Detailed descriptions can be found in the supplemental material. Two replicate dilutions of plasma were incubated with MagPlex beads conjugated with SARS-CoV-2 spike, RBD, nucleoprotein, and tetanus toxoid followed by incubation with anti-human IgG Fc-phycoerythrin (Fc-PE) (Southern Biotech, Birmingham, AL). Background was established by measuring the mean fluorescence intensity (MFI) of beads conjugated to antigens incubated in assay buffer and was subtracted from all readings. Pooled sera collected in 2015 to 2016 from normal human donors was included as the negative control for SARS-CoV-2 antigens. Convalescent plasma from a subject with PCR-confirmed severe COVID-19 was used as a positive control.
Concentration of antigen-specific IgG was estimated using a standard curve based on the measurement of MFI for serial dilutions of standard IgG (Sigma, St. Louis, MO) captured by MagPlex beads conjugated with goat anti-human Ig Fab-specific antibody (Southern Biotech). MFI readings and associated IgG concentrations were fitted to a four-parameter logistic curve (4PL) using the R packages nCal and drc.
Concentration of antigen-specific IgG was estimated using a standard curve based on the measurement of MFI for serial dilutions of standard IgG (Sigma, St. Louis, MO) captured by MagPlex beads conjugated with goat anti-human Ig Fab-specific antibody (Southern Biotech). MFI readings and associated IgG concentrations were fitted to a four-parameter logistic curve (4PL) using the R packages nCal and drc.