Figure 2

Characterization of anti-SARS-Cov-2 antibodies with different valencies by surface plasmon resonance. The binding potencies of the anti-SARS-Cov-2 tetrameric Quad proteins was determined using surface plasmon resonance. Purified proteins were captured using immobilized SARS-Cov-2 RBD, carried out with a Biacore T200 instrument. (A) Anti-SARS-Cov-2 antibodies were expressed by transfection and secretion from Expi293 suspension cells and characterized by SDS-PAGE following purification by Ni-affinity chromatography and gel filtration. Gels were stained with InstantBlue Ultrafast Protein Stain and Mw size markers included (left hand lanes). Uncropped gels are shown in Supplementary Fig. 7. (C–I) Antibody binding to SARS-Cov-2 RBD was evaluated using SPR. Biotinylated SARS-CoV-2 RBD was captured on a streptavidin chip and antibodies flowed over the surface at different concentrations. CR3022 and CR3014 antibody concentrations were 10, 5, 2.5, 1.25, 0.625 and 0.312 nM, while those for H4 and B38 antibodies were 12.5, 6.25, 3.125, 1.56 and 0.78 nM (the H4-Fab-TD has four curves 6.25, 3.125, 1.56 and 0.78 nM). Sensorgrams representative of two independent experiments are shown for (C) CR3022-Fab-TD, (D) CR3022-IgG, (E) CR3022-Fab, (F) H4-Fab, (G) H4-Fab-TD, (H) H4-scFv-TD and (I) B38-Fab-TD. Kinetic parameters are shown in Supplementary Table S1. Tetramerization decreases dissociation rates for the multimeric species compared to the monomeric proteins. This results in increased affinity for SARS-Cov-2 RBD for CR3022-Fab-TD compared to CR3022-Fab, and for H4-Fab-TD and H4-scFv-TD compared to H4-Fab.