Tmb solution

The binding activity of the purified plant-produced ACE2-Fc to SARS-CoV-2 RBD was analyzed by ELISA. 96-well plate (Greiner Bio-One GmbH, Austria) was coated with 100 ng of plant-produced ACE2-Fc and incubated overnight at 4°C. The wells were blocked using 5% skim milk in 1xPBS for 2 h at 37°C. The plate was washed three times with 1xPBST and incubated with various dilutions of proteins including the RBD of SARS-CoV-2 produced from Sf9 cells (Genscript Biotech, United States), S1 protein of porcine epidemic diarrhea virus (PEDV) (Supplementary Figure S1), and PBS as negative controls for 2 h at 37°C. After washing, an anti-6X His tag-HRP fusion (Abcam, UK) diluted in 1xPBS was added into the wells and incubated for 2 h at 37°C. For detection, a TMB solution (Promega, United States) was added into the plate. The enzymatic reactions were stopped by adding 1M H₂SO₄. The absorbance at 450 nm was measured using a 96-well microplate reader (Molecular Devices, United States).

Total chicken and penguin IgY were purified using the Pierce™ Chicken IgY Purification Kit kit (Thermo Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Purified chicken or penguin IgY were loaded on a 4-15% Mini-protean TGX stain-free gel (Bio-Rad, Hercules, USA) and separated by SDS-electrophoresis 1 hour at 120V by duplicated. One of the gels was used for protein staining with Coomassie blue. The other gel was used for protein transference onto Trans-Blot Turbo Transfer Packs membrane (Bio-Rad, Hercules, USA). The membrane was blocked 2 hours with 1% HSA (Sigma-Aldrich, St. Louis, MO, USA)/PBS at RT, washed three times with PBST, and incubated at 4°C overnight with HRP-conjugated goat anti-turkey IgY (Mybiosource, San Diego, CA, USA), or goat anti-chicken IgY (Sigma-Aldrich, St. Louis, MO, USA) antibodies dilution 1:400 in PBS. Immunoreactive proteins were visualized with TMB solution (Promega, Madison, WI, USA).

The binding activity of plant-produced SARS-CoV-2 RBD-Fc fusion protein to ACE2 protein was demonstrated by ELISA. Briefly, 96-well ELISA plate was coated by 100 ng of two different ACE2 protein derived either from HEK293-cells (Abcam, United Kingdom) or CHO-cells (InvivoGen, California, United States). For blocking, 5% skim milk in 1xPBS was added into the wells and incubated for 2 h at 37°C. After blocking, the plate was washed three times with 1xPBST (1xPBS plus 0.05% Tween-20) and incubated with various concentrations of plant-produced SARS-CoV-2 RBD-Fc fusion protein in 1xPBS. The SARS-CoV-2 RBD proteins in the wells were detected by addition of 1: 100 dilution of plant-produced anti-SARS-CoV-2 (H4) mAb (Shanmugaraj et al., 2020b (link)) and followed by a 1:1,000 dilution of anti-human Kappa chain-HRP fusion (SouthernBiotech, United States) in 1xPBS for 1 h at 37°C. For colorimetric development, a TMB solution (Promega, United States) was added into the wells followed by addition of 1M H₂SO₄ for terminating the enzymatic reaction. The absorbance at 450 nm was measured using a 96-well microplate reader (Molecular Devices, United States).