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.
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.
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