Figure 2.

Screening by ELISA and expression of anti-Spike scFvs for analysis of their binding to Spike RBD and their competition with ACE2. (a) Representative image of scFv-phages screening by ELISA to test their binding to Spike-RBD recombinant protein. (b) Screening of positive phage clones by ELISA assay on human Spike RBD-Fc recombinant protein (black bars) or human recombinant IgG Fc used as a negative control in parallel assays (grey bars). (c) Western blotting analysis with the anti-c-myc antibody of the periplasmic extracts of the cells transformed with the two selected positive clones, D3 and F12, expressed in the absence or in the presence of IPTG, used for induction (The blot was obtained by grouping two different parts of the same blot and the black line has been inserted to indicate the two distinct parts. The corresponding full-length blot has been inserted in Supplementary Data set as full-length blot of Figure 2. The samples were processed in parallel in the same experiment). (d) Representative image of soluble scFvs binding to Spike-RBD recombinant protein by ELISA (e) at two different concentrations: 45 nM (grey bars) or 90 nM (black bars). (f) Representative image of soluble scFvs interference in Spike-ACE2 interaction tested by ELISA (g). The binding of ACE2-His to immobilized Spike RBD protein in the absence (dark grey bars) or in the presence (light grey bars) of the indicated soluble scFvs (90 nM). (h) Competitive ELISA assay was performed by measuring the binding of Spike RBD-Fc protein on ACE2-positive VERO E6 cells in the absence or in the presence of D3 and F12 scFvs.