Mers cov s1 protein

Surface plasmon resonance measurements were performed using a BIAcore X100 instrument (GE Healthcare). MERS-CoV S1 protein (Sino Biological Inc.) diluted in 10 mM sodium acetate buffer (pH 5.5) was immobilized on a CM5 biosensor chip using a primary amine coupling method. The running buffer was allowed to flow through the cells at a rate of 30 μl min⁻¹. The analytes consisted of serial dilution of proteins between 500 to 0.05 nM (500, 50, 5, 0.5 and 0.05 nM).

The end-point titers of anti-S1 total IgG Ab as well as IgG1, IgG2a and IgG2b isotypes from immunized mice were determined by ELISA as described previously29 (link)50 (link) with minor modifications. Briefly, 96-well plates (EU Immulon 2 HB, Thermo Scientific) were coated with the MERS-CoV S1 protein (Sino Biological) at 2 μg/ml in PBS at 4 °C overnight. Plates were then washed 6 times with PBS containing 0.05% Tween-20 (PBS-T), followed by blocking with 5% skim milk in PBS-T for 1 h at 37 °C. After washing, plates were incubated with a 2-fold serial dilution of mouse sera starting from 1:100 and incubated for 1 h at 37 °C. Then, plates were washed and incubated with peroxidase-conjugated rabbit anti-mouse IgG, IgG1, IgG2a or IgG2b secondary Abs (Jackson Immunoresearch Laboratories) at concentrations recommended by the supplier and incubated for additional 1 h at 37 °C. After extensive washing, Tetramethylbenzidine (TMB) substrate (KPL) was added for 30 min for colorimetric development and the reaction was stopped with 0.16 M sulfuric acid. Absorbance was read spectrophotometrically at 450 nm. End-point titers were determined and expressed as the reciprocals of the final detectable dilution with a cut-off defined as the mean of pre-bleed samples plus three SD.