Human ace2 protein

Human ACE2 protein was ordered from Sino Biological (Beijing, China). Recombinant SARS-CoV-2 spike RBD proteins with His-tag from wild-type virus (wt RBD) as well as the delta variant (delta RBD) were produced by ISAR Bioscience (Planegg, Germany) with the wt RBD being taken from the S protein nucleotide sequence of the SARS-CoV-2 Wuhan Hu-1 genome (GenBank accession number MN908947, positions 22517 to 23183), while the delta RBD contained the following mutations: L452R and T478K. Details on the procedures were published before [45 (link), 46 (link)]. Shortly, CHO cells were transfected with plasmid vectors containing the DNA sequences for the RBD proteins with an added His-tag and subsequently grown at 37 °C. The supernatants were centrifuged and filtered and subsequently purified using HisTrap columns. Protein content after elution was determined by OD₂₈₀ measurement and the relevant fractions were dialyzed.
The biotinylation of wt RBD and human ACE2 was done using the EZ-Link Micro Sulfo-NHS-LC biotinylation kit (Thermo Scientific #21935 or #A39257) with 20-fold molar excess according to the standard procedure instruction, followed by removal of excess biotin by dialysis against 1 L PBS for 16 h at 4 °C, using Slide-A-Lyzer™ Dialysis Cassettes, 7 K MWCO, 0.5 mL (Thermo Scientific #66373).

A 96-well ELISA plate (Corning, New York, USA) was coated with 25 μl of 2 μg/ml human ACE2 protein (Sino Biological, Beijing, China) and incubated overnight at 4°C. The coated plate was washed three times with 1xPBST (1xPBS with 0.05% Tween20) and blocked with 5% skim milk for 1 h at 37°C. Then, the plate was washed three times with 1xPBST and incubated with 0.02–5.00 μg/ml of purified plant-produced SARS-CoV-2 RBD-Fc or Alpha RBD-Fc or Beta RBD-Fc or Fc protein for 2 h at 37°C. The plate was washed three times with 1xPBST and incubated with HRP-conjugated goat anti-human IgG diluted 1:2,000 in 1xPBS for 1 h at 37°C. The plate was washed and the signal was detected with 25 μl of TMB substrate (Promega, Wisconsin, USA) and stopped with 25 μl of 1M H₂SO₄. The absorbance was measured at 450 nm using SpectraMax M5 (Molecular devices, California, USA). The experiment was performed in three replicates and the data are presented as mean±SD.