Ace2 assay kit

To evaluate the specificity of ACE2 substrate cleavage activity by the plasma, we designed tiled SARS-CoV-2 spike protein RBD peptides which covered RBD amino acid sequence from residues Arg319 to Phe541 including receptor-binding motif from Ser438 to Gln506 based on previous comprehensive structure analysis on viral-host interaction (37 (link)) and had them synthesized by SB-PEPTIDE (SmartBioscience SAS, France; Table 2). Plasma samples and positive control, purified recombinant human ACE2 protein provided in the ACE2 assay kit (Abcam, Cat. #ab273297), were incubated with or without fivefold serially diluted peptide pools at room temperature for 15 min before adding the substrate. Samples incubated with ddH₂O and dimethyl sulfoxide (DMSO) which were equivalent to the concentrations used in the peptide pool reconstitution served as negative controls. Then 50 µL of ACE2 substrate (pre-diluted according to the manufacturer’s protocol) was added into the wells, and the samples were assayed. RFUs were measured, and data were analyzed as described below.

To evaluate whether native ACE2 shed into blood (38 (link), 39 (link)) could contribute to the catalytic activity observed in the patient plasma samples, we investigated whether EDTA present in the plasma samples inhibited the activity of recombinant ACE2 spiked into the samples. EDTA can inhibit the catalytic activity of ACE2, a Zn²⁺ metaloprotease (40 (link), 41 ).
We conducted serial 10-fold dilutions of the normal donor plasma in ACE2 assay buffer from the assay kit and then added 2 µL recombinant positive control ACE2, provided as a positive control in the ACE2 assay kit (Abcam, Cat. #ab273297) to each well to the samples to the samples according to the manufacturer’s protocol, conducting the ACE2 assays on the serially diluted samples with the added recombinant ACE2. We also added ZnCl₂ to some samples to achieve final concentrations ranging from 0 to 2 mM. Samples containing plasma, recombinant ACE2, and ZnCl₂ mixture with pre-diluted ACE2 inhibitor served as negative comparisons. Then, 50 µL of the substrate (pre-diluted according to the manufacturer’s protocol) was added into the wells. RFUs were measured, and data were analyzed as described below.