Fig. 2

Identification of the C5 clone as a spike protein/RBD binder and competing with both Spike and RBD binding to ACE2 Phage panning of the ASyNAL library against antigens RBD, S1 and spike protein respectively generated eight nanobody clones. Clone B7 is for RBD, A11 is for S1 and C5, D3, D8, D9, F12, and G6 are for spike protein. The cross-reactivities of the eight clones to Spike A, S1 B and RBD C were determined by phage ELISA. Milk or Fc were applied as negative control in ELISA. Two replicates were performed, and the p-value was performed with Student’s t-test (ns, no significant; *p < 0.05; **p < 0.01; ***p < 0.001). Competitive ELISA showed that C5 phage and ACE2 competed with each other to bind RBD D and the full-length spike protein E. As controls, B7 phage and D3 phage had no competition. F Binding curve of C5 protein to RBD in BLI assay. The affinity is 18 nM