Neutralization assays were carried out essentially as described previously (18 (link)). Serum antibodies were diluted 4-fold in negative serum and 10-point 3-fold titrations in 25% negative serum were performed in 384 well polystyrene plates in duplicate using a Beckman (Biomek i5) liquid handler. Positive and negative control antibodies and an unrelated control (hIgG1 isotype) were tested in a 10-point, 3-fold serial dilution starting at 8 µg/mL, 2 µg/mL and 8 µg/mL, respectively, in 25% negative serum. An empirically pre-determined fixed amount of pseudovirus (Wuhan, E484Q, E484K, or the B.1.351 spike) was dispensed by WDII liquid dispenser on titrated serum antibodies and controls and pre-incubated for 20 minutes at 37°C. Following pre-incubation, the virus-antibody complexes were transferred by Biomek i5 to 8,000/well VeroE6 cells in white, opaque, tissue culture treated 384W plates, and incubated for 16-20 hours at 37°C. Control wells included virus only (no antibody; 14 replicates) and cells only (14 replicates). Following infection, cells were lysed with Promega BrightGlo and luciferase activity was measured on the Biotek Synergy Neo2 Multimode Reader.
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