Streptavidin-coated beads with different intensities of phycoerythrin (PE)-channel fluorescence (Spherotech SVFA-2558–6K and SVFB-2558–6K) were incubated with the following biotinylated antigens: 10 μg/mL MERS spike (Sino Biological 40069-V08B), NL63 spike (Sino Biological 40604-V08B), 229E spike (Sino Biological 40605-V08B), HKU1 spike (Sino Biological 40606-V08B), OC43 spike (Sino Biological 40607-V08B), SARS-CoV-2 spike, SARS-CoV-2 RBD, SARS-CoV-2 NTD, SARS-CoV-1 spike, SARS-CoV-1 RBD or 10 μg/mL CD4 (56 (link)) as a control. SARS-CoV-2 and SARS-CoV-1 antigens were produced in-house as described above. Excess streptavidin sites were blocked with 10 μg/mL of CD4 and the beads were washed and mixed. The beads were stained with 1:50, 1:250 or 1:1250 plasma for 30 minutes at room temperature, washed, and stained with 2.5 μg/mL goat anti-human IgG Alexa Fluor 647 (Jackson Immunoresearch 109-606-170), anti-human IgA Alexa Fluor 647 (Jackson Immunoresearch 109-606-011) or anti-human IgM Alexa Fluor 647 (Jackson Immunoresearch 109-606-129). The samples were read with the iQue Screener Plus (Intellicyt) high-throughput flow cytometer and FACS data were analysed with Flowjo. Data from titrations were analysed by calculating area under the curve (AUC) for the titration and subtracting the AUC of the negative control antigen.