Fc γ specific goat anti human igg f ab 2 fragment

Serum or plasma samples were tested at 1:100 dilution in 0.05% PBS-Tween supplemented with 1% (w/v) bovine serum albumin (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. 45 μl of the 1:100 diluted sera were transferred into the 384-well plate containing the bead array. Samples were incubated for 60 min 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 1:500 diluted R-phycoerythrin (R-PE) conjugated Fc-γ-specific goat anti-human IgG F(ab’)2 fragment (Jackson ImmunoResearch) was added to the 384-well plate for detection of bound human IgG. After incubation with the secondary antibody for 30 min, the plate was washed with 3 × 60 μl PBS-Tween and re-suspended in 50 μl PBS-Tween prior to analysis using a FlexMap3D^™ instrument (Luminex Corp.). Binding events were displayed as Mean Fluorescence Intensity (MFI). All samples were run in duplicate in each experiment. Longitudinal samples which 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^{45 (link)}.

Serum or plasma samples were diluted at 1:100 in 0.05% PBS-Tween supplemented with 1% (w/v) bovine serum albumin and transferred into 96-well plates. The bead array was distributed into a 384-well plate (Greiner BioOne) by transfer of 5 µL of bead array per well. Then 45 µL of the 1:100 diluted sera were 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 3 times with 60 µL PBS-Tween on a plate washer (EL406, Biotek), and 50 µL of 1:1000 diluted R-phycoerythrin (R-PE) conjugated Fc-γ-specific goat anti-human IgG F(ab’)2 fragment (Jackson ImmunoResearch) was added to the 384-well plate for detection of bound human IgG. After a 30-minute incubation, the plate was washed 3 times with 60 µL PBS-Tween and re-suspended in 50 µL PBS-Tween prior to analysis using a FlexMap3D™ instrument (Luminex Corp.). Binding events were displayed as Mean Fluorescence Intensity (MFI). All samples were run in duplicate in each experiment. A static cutoff of 3000 MFI was used to determine positive autoantibody reactivities.