ELISA assays for SARS-CoV-2 and ACE2 antibodies

Fifty microliters of a solution of recombinant SARS-CoV-2 receptor binding domain protein (2 μg/mL, plasmid from BEI) or recombinant ACE2 protein (2 μg/mL, SinoBiologicals) in carbonate buffer (0.0125 M Na₂CO₃, 0.0875 M NaHCO₃, pH 9.4) was added to each well of a high binding ImmunoGrade 96-well plate (MidSci) and coated overnight. To determine the presence and concentration of antibodies in plasma or serum, samples were diluted 1:50 in 1% dry milk PBS-T (1X PBS, 0.1% Tween-20) and added to duplicate wells for 2 hours, followed by peroxidase-conjugated goat anti-human IgG + IgM antibody (JacksonImmuno) diluted at 1:5000 in 1% dry milk PBS-T. Seventy-five microliters of a solution containing tetramethyl benzidine (SeraCare—SureBlue TMB Solution) was added and stopped after 5 minutes with 75 μL of 1% HCl solution (SeraCare-TMB stop solution). The optical density at 450 nm was determined. The value of a blank well control was subtracted to obtain the final value and reported as OD (450 nm). All measurements were made in duplicate and the mean value of the two wells was used for the analysis. Cutoff values for a positive test were defined as mean of negative controls plus 3 standard deviations. The cutoff values are 0.60 for the RBD antibody and 0.1106 for ACE2 antibody.