Serum or plasma samples were tested at 1:100 dilution in 0.05% PBS-Tween supplemented with 1% (w/v) BSA and transferred into 96-well plates in a randomized layout. The bead array was distributed into a 384-well plate (Greiner BioOne) by transfer of 5 μL bead array per well. A total of 45 μL of the 1:100 diluted sera was transferred into the 384-well plate containing the bead array. Samples were incubated for 60 minutes on a shaker at room temperature. Beads were washed with 3 × 60 μL PBS-Tween on a plate washer (EL406, Biotek), and 50 μL of 1:1,000 diluted R-phycoerythrin–conjugated (R-PE–conjugated) Fc-γ–specific goat anti–human IgG F(ab’)2 fragment (Jackson ImmunoResearch, 106-116-098) was added to the 384-well plate for detection of bound human IgG. After incubation with the secondary antibody for 30 minutes, the plate was washed with 3 × 60 μL PBS-Tween and resuspended in 50 μL PBS-Tween prior to analysis using a FlexMap3D instrument (Luminex Corp.). Binding events were displayed as MFI. All samples were run in duplicate in each experiment. Longitudinal samples that showed new-onset autoantibodies were reanalyzed in duplicate on new bead arrays to confirm results. Samples from patients with COVID-19 were heat inactivated prior to analysis, as previously described (41 (link)).
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