Kinetic Characterization of SARS-CoV-2 S-RBD Binding to hACE2

The recombinant S-RBD (Sino Biological, Beijing, China) and hACE2 protein (Sino Biological, Beijing, China) were used for surface plasmon resonance (SPR) analysis using a Biacore 8K instrument (Biacore, Uppsala, Sweden) as previously described (Chu et al., 2018a (link); Chu et al., 2018b (link)). Each target was immobilized onto flow cells in a CM5 sensor chip (GE Healthcare) via the amine-coupling method. Briefly, S-RBD was diluted in 10 mM pH 5.5 acetate to 20 µg/ml, while hACE2 was diluted in 10 mM pH 4.5 acetate to 20 µg/ml. Then the protein solutions were injected individually on the carboxyl modified sensor surface to form amine bonds. Both S-RBD and hACE2 immobilized levels were about 10,000 RU. Binding analyses were carried out at 25 °C and a flow rate of 30 μl min⁻¹. The retrieved drugs in a running buffer (1×PBS, 0.05% Tween 20 and 5% dimethyl sulfoxide, pH 7.4) were run over each target at gradient concentrations as indicated. An empty flow cell, without any immobilized protein, was used as a deducted reference. The binding curves were analyzed using a kinetic binding model supplied with Biacore Evaluation Software (GE Healthcare).