High binding microplates

Ninety-six-well high-binding microplates (Corning) coated with a capture antibody were blocked using 3% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 2 hr at 37 °C. Next, 100 μL of increasing concentrations of the RBDs of wild-type SARS-CoV-2 or mutants (A435S; Sino#40592-V08H4, G476S; Sino#40592-V08H8, F342L; Sino#40592-V08H6, N354D; Sino#40592-V08H2, V483A; Sino#40592-V08H5, V341I; Sino#40592-V08H11, B.1.1.7; Sino#40592-V08H82, and B.1.617.2; Sino#40592-V08H90) were added to the wells, and the microplate was incubated for 2 hr at 37 °C. The plates were washed thrice with PBS containing 0.05% (v/v) Tween 20 (PBST) and incubated with HRP-conjugated anti-HA antibody (1:3000, Bethyl Laboratories, Montgomery, TX, USA) for 1 hr at 37 °C. Following three washes with PBST, 3,3′,5,5′-tetramethylbenzidine substrate solution (Thermo Fisher Scientific) was added to each well and allowed to react with HRP for 5 min. The reaction was terminated by adding 100 μL of 1 M H₂SO₄. The absorbance of each sample was measured at 450 nm using a microplate reader.

To detect anti-PR8 antibodies, high-binding microplates (Corning) were coated with UV-inactivated PR8 in PBS at 4 °C overnight. To quantify the absolute antibody concentration, a standard curve for mouse isotype was established for each plate by coating with goat anti-mouse Ig (1 µg/mL in PBS; Southern Biotech) overnight at 4 °C. Plates were blocked with 1% bovine serum albumin (Fisher Scientifics) in PBSTE (Phosphate buffered saline with 0.05% Tween-20 and 1 mM EDTA) for 1 h at 37 °C. After washing with PBSTE, samples and standard Ig (Southern Biotech) were added to respective wells and incubate for 1 h at room temperature (RT). For total IgG standard, a mixture of purified mouse IgG1, IgG2b, IgG2c, and IgG3 were combined to emulate WT antibody titers^{74 (link)}. Biotinylated goat-anti-mouse isotype-specific (IgM, IgG) antibodies (0.1 µg/mL; Southern Biotech) were added and incubated at RT for 1 h. Horseradish-peroxidase-conjugated streptavidin (ThermoFisher) was added and incubated at RT for 30 min. The plates were washed with PBSTE between steps for the procedures above. TMB High Sensitivity Substrate (BioLegend) was used for detection; reactions were stopped by adding 1 M sulfuric acid. Absorbances were measured at OD450 with OD540 as background using M2e SpectraMax 500 (Molecular Devices) and antibody concentrations were calculated using the generated standard curve.