Goat anti mouse igg antibody conjugated with hrp

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).