Affinity Measurement of SARS-CoV-2 RBD Variants

The human ACE2 protein was purchased from Sino Biological (Beijing, China; Cat# 10108-H08H) and the human IgG1 Fc-tagged RBD proteins were made in-house using a method as previously described^{28 (link)}. The affinity measurement was performed on the Forte bio Octet RED 96 system (Sartorius, Goettingen, Germany). Briefly, wt or N501Y mutant RBD proteins (20μg/ml) were captured onto protein A biosensors for 300s. The loaded biosensors were then dipped into the kinetics buffer for 10s for adjustment of baselines. Subsequently, the biosensors were dipped into serially diluted (1.23~300nM) human ACE2 protein for 200s to record association kinetics and then dipped into kinetics buffer for 400s to record dissociation kinetics. Kinetic buffer without ACE2 was used to correct the background. The Octet Data Acquisition 9.0 software was used to collect affinity data. For fitting of K_D values, Octet Data Analysis software V11.1 was used to fit the curve by a 1:1 binding model and use of the global fitting method.