Ab273687

ELISA microplates (Greiner bio-one) were coated with 4 μg/mL (50 μL/well) of recombinant human ACE2 (ab273687, Abcam). Following an overnight incubation at 4 °C, plate was washed 3 times with PBST and blocked with 100 μL 5% FBS/PBST for 1 h at RT. After washing, RBD protein (2.5 μg crude protein in DMEM) prepared in a final volume of 100 μL in 3 different conditions (i) RBD alone, (ii) RBD mixed with monkey serum (diluted 1:500), and (iii) RBD mixed with monkey serum and peptide, was added into each well and incubated for 1 h. The experiment was conducted in triplicate. After a 3-time wash, mouse anti-V5 antibody (Invitrogen) diluted 1:2,000 in 1% FBS/PBS was added and the plate was incubated for 1 h at RT. After washing, goat-anti mouse IgG-HRP (Abcam) diluted 1:100,000 was added to the well (100 μL/well) and incubated for one hour. The following steps followed ELISA method as described above. Percent change was calculated based on the formula: 100 x (OD of RBD with serum and peptide - OD of RBD with serum)/(OD of RBD – OD of RBD with serum).

The binding affinity of the plant-produced RBD to ACE2 was analyzed by ELISA. Briefly a 96-well plate (Greiner Bio-One GmbH, Germany) was coated with 2 μg/ml of commercially available ACE2 (ab273687, Abcam, UK) and incubated overnight at 4ºC. After incubation, the coating buffer was discarded and the plates were blocked with 5% skim milk in 1XPBS for 2 h at 37 °C. The plate was then washed three times with 1X PBST and incubated with the plant-produced RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD protein (R & D Systems, USA) for 2 h at 37 °C and non-infiltrated (WT) plant protein was used as a control. Then the plates were washed three times with 1X PBST followed after which an anti 6xHis antibody (ab1187, Abcam, UK) diluted (1:1000) in 1X PBS was added to the plate which was then incubated for 1 h at 37 °C. Finally the plate was washed with 1X PBST, and the signal developed with TMB substrate (Promega, USA) and the absorbance read at 450 nm.