Nr 52309

ELISAs were performed as described (Bates et al., 2021b (link)). Plates were coated overnight at 4°C with 1 mg/mL recombinant SARS-CoV-2 spike receptor binding domain (RBD) protein (Bates et al., 2021c ) (BEI Resources NR-52309) or recombinant SARS-CoV-2 nucleocapsid (N) protein (BEI Resources NR-53797). Serum dilutions (6 × 3-fold for RBD, 6 × 4-fold for N) in duplicate were prepared in 5% milk powder, 0.05% Tween-20, in phosphate buffered saline (PBS), starting at 1:1600 (pan-Ig), 1:50 (IgA), 1:200 (IgG). The secondary antibodies used were pan-Ig (1:10,000 anti-human GOXHU IgG/A/M-HRP, A18847 Invitrogen), IgA (1:3,000 anti-human IgA-HRP, 411002 Biolegend), and IgG (1:3,000 anti-human IgG-HRP 555788, BD Biosciences). Plates were developed with o-phenylenediamine (OPD) (ThermoScientific). Absorbance at 492nm was measured on a CLARIOstar plate reader and normalized by subtracting the average of negative control wells and dividing by the highest concentration from a positive control dilution series. ELISA EC50 values were calculated by fitting normalized A492 as described (Bates et al., 2021b (link)). The limit of detection (LOD) was defined by the lowest dilution tested for RBD and half of the lowest dilution for N. Values below the LOD were set to LOD – 1.

ADNKA was performed as described (26 (link), 133 (link)). ELISA plates were coated with recombinant RBD (300 ng/well) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serially diluted samples (1:10, 1:100, 1:1000) in duplicate for 2hours at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10⁴ cells/well) for 5hours with brefeldin A (Biolegend), Golgi Stop (BDBiosciences) and anti-CD107a (clone H4A3, BDBiosciences). Cells were stained with anti-CD56 (clone 5.1H11, BDBiosciences) and anti-CD16 (clone 3G8, BDBiosciences) and fixed with 4%PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BDBiosciences) and TNFα (clone Mab11, BDBiosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed. NK cell degranulation and activation were calculated as %CD56+CD107a+, IFNγ+ or TNFα+. Representative data from one dilution was chosen by the highest signal-to-noise ratio. Experiments were conducted two independent times.