G18 145

We diluted the antigens for antibody detection in PBS and coated the plates (Nunc MaxiSorp, Thermofisher Scientific) with 250 ng/mL WT (AcroBiosystems) or Omicron BA.1 (AcroBiosystems) SARS-CoV-2 S protein for IgG and IgG avidity assessment and 500 ng/mL ancestral (Sinobiological) or Omicron BA.1 (AcroBiosystems) S for FcγRIIIa-binding detection. ORF8 protein of 300 ng/mL was coated at 37 for 2 hours. The plates were blocked with 1% FBS in PBS for 1 hour, followed by incubation with 1:100 HI sera diluted in 0.05% Tween-20/0.1% FBS in PBS for 2 hours for IgG detection, and 1:50 for 1 hour at 37 for FcγRIIIa-binding detection, prior to rinsing. For avidity, plates with 8M were washed with urea 3 times. IgG was measured after 2 hours of incubation period with anti-IgG-HRP (1:5000; G18-145, BD), HRP revealed with addition of stabilized hydrogen peroxide and tetramethylbenzidine (R&D systems) for 20 minutes. The reaction was terminated with 2N H2SO4, which was then analyzed at 450 nm wavelength with an absorbance microplate reader (Tecan Life Sciences). Similarly, FcγRIIIa-binding antibodies were assessed after incubation with biotinylated FcγRIIIa-V158 at 100 ng/mL for 1 hour at 37 after streptavidin-HRP (1:10000, Pierce).

Column bound or flow-through samples were stained with a Ghost Red 710 viability dye (TonBo, 13-0871-T100) and fluorophore-conjugated antibodies specific for CD3 (OKT3, Invitrogen, 47-0037-42)), CD14 (61D3, Invitrogen, 47-0149-42), CD16 (CB16, Invitrogen, 47-0168-42), CD19 (HIB19, Biolegend, 302242), CD20 (2H7, BD, 563782), CD21 (B-ly4, BD, 562966), CD27 (O323, Biolegend, 302834), CD38 (HB-7, Biolegend, 356620), CD11c (B-ly6, BD, 526393), CD79b (CB3-1, Biolegend, 341404), IgD (IA6-2, Biolegend, 348210), IgM (MHM-88, Biolegend, 314524), IgA (Southern Biotech, 2050-02), and IgG (G18-145, BD, 564230); washed in sorter buffer; and fixed in 250 μL BD Cytofix/Cytoperm. Flow cytometry was performed on a BD Fortessa X20 and analyzed with FlowJo software (Tree Star).

PBMCs were first stained with fluorophore-labeled antibodies against cell surface markers CD3 (OKT3, BioLegend), CD19 (SJ25-C1, Thermo Fisher), CD20 (SI Appendix, Table S2), and CD27 (O323, BioLegend), fixed (Lysing Solution, BD Biosciences), permeabilized (Permeabilizing Solution 2, BD Biosciences), and stained with fluorophore-labeled antibodies against IgG (G18-145, BD Biosciences), IgA (8E10, Miltenyi Biotec), IgD (IA6-2, BioLegend), and IgM (MHM-88, BioLegend). The stained cells were acquired on a FACS Canto II flow cytometer (BD Biosciences) and analyzed in FlowJo software v9.9.6 (BD Biosciences).