Fig. 2. Cross-reactivity of anti-SARS-CoV-2 S V_HHs.

Data are organized based on V_HH subunit/domain specificity and epitope bin designation (see Fig. 1d). a ELISA showing the cross-reactivity of V_HHs against various coronavirus spike glycoprotein fragments, S, S1, S2, RBD and NTD. Shades of red represent binding, colorless boxes represent no binding. Assays were performed at a single V_HH-Fc concentration. The “isotype” control (A20.1 V_HH-Fc) shows no binding to S. Anti-SARS-CoV VHH-72 and ACE2-Fc were included as references. The phylogenetic tree of spike glycoproteins was constructed using MEGA11⁹⁸. Source data used to generate the figure are included in Supplementary Data 1. b, c On-/off-rate maps summarizing V_HH kinetic rate constants, k_as and k_ds determined by SPR for the binding of V_HHs to SARS-CoV S (b) and SARS-CoV-2 Alpha and Beta S (c). Diagonal lines represent equilibrium dissociation constants, K_Ds (see also Table 1). S2F3 cross-reacted to Alpha and Beta, as did S2G4 and MRed22 to Beta; however, poor fitting of SPR data precluded determining their k_as, k_ds and K_Ds, hence their exclusion from relevant graphs. Maps were constructed using the V_HH binding data from Supplementary Fig. 7 and Supplementary Table 4. Anti-SARS-CoV S VHH-72 and the monomeric ACE2 (ACE2-H₆) are included as benchmark/reference binders.