Diluent 100

IgG titers in B cell culture supernatants to mutant RBDs were measured using the U-PLEX kit (Meso Scale Discovery) as described previously (30 (link)). Briefly, biotinylated RBDs were conjugated to linker proteins from the kit by incubation at room temperature for 30 min, followed by another 30-min incubation with stop solution. All the linker-conjugated RBDs were mixed, transferred into U-PLEX plates, and incubated at 4°C overnight. The plates were washed with washing buffer (PBS supplemented with 0.05% Tween 20) three times and filled with MSD Blocker A reagent, followed by 1 hour of incubation at room temperature with rotation for blocking. The plates were washed with the washing buffer three times and were incubated with samples diluted in Diluent 100 (Meso Scale Discovery) at room temperature for 2 hours with rotation. The plates were washed three times and incubated with SULFO-TAG–conjugated anti-human IgG (Meso Scale Discovery) at room temperature for 1 hour with rotation. The plates were washed with the washing buffer and filed with MSD Gold Read Buffer B (Meso Scale Discovery), and electrochemiluminescence was immediately measured with MESO QuickPlex SQ120 (Meso Scale Discovery). Signals were normalized to those of CR3022-IgG1, and a threshold of susceptibility to the mutated RBDs was set as a <0.25 ratio to Wuhan RBD.

Maxisorp 384 well plates (Nunc, #P6366) were coated with 1 µg/mL of Wuhan NTD (amino acids 1 to 162 of spike) or trimeric Wuhan spike proteins prepared as described previously^{70 (link)} at 4 °C overnight. After washing with wash buffer (PBS supplemented with 0.05% Tween-20), the plates were blocked with 1% BSA (Roche, #10735086001)/PBS at room temperature for 1 h. After washing with the wash buffer, the plates were incubated with serially diluted plasma (6-fold serial dilutions starting from 1:500) in Diluent 100 (Meso Scale Discovery) for 2 h at room temperature. The plates were washed with the wash buffer and incubated with HRP-conjugated goat anti-human IgG (1:5000, polyclonal, Southern Biotech, #2040-05) for 1 h at room temperature. After washing with the washing buffer, HRP activity was visualized with OPD substrate (Sigma, #523121), followed by the addition of HCl to stop the reaction. Absorbance at 490 nm was measured using Epoch2 (Biotek). Binding curves were analyzed with GraphPad Prism software with 4-parameter nonlinear regression to calculate the EC₅₀ values.