Ace2 his

RBD-Fc (0.2 μg/well) was coated in a microplate in 100 μL of coating buffer (pH 9.6) at 4 °C overnight. Then, 100 μL/well 2% BSA-PBST was incubated for 1.5 h for blocking. After washing 3 times, a serial phage solution was added and incubated with RBD-Fc for 1 h. Anti-M13/HRP conjugate (diluted 1/10,000 in 2% BSA/PBS dilution) and TMB were used to amplify the signals and to develop color, respectively. After stopping the reaction via 1.0 M HCl, the absorbance was measured at 450 nm. After that, the sub-saturation (80% of maximal effect) concentration of the phage solution was used for competitive ELISA.
RBD-Fc (0.2 μg/well) was coated for competitive phage ELISA. After blocking for 1.5 h and washing 3 times, the sub-saturation concentration of VHH-phage solutions was mixed with 4 μg/mL ACE2-His (SinoBiological). The mixture of phage and ACE2-His was added to wells coated with RBD-Fc and incubated for 1 h, and the following steps were as described previously.

Human chimeric SARS-CoV-2 (2019-nCoV) Spike-RBD/Fc, SARS-CoV-2 (2019-nCoV) Spike-RBD/His and ACE-2/His proteins were purchased from Sino Biological, Dusseldorfer Eschborn, Germany. Human chimeric SARS-CoV-2 Omicron-RBD/Fc and IgG1 Fc proteins were purchased from R & D Systems, Minneapolis, MN, USA. Human chimeric ACE-2/Fc and SARS-CoV-2 Omicron-RBD/His proteins (from GenScript, Piscataway, NJ, USA) were also used.
Sotrovimab (from GlaxoSmithKline and Vir Biotechnology), Casirivimab and Imdevimab (from Regeneron Pharmaceuticals) mAbs were used. D3 mAb was expressed and purified as previously described [22 (link)].
HRP-conjugated anti-His antibody (Proteintech, Deansgate, Manchester, UK), anti-human IgG (Fab’)2 goat polyclonal antibody (Abcam, Banzarate, MI, Italy), anti-human Fc antibody (Sigma, St. Louis, MO, USA), and HRP-conjugated streptavidin (Biorad, Segrate, MI, Italy) were used for the detection of primary mAbs.
Human peptides derived from RBD portion of human SARS-CoV-2 Spike-RBD protein were synthesized from GenScript (Piscataway, NJ, USA) with the following aa sequences:
Peptide 1 Sequence: RKSNLKPFER;
Peptide 2 Sequence: GVEGFNCYFP;
Peptide 3 Sequence: RFASVYAWNRK;
Peptide 4 Sequence: RVQPTESIVR.

Compounds were diluted to the appropriate concentrations (1–100 μM) with PBS. Purified SARS-CoV-2 RBD peptide (Sino Biological, Beijing, China) was conjugated with biotin using EZ-LinkTM Sulfo-NHS-Biotin (Genemore, Beijing, China) following the protocol reported by the manufacturer. The biotinylated SARS-CoV-2 RBD peptide was immobilized onto Super Streptavidin (SSA) biosensors (Fortebio, Fremont, CA, USA). ACE2-His (Sino Biological, China) was immobilized onto the biosensor coated with nickel-nitrilotriacetic acid (Ni-NTA, Fortebio, USA). After washing (60 s) and baseline step with PBS containing 2% DMSO (Merck, Darmstadt, Germany), the biosensor tips were immersed into the wells containing serial dilutions and allowed to associate (300 s). A dissociation step was then performed (300 s). K_D values were calculated using a 1:1 binding model in Data Analysis Software 9.0 (Fortebio, CA, USA), using k_dis as the dissociation constant (s⁻¹) and k_on as the association constant (1/Ms). K_D is computed from k_dis/k_on.