Attune next flow cytometer

For the measurements of SARS‐CoV‐2 receptor‐binding domain (RBD) and full‐length Spike IgG antibodies in sera of patients and controls, a bead‐based multiplexing assay was used. Briefly, purified His‐tagged SARS‐CoV‐2 RBD and full‐length Spike proteins (production see Methods S1) were biotinylated at a biotin to protein ratio of 1:1 and bound to neutravidin‐conjugated polymer microspheres with fluorescent barcodes.³⁰, ³¹ Beads with different proteins and barcodes were mixed in assay buffer (phosphate‐buffered saline [PBS] with Tween‐20, bovine serum albumin, Neutravidin, D‐biotin and NaN₃) and kept at 4°C until use. Bead‐multiplexes were added to serum diluted 1:100 in assay buffer. After a 1‐h incubation with constant agitation, the beads were washed with PBS‐Tween 20 (1%), labelled with R‐phycoerythrin (PE)‐goat‐anti‐human IgG‐Fc (Jackson Immunoresearch; 1:600) and analysed with an Attune Next flow cytometer (Thermo). Flow cytometry data were analysed with WinList 10.0 and median fluorescence intensity (MFI) values exported to Excel. Results are reported as: arbitrary units (au) = (MFI_{viral protein beads})/(MFI_{no protein beads}). A double cut‐off of 5 au for RBD and Spike was used to classify positives.