Tmb solution

To confirm the proper folding of RBD-FR and its variant (RBD-[SSG]-FR), the binding of the purified proteins with the MERS-Cov receptor hddp4 was performed by ELISA. FR only and phosphate-buffered saline (PBS) were used as negative controls. Nunc 96-well microtiter immunoplates (Thermo Fisher Scientific) were coated with 100 ng/well of hDPP4 proteins (Abcam) and incubated at 4°C overnight. The plates were washed and blocked with 150 µl/well of blocking buffer (1% BSA) for 1 h at room temperature. RBD (SSG linker, WT, 2 m, or 9 m)-FR (100 ng/well) were added for 2 h at 37°C. An anti-penta His antibody (100 µl/well; Qiagen) was serially diluted (1/100 to 1/12,800) in TBST [50 mM Tris–Cl (pH 7.4), 0.05% Tween-20], added to the wells, and incubated for 1 h at 37°C. A secondary goat anti-mouse IgG antibody conjugated with HRP in a 100-µl volume (1:5,000, Sigma-Aldrich) was added and incubated for 1 h at 37°C. The plates were washed three times with TBST at the end of each step. After washing, 100 µl/well of substrate TMB solution (BD Biosciences) were added to the well and the plates were incubated at 37°C for 30 min in the dark. 50 µl of stop solution (2 N H₂SO₄) was added to the well to stop the colorimetric reaction, and the absorbance at 450 nm was measured using an ELISA reader, FLUOstar OPTIMA (BMG LABTECH).

96-well ELISA plates were coated with RBD protein of 2 μg/mL at a volume of 100 μL/well overnight at 4 °C. The plates were washed three times with PBST (PBS with 0.1% Tween-20) and then blocked with 3% BSA at 37 °C for 1 h. Gradient-diluted sera were added to each well, and the plates were incubated at room temperature for 1 h. Then the plates were washed three times with PBST, and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. After the plates were washed three times as described above and added 100 μL/well of TMB solution (BD, USA) to all the wells. After 0.5 h at room temperature, the plates were added 100 μL of 2 M HCl to all the wells and the absorbance at 450 nm was measured with LUX 3020 microplate reader (Thermo, USA), and samples with values greater than twice those of the controls were considered positive.

Ten-fold serial dilutions of virus inoculum were absorbed into the well of confluent Vero cells for 90 min in 37°C. After the removal of virus solution, cells were overlaid with 0.75% Avicel–DMEM and incubated in a humidified incubator at 37°C and 5% CO₂ for 6 days. To visualize the plaques, the cells were fixed by the use of 4% formaldehyde and stained by the use of 0.2% crystal violet solution.
ELISA plates were coated with bovine serum antipoliovirus antibody (National Institute for Biological Standards and Control; catalog no. 234) at a concentration of 1:100. The sIPV was loaded at 2-fold dilutions, and a WHO standard IPV (National Institute for Biological Standards and Control; catalog no. 12/104) was used to produce a standard curve. Mouse monoclonal antibody against type 1 PV (Abcam; catalog no. ab47802) diluted at 1:1,000 was added for detection, and secondary anti-mouse horseradish peroxidase (HRP) antibody (Cell Signaling Technology; catalog no. 7076) diluted at 1:1,000 was loaded. TMB solution (BD Biosciences; catalog no. 555214) was used for quantification on a FilterMax F5 microplate reader (Molecular Devices; F5) after the reaction was stopped using hydrosulfuric acid.