Figure 5. Epitope‐binning with the indicated VHH antibodies.

1. HeLa cells were transiently transfected to express the SARS‐CoV‐2 Spike protein. Following fixation, cells were stained for 1 h with fluorophore‐labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.
2. ACE2 competition experiments with selected VHHs from A. 50 nM RBD was mixed with indicated nanobodies (at 500 nM). Binding to ACE2 (immobilized on the sensors) was monitored by BLI.