Enzyme-linked immunosorbant assay (ELISA)
To determine the total IgG antibodies against the candidate vaccine mCuMVTT-RBM in sera of vaccinated mice, ELISA plates were coated with SARS-CoV-2 RBD wildtype or with SARS-CoV-2 Spike (Sinobiological, Beijing, China) at concentrations of either 1µg/ml or 0.1 µg/ml overnight. ELISA plates were washed with PBS-0.01% Tween and blocked using 100µl PBS-Casein 0.15% for 2h in RT. Sera from vaccinated mice serially diluted 1:3 starting with a dilution of 1:20 and incubated for 1h at RT. After washing with PBS-0.01%Tween, goat anti-mouse IgG conjugated to Horseradish Peroxidase (HRP) (Jackson ImmunoResearch, West Grove, Pennsylvania) was added at 1/2000 and incubated for 1h at RT ELISA was developed with tetramethylbenzidine (TMB), stopped by adding equal 1 M H2SO4 solution, and read at OD450 nm or expressed as Log OD50. Detecting RBD-specific IgGs against mutated RBDs was carried out in a similar way.
IgG subclasses were measured from sera collected on day 42 following the same described ELISA protocol. The following secondary antibodies were used: goat anti-mouse IgG1-HRP and goat anti-mouse IgG2a-HRP (1:1000) (Thermo Fischer Scientific, Waltham, Massachusetts), goat anti-mouse IgG2b-HRP (SouthernBiotech, Birmingham, Alabama) 1:4000, rat anti-mouse IgG3-HRP (Becton, Dickinson, Franklin Lakes, New Jersey) 1:2000.
To detect IgA antibodies the plates were coated with 1µg/ml SARS-CoV-2 RBD wildtype protein and goat anti-mouse IgA POX (ICN 55549, ID 91, 1:1000 dilution) as the secondary antibody was used. An additional step prior to serum incubation was added in order to deplete IgG. 10µl of Protein G beads (Invitrogen, USA) were transferred into a tube and placed into a magnet. The liquid was removed and the 75.6µl diluted sera in PBS-Casein 0.15% was added to the beads and mixed. The tube was incubated on a rotator at RT for 10 minutes. The tubes were placed back into the magnet and ELISA was carried out as described above. Analysis and graphs were created using GraphPad PRISM 9, Version 9.1.0 (216), March 15, 2021.
To determine RBD-specific IgG antibodies against the candidate vaccine mCuMVTT-RBM in sera of vaccinated rabbits, ELISA plates were coated with 2µg/ ml SARS-CoV-2 RBDwildtype in PBS overnight at +4°C. ELISA plates were washed 4 times with PBST (1x PBS / 0.05 % (v/v) Tween 20) and plates were blocked with 200 μL/well Blocking buffer (ThermoFisher, Life Technologies Europe BV, Zug Switzerland). Plates were incubated at +22°C between 2-4 h. After removal of the blocking buffer and a washing step, diluted internal standard (1 µg/mL) and the pre-diluted immune sera (1:50) were added to the wells of the first column on the ELISA plate (150 µL/well). The internal standard and the immune sera were then serially 3-fold diluted in dilution buffer (2% BSA/ PBST). Plates were incubated for 2 h at +22°C and washed. 100 µL/well of detection antibody (1:10’000 diluted in dilution buffer, Peroxidase-conjugated polyclonal goat anti- rabbit IgG (H+L) (Jackson Immuno Research, cat no 111-035-144) was added and incubated for 2h at +22°C. After rigorous washing, 100 µl /well of the OPD detection solution (POD tablets (Sigma cat. no. P6912), phosphate citrate tablets (Sigma cat. no. P4809), 30% H2O2 (Sigma cat. No. 1009)) was added and reaction was stopped by addition of 50µL/well 2 M H2SO4. Absorption at 490 nm was quantified in each well using the plate reader Spark 10M (Tecan).