SARS-CoV-2 S1 Antibody ELISA Assay

End-point titers of anti-S1 total IgG or its isotypes (IgG1, IgG2a and IgG2b) from immunized mice were determined by ELISA, as previously described [29 (link)]. Briefly, 96-well plates were coated with 1 μg/mL SARS-CoV-2 S1 protein (Sino Biological, Beijing, China) at 4 °C overnight. Plates were washed three times with PBS containing 0.1% Tween-20 (PBS-T) before blocking with 5% skim milk in PBS-T for 1 h at room temperature. After washing, 2-fold serial dilutions of mouse sera, starting from 1:100, were added to wells and incubated for 1 h at 37 °C. Then, peroxidase-conjugated rabbit anti-mouse IgG secondary antibodies (Sigma, Germany) were added at the recommended concentrations and incubated for 1 h at 37 °C. After extensive washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD, USA) was added for 30 min to develop a colorimetric reaction. Finally, the reaction was stopped with 0.16 M sulfuric acid, and absorbance was read spectrophotometrically at 450 nm on a Synergy 2 Multi-Detection Microplate Reader (BioTek, Winooski, VT, USA). End-point titers were determined as the reciprocals of the highest dilution with an OD above the cut-off value which was defined as the OD mean from the control group plus three standard deviations (SDs).