assay for SARS-CoV-2 Antibodies

Simoa assays for IgG, IgA, and IgM against four SARS-CoV-2 targets (spike, S1, nucleocapsid, and RBD) were performed as previously described (Norman et al., 2020 (link)). Reference materials were diluted 1:250-, 1:1,000-, 1:4,000-, and 1:16,000-fold in Homebrew Detector/Sample Diluent (Quanterix Corporation, Product code: 101359, Billerica, Massachusetts, USA). Four antigen-conjugated capture beads were mixed and diluted in Bead Diluent (Quanterix Corporation, Product code: 101362, Billerica, Massachusetts, USA), with a total of 500,000 beads per reaction (125,000 of each bead type). Biotinylated antibodies were diluted in Homebrew Detector/Sample Diluent to final concentrations of IgG (Bethyl Laboratories A80-148B; Montgomery, Texas, USA): 7.73 ng/ml, IgA (Abcam ab214003, Waltham, Massachusetts, USA): 150 ng/ml, and IgM (Thermo Fisher Scientific, MII0401, Pittsburgh, Pennsylvania, USA): 216 ng/ml: Streptavidin-β-galactosidase (SβG) concentrate (Quanterix Corporation, Product code: 1013397, Billerica, Massachusetts, USA) was diluted to 30 pM in SβG Diluent (Quanterix Corporation, Product code: 100376, Billerica, Massachusetts, USA). The serology assay was performed on the HD-X Analyzer (Quanterix) in an automated three-step assay. Average enzymes per bead (AEB) values were calculated by the HD-X Analyzer software (Norman et al., 2020 (link)).