Msd gold read buffer b

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.

Plasma antibody titers for mutant RBDs were measured using U-PLEX kits (Meso Scale Discovery), according to the manufacturer’s instructions. Briefly, recombinant biotinylated RBDs were incubated with the linker proteins. After mixing with the stop solution, the linker-conjugated RBDs were added to U-PLEX plates, and the plates were incubated at 4 °C overnight. After washing with wash buffer, the plates were incubated with MSD Blocker A reagent (Meso Scale Discovery) to reduce non-specific binding, and diluted samples were added after washing. After incubation at room temperature for 2 h with rotation, the plates were incubated with SULFO-TAG-conjugated anti-human IgG (1:200 dilution, Meso Scale Discovery). After washing, MSD Gold read buffer B (Meso Scale Discovery) was added to the plates, and electrochemiluminescence was determined using MESOQuickPlex SQ 120 (Meso Scale Discovery).