Fig. 2: Biochemical characterization of designed VHHs.

a,b, Nine thousand designed VHHs were screened against RSV site III (a; VHH_RSV_01) and influenza haemagglutinin (b; VHH_flu_01) with yeast surface display, before soluble expression of the top hits in E. coli. SPR demonstrated that the highest affinity VHHs to RSV site III and influenza haemagglutinin bound their respective targets with 1.4 μM and 78 nM, respectively. c, Nine thousand VHH designs were tested against the SARS-CoV-2 RBD, and after soluble expression, SPR confirmed an affinity of 5.5 μM to the target for design VHH_RBD_D4 (left). Binding was to the expected epitope, confirmed by competition with a structurally confirmed de novo binder (AHB2 (PDB ID 7UHB), right). d, Ninety-five VHH designs were tested against C. difficile TcdB. The highest affinity VHH, VHH_TcdB_H2, bound with 262 nM affinity (left), and also competed with a structurally confirmed de novo binder (FZD48, PDB ID 9CM5 (ref. ²⁹)) to the same epitope (right). See also Extended Data Fig. 4a–c for quantification of the competition shown in panels c,d. For all panels, the measured binding response is indicated in a solid blue line, and the global fit using a 1:1 binding interaction model is indicated with a black dashed line.