S1 s2 trimer his recombinant protein

Binding ELISAs were used to confirm the epitope specificities of DMAbs 2130, 2196 and 2381. NUNC 96-well MaxiSorp plates were coated with recombinant RBD proteins (3 µg ml⁻¹ in 1× PBS) containing mutations at residues F444A (RBD-F444A) or F486 (RBD-F486A) (AstraZeneca), which are key residues required for the binding of clones 2130 and 2196/2381, respectively. To evaluate the relative binding of each construct to different VoC, the following coating antigens were used (0.5–1 µg ml⁻¹ in 1× PBS): SARS-CoV-2 Spike RBD-His Recombinant Protein (Sino Biologicals; 40592-V08B), Spike S1(D614G)-His Recombinant Protein (Sino Biologicals; 40591-V08H3), RBD-His K417N Recombinant Protein (Sino Biologicals; 40592-V08H59), RBD-His E484K Recombinant Protein (Sino Biologicals; 40592-V08H84), RBD-His N501Y (Sino Biologicals; 40592-V08H82), Spike S1-K417N/E484K/N501Y/D614G Recombinant Protein (Sino Biologicals; 40591-V08H10), B.1.1.529(BA.1) S1 + S2 Trimer-His Recombinant Protein (Sino Biologicals; 40589-V08H26). ELISA procedure was completed as described above.

B.1.1.529 (BA.1) S1 + S2 trimer-His Recombinant Protein (Sino Biological; cat. number: 40589-V08H26) was reconstituted in sterile water (100 μL) to prepare a stock solution (1 mg/mL). Buffer exchange to 10 mM Tris pH 7.6 was performed twice through Zeba Spin Desalting Columns (Thermo Fisher Scientific; cat. number: 899882). BA.1 spike was mixed with the Fab 17T2 (1:1.5 molar ratio of spike monomer: Fab, i.e., 0.59:0.71 mg/mL, respectively) and 3 μL were kept 5 min at RT until their application onto glow-discharged holey carbon grids (Quantifoil, Au 300 mesh, R 0.6/1; cat. number: 4N1-C11nAu30). The grids were blotted and then plunged into liquid ethane using a FEI Vitrobot Mark IV at 20 °C and 95% relative humidity. Data were collected at a FEI Talos Arctica electron microscope operated at 200 kV and equipped with a Falcon III electron detector. A total of 9,615 movies (Supplementary Fig. 4a) were recorded at a defocus range of −1 μm to −2.5 μm with a pixel size of 0.855 Å; exposure time was 40 s, with a total exposure dose of 32 e/Å2 over 60 frames.