SPR Analysis of RBD-ACE2 Binding Affinity

The binding affinity between recombinant His-tagged RBD and Fc-tagged ACE2 was measured by SPR using the Biacore S200 system (Cytiva) as previously described [16 (link),17 (link)]. Each ACE2 protein (in the amount of 4.85 μg) was immobilized onto the Sensor Chip Protein A (Cytiva). Then, the RBD protein at concentrations of 40, 80, 160, 320 and 640 nM was injected over the sensor surface, bound to immobilized ACE2 protein and washed off the surface. The running buffer used for all proteins in the SPR assays consisted of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20, adjusted to pH 7.4. The apparent equilibrium dissociation constant (Kd) was measured using the Biacore analytical software (Cytiva). For this, a one-to-one Langmuir binding model was used for Fc-tagged proteins that were immobilized, with monomeric proteins flown over them. All measurements were independently repeated for biological triplicates.