Evaluation of nAb-binding activities

The binding activities of the recombinant mutI-tri-RBD and homo-tri-RBD proteins with two monoclonal nAbs, including MM43 and R117 (Sino Biological Inc., China. Cat: 40591-MM43 and 40592-R117), were evaluated by using ELISA. As controls, the binding activities with the monoclonal nAbs for the monomeric his-tagged RBDs from the prototype, Beta (B.1.351) and Kappa (B.1.617.1) SARS-CoV-2 strains were also measured. To detect the binding activity with the anti-RBD monoclonal nAbs, the protein samples were prepared at the starting concentration of 1.0 µg/mL, which were then subjected to 2-fold serial dilutions and coated on the 96-well microplate with 100 µL per well at 2–8 °C overnight. Subsequently, the plate was washed 3 times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and then blocked with 100 µL blocking buffer per well, followed by incubation at 37 °C for 2 h. After washing the plate 3 times with PBST, the nAb sample was diluted to 1.0 µg/mL and added into the wells with 100 µL per well, which was then incubated at 37 °C for 1 h. After that, the plate was washed 3 times with PBST and the horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit IgGs at 1:10,000 dilution was added into the wells of the plate. The plate was again incubated at 37 °C for 1 h and washed 3 times with PBST. Subsequently, 50 µL tetramethylbenzidine (TMB) and 50 µL hydrogen peroxide solutions were added to start the color reaction. After color development for 5 min, the reaction was stopped using 0.2 M sulfuric acidic solution with 50 µL per well, and absorbance at 450 nm was read by plate reader. The background absorbance at 630 nm was also measured, and then the difference between absorbance at 450 nm and 630 nm (OD_450/630nm) was obtained to detect the specific binding of the mutI-tri-RBD and homo-tri-RBD proteins with the nAbs.