Polyclonal serum igg

Characterization of the phagocytic activity of serum antibodies was performed^{80 (link)}. Briefly, 1 μM yellow-green fluorescent beads (Thermo Fisher, F8813) were covalently conjugated to spike or RBD antigens. Beads were then incubated with serum samples for 4 h with THP-1 cells (ATCC TIB-202) at 37 °C in 5% CO₂. Cells were fixed and analyzed by flow cytometry (Supplemental Fig. 6) using a MACSQuant Analyzer (Miltenyi Biotec). Scores were calculated as the percentage of cells that phagocytosed one or more fluorescent beads multiplied by the MFI of this population. S309 and VRC01 antibodies were included as positive and negative controls, respectively. Additional control wells with no added antibody were used to determine the level of antibody-independent phagocytosis. Serum samples were assayed at three different dilutions, which were determined by an initial pilot experiment to determine the optimal dilution series for measuring signal compared to the background. Concentrated pooled polyclonal serum IgG (Sigma Aldrich I4506) was used as a positive control for endemic CoV (Supplemental Fig. 7), and samples were run in three biological replicates.

A surrogate for antibody-mediated cellular cytotoxicity was measured using a CD16 reporter assay system^{81 (link)}. Jurkat Lucia NFAT (Invivogen, jktl-nfat-cd16) cells were cultured according to manufacturer’s instructions. Cultured cells express CD16 (FcγRIIIa), which, when engaged on the cell surface, leads to luciferase secretion from the cell. First, high-binding 96-well plates were coated overnight at 4 °C with 1 μg/mL of spike or RBD antigen. Following incubation, plates were washed (PBS + 0.1% Tween20) and blocked (PBS + 2.5% BSA) at room temperature (RT) for 1 h. Following plate washing, 100,000 cells per well and dilute serum samples were added to each well in cell culture media lacking antibiotics in a 200 μL volume. Following 24 h incubation, 25 μL of supernatant from each well was transferred into a white 96-well plate in which 75 μL of quantiluc substrate was immediately added. Following 10 min incubation, plates were read on a SpectraMax plate reader (Molecular Devices). VRC01 was used as a negative control; cell stimulation cocktail (Thermo Fischer Scientific, 00-4970-93) and ionomycin and S309 served as positive controls. Concentrated pooled polyclonal serum IgG (Sigma Aldrich I4506) was used as a positive control for endemic CoV (Supplemental Fig. 7), and samples were run in three biological replicates.