Fig. 3. Binding to hACE2 by human anti-hACE2 antibodies.
a, Parental A549 cells (top) or A549 cells stably expressing hACE2 (bottom) were incubated in the presence (solid lines) or absence (dotted lines) of the indicated hACE2 antibodies. The cells were then incubated with Alexa Fluor 488 conjugated goat anti-human IgG and then analysed by flow cytometry. b, Four anti-hACE2 mAbs (2G7A1, 05H02, 05B04 and the hybrid antibody 05B04LC/05D06HC) were immobilized onto a Protein G Sensor chip. His-tagged hACE2 1–740aa proteins (7.8 nM, 31.2 nM, 125 nM or 500 nM) were injected at 30 µl min−1 for 240 s followed by a dissociation phase of 2,400 s at a flow rate of 30 µl min−1. KD values were calculated from the ratio of association and dissociation constants (KD = kd/ka), derived using a 1:1 binding model. RU, resonance unit. c, Schematic representation of the spike–hACE2 binding assay in which NanoLuc luciferase was appended to the C-termini of a conformationally stabilized SARS-CoV-2 spike trimer, S-6P-NanoLuc (based on Wuhan-hu-1, or Omicron BA.1 variants). The fusion protein was incubated with His-tagged hACE2 (1–740aa) that was pre-incubated in the presence or absence of anti-hACE2 mAbs, and complexes were captured using His-tag magnetic beads. d, Serially diluted mAbs (2G7A1, 05B04, 05H02 or 05B04LC/05D06HC) were mixed with 100 ng of His-tagged hACE2 1–740aa proteins. After incubation, the mixture was then incubated with Wuhan-hu-1 or Omicron S-6P-NanoLuc proteins (10 ng) followed by capture on His-tag magnetic beads. Bound NanoLuc activity was measured after washing. Mean and range of four independent experiments is plotted.