Quantifying SARS-CoV-2 Spike RBD Affinities

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^{35 (link)}. The affinity measurement was performed on the ForteBio Octet RED 96 system (Sartorius, Goettingen, Germany). Briefly, the RBD proteins (20 μg/ml) of Alpha or Delta RBDs were captured onto protein A biosensors for 300s. The loaded biosensors were then dipped into the kinetics buffer for 10 s for adjustment of baselines. Subsequently, the biosensors were dipped into serially diluted (from 1.23 to 300 nM) human ACE2 protein for 200 s to record association kinetics and then dipped into kinetics buffer for 400 s 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 using the global fitting method.