The general format of the ELISA assay is described below with specifics reported in the individual figure legends. Proteins were diluted in 0.1 M NaHCO3 and added at a fixed or range of concentrations to Nunc Maxisorp™ flat-bottom 96 well plates (ThermoFisher), at 100 μL per well, in triplicate. After incubation at room temperature for 45 min, the wells were washed once in PBS-0.1 % Tween 20 (PBST), and non-specific binding sites on the plate surface were blocked with 5% skim milk in 0.1 M NaHCO3 for 30 min. The wells were then washed once in PBST, and proteins under study were added at a fixed or increasing concentrations to the wells.
After 45 min incubation, the wells were washed 3x in PBST before adding streptavidin-horseradish peroxidase conjugate (HRP; Sigma-Aldrich, St. Louis, MO), anti-human IgG-HRP (Abcam) or ACE2-Fc-HRP (see above) to the wells for 1 h incubation. After washing the wells 3x in PBST, 100 μL of 2’,2’-Azino-Bis 3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS, Sigma-Aldrich) or 3,3′,5,5′-Tetramethylbenzidine (TMB, Fast Kinetic Rate; Abcam) was added to each well, optical absorbance of the wells measured at 405 or 485 nm, respectively, on a POLARstar OPTIMA microtiter plate reader (BMG Labtech, Ortenberg, Germany).
After 45 min incubation, the wells were washed 3x in PBST before adding streptavidin-horseradish peroxidase conjugate (HRP; Sigma-Aldrich, St. Louis, MO), anti-human IgG-HRP (Abcam) or ACE2-Fc-HRP (see above) to the wells for 1 h incubation. After washing the wells 3x in PBST, 100 μL of 2’,2’-Azino-Bis 3-Ethylbenzothiazoline-6-Sulfonic Acid (ABTS, Sigma-Aldrich) or 3,3′,5,5′-Tetramethylbenzidine (TMB, Fast Kinetic Rate; Abcam) was added to each well, optical absorbance of the wells measured at 405 or 485 nm, respectively, on a POLARstar OPTIMA microtiter plate reader (BMG Labtech, Ortenberg, Germany).