ELISA assay for soluble ACE2 protein in plasma

We used an ELISA from Raybiotech (Norcross, GA) to measure the concentration of soluble ACE2 protein in plasma following the manufacturer’s instruction. Briefly, samples were diluted 8-fold and loaded in duplicates on 96 well plates precoated with an antibody against human ACE2. Plates were incubated for 2.5 hours, washed, and incubated with a biotinylated detection antibody for 1 hour. Plates were washed and incubated with HRP conjugated streptavidin solution for 45 min. Plates were washed and incubated with TMB (tetramethylbenzidine) One Step Substrate Reagent for 30 min. Reaction was stopped with 0.2M sulfuric acid and absorbance measured at 450 nm. A serial dilution of recombinant ACE-II was loaded in duplicates and served as standard curve. ACE2 concentrations (ng/ml) were calculated based on a standard curve using 4 parametric nonlinear regression.