The antigen-specific IgG titres in mouse sera were assessed by a semi-quantative ELISA. MaxiSorp high binding ELISA plates (Nunc) were coated with 100 μL per well of 1 μg/mL recombinant SARS-CoV-2 protein with the pre-fusion stabilized conformation in phosphate-buffered saline (PBS). For the standard IgG, three columns on each plate were coated with 1 in 1000 dilution each of goat anti-mouse κ- and λ-light chains (Southern Biotech).
After overnight incubation at 4 °C, the plates were washed four times with PBS–Tween 20 0.05% (v/v) and blocked for 1 h at 37 °C with 200 μL per well blocking buffer (1% bovine serum albumin (w/v) in PBS–Tween-20 0.05%(v/v)). The plates were then washed and the diluted samples or a fivefold dilution series of the standard IgG added using 50 μL per well volume. Plates were incubated for 1 h at 37 °C, then washed and secondary antibody added at 1 in 2000 dilution in blocking buffer (100 μL per well), and incubated for 1 h at 37 °C. After incubation and washes, plates were developed using 50 μL per well SureBlue TMB (3,3′, 5,5′-tetramethylbenzidine) substrate and the reaction stopped after 5 min with 50 μL per well stop solution (Insight Biotechnologies). The absorbance was read on a Versamax Spectrophotometer at 450 nm (BioTek Industries). Statistical analyses were performed on log-transformed data.
After overnight incubation at 4 °C, the plates were washed four times with PBS–Tween 20 0.05% (v/v) and blocked for 1 h at 37 °C with 200 μL per well blocking buffer (1% bovine serum albumin (w/v) in PBS–Tween-20 0.05%(v/v)). The plates were then washed and the diluted samples or a fivefold dilution series of the standard IgG added using 50 μL per well volume. Plates were incubated for 1 h at 37 °C, then washed and secondary antibody added at 1 in 2000 dilution in blocking buffer (100 μL per well), and incubated for 1 h at 37 °C. After incubation and washes, plates were developed using 50 μL per well SureBlue TMB (3,3′, 5,5′-tetramethylbenzidine) substrate and the reaction stopped after 5 min with 50 μL per well stop solution (Insight Biotechnologies). The absorbance was read on a Versamax Spectrophotometer at 450 nm (BioTek Industries). Statistical analyses were performed on log-transformed data.