Figure 5.

Competitive binding assay in cells. The binding affinity of the sACE2-Fc’s in a cellular condition was estimated by a competitive assay. The wild-type (a) and the M2 mutant (b) sACE2-Fcs were added to the spike protein/ACE2 binding assay (see the text). The Y-axis represents the percentage of the spike protein bound to the surface ACE2 on the cell. The X-axis represents different concentrations of sACE2-Fc used in the assay. Bars 1–8 in (a) and (b) represent 0.1, 0.3, 0.8, 2.5, 7.4, 22.2, 66.7, and 200 nM of the wild-type (a) and the mutant sACE2-Fc (b) proteins, respectively. The amount of the spike protein bound to sACE2-Fc decreased in a dose-dependent manner. The assay was triplicated, and the standard deviation is indicated in each bar. In the assay, we prepared the 200 nM protein solution and serial-diluted it three-fold each time. Thus, the lowest concentration was 0.1 nM (bar 1), not 0.0.