Elisa maxisorp plates

Speci c antibodies were determined by an optimized indirect ELISA method. Brie y, 100µl of 5µg/ml of inactivated SARS-COV-2 in PBS were added into 96-well ELISA Maxisorp plates (Greiner, Germany) and incubated overnight at 4°C. The wells washed with PBS containing 0.1% Tween 20 (washing buffer) and blocked 1 hr at 37°C with 2% skimmed milk in PBS + 0.1% Tween 20 (blocking buffer). The plates were washed with washing buffer and 100 µl of 1/25 diluted sera up to 16 serial dilutions were added into each wells and incubated at 37°C for 90 min. The wells washed ve times with washing buffer and incubated for 2 hrs with 100 µl of 1/8000 dilution of anti-mouse conjugated to HRP (Razirad, Iran). The wells washed ve times and incubated 30 min with 100 µl of TMB substrate in the dark and the reaction was stopped with 100 µl of 2N H2SO4 and colour density was measured at 450 nm with ELISA plate reader. The cut off of ELISA for each titer was calculated on 10 sera samples of sham group by formula: Mean+3SD of these samples and based on the cut off the titer of each mouse of the experiments was earned. The nal results of antibody titer for each mouse presented as Log10 of antibody titer. In addition, detection of speci c IgG1 and IgG2a subclasses were carried out using goat anti-mouse IgG1and IgG2a secondary antibodies (Sigma, USA) according to the manufacture's instruction.

The speci c IgG antibody response against RBD protein was determined by a laboratory-optimized indirect ELISA. At the rst for antigen coating, 100 µl of 2µg/ml of RBD protein (The Native Antigen Company, UK) in carbonate-bicarbonate buffer with pH 9.6 was added into 96-well ELISA Maxisorp plates (Greiner, Germany) and incubated overnight at 4°C. The wells were washed with PBS containing 0.1% tween 20 (washing buffer) and then blocked 1 hr at 37°C with 1.5% BSA in PBS + 0.05% tween 20 (blocking buffer). The plates were washed 5 times with washing buffer and 100 µl of 1/50 diluted sera of experimental vaccinated and sham group of mice were added into each well and incubated at 37°C for 90 min. The wells were washed 5 times with washing buffer and incubated for 90 min with 100 µl of 1/8000 dilution of anti-mouse HRP-conjugate (Razirad, Iran). The wells washed 5 times and incubated 10min with 100 µl of TMB substrate in the dark and the reaction was stopped by adding 100 µl of 2N HCL and colour density was measured at 450 nm with ELISA plate reader. The cutoff of RBD-ELISA was calculated on the sera samples of sham group by formula: Mean+3SD and then IgG response to RBD for individual mouse was reported by formula; OD of RBD ELISA of individual mouse/cutoff.