Measuring SARS-CoV-2 Spike ADCP Activity

Assays to measure spike-specific ADCP were performed using a protocol reported previously (25 (link), 27 (link)). Briefly, FluoSpheres carboxylate-modified yellow-green fluorescent microspheres (Thermo Fisher, #F8823) were coupled with SARS-CoV-2 spike or RBD proteins using the xMAP Antibody Coupling Kit (5 µg protein/~36.4x10⁹ beads, Luminex #40-50016). Spike-conjugated microspheres were incubated with diluted plasma for 2 hours at 37°C in the dark. After washing and centrifugation (2,000 g, 10 min), the beads (~3x10⁸ beads, 10 µL/well) were incubated with THP-1 cells (0.25x10⁵ cells, 200 µL/well) for 16 hours. The samples were analyzed on an Attune NxT flow cytometer (Thermo Fisher, #A24858). Data analysis was performed using FCS Express 7 Research Edition (De Novo Software). ADCP scores were calculated as follows: (% microsphere positive cells) x (geometric mean fluorescent intensity of the microsphere positive cells)/1000.