1 n sulfuric acid

Microtiter plate wells were coated with purified recombinant WT or mutant GP_{1,2ΔTM/ΔMLD} and incubated at room temperature for 1 h before blocking with 3% bovine serum albumin (BSA; Millipore Sigma) in DPBS containing 0.05% TWEEN-20 (Fisher Scientific) for 1 h. Serial dilutions of mAb were applied to the wells and incubated for 1 h at room temperature. The bound antibodies were detected using Jackson Immuno Research Labs peroxidase-conjugated goat anti-human IgG (Thermo Fisher Scientific; #109036006) with horseradish peroxidase (diluted 1:4,000) and 3,3’,5,5”-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) before 50 μl of 1 N sulfuric acid (Fisher Scientific) was added to stop the reaction. Absorbance at 450 nm was then measured using a Spark microplate reader (Tecan, Männedorf, Switzerland). Half-maximal response (EC₅₀) values for mAb binding were determined using Prism 7 (GraphPad Software, Boston, Massachusetts, USA) after log-transformation of antibody concentrations using EC₅₀ shift nonlinear regression analysis.

The streptavidin-coated plates (R&D Systems, Minneapolis, MN) were coated with 50 μl of 0.5 μg/ml of biotinylated KRAS^G12V[7–16]/HLA-A3 monomer, 0.5 μg/ml biotinylated KRAS^WT[7–16]/HLA-A3 monomer, or 0.4 μg/ml biotinylated recombinant human CD3ε/CD3δ heterodimer (CDD-H82W6, Acro Biosystems, Newark, DE) in BAE buffer (PBS containing 0.5% bovine serum albumin [Sigma], 2 mM EDTA [Thermo] and 0.1% sodium azide) for 1 h at room temperature. Plates were washed 6 times with 1× TBST (J77500-K8, Thermo Fisher Scientific) using a 405 TS microplate washer (BioTek, Winooski, VT). scDb samples were diluted to 200 ng/ml in PBS and incubated in the coated plates for 1 h at room temperature. Plates were washed 6 times with TBST. 50 μl of 0.5 μg/ml recombinant Protein L (Pierce) was added to the well and incubated for 1 h at room temperature. Plates were washed 6 times with TBST. Plates were incubated with 50 μl of 0.2 μg/ml chicken anti-Protein L HRP antibody (ab63506, Abcam) for 1 h at room temperature and then washed 6 times with TBST. 50 μl of TMB substrate (Biolegend) was added to each well and quenched with 50 μl 1 N sulfuric acid (Fisher Scientific). Absorbance was measured at 450 and 540 nm using a Synergy H1 Multi-Mode Reader (BioTek). Reported A₄₅₀ values include an A₅₄₀ correction (subtract A₅₄₀ from A₄₅₀ for each well). All samples were measured in triplicate.

Enzyme-linked immunosorbent assay (ELISA) was used to test antibody binding to SARS-CoV-2 receptor binding domain (RBD) (catalog numbers 40592-V08H, Sino Biological US Inc., Wayne, PA, USA) (31 (link), 32 (link)). High-binding 96-half-well microplates (Corning Life Sciences, Tewksbury, MA, USA) were coated with 50ng RBD protein in PBS overnight at 4°C. The next day, plates were washed five times with PBS + 0.5% Tween 20 and treated with blocking buffer (PBS + 0.5% Tween 20 + 2% BSA + 2% normal goat serum [Jackson ImmunoResearch Inc., West Grove, PA, USA]) for two hours. Different dilutions of human plasma in blocking buffer were added and incubated for one hour. Plates were washed and incubated for one hour with HRP-conjugated anti-human IgG antibody (Jackson ImmunoResearch Inc.) (1:10,000 dilution). The process was stopped by adding 50µL of 1N sulfuric acid to 100L TMB substrate (Thermo Fisher Scientific, Rockford, IL, USA). BioTek Synergy 2 plate reader was used to measure OD at 450 nm (BioTek Instruments Inc., Winooski, VT, USA).