Sigmafast o phenylenediamine dihydrochloride

Assays were performed in 96-well plates (MaxiSorp; Thermo) coated with 100 μL of recombinant spike or RBD protein^{12 (link)} in PBS, and plates were incubated at 4 °C overnight. Plates were then blocked with 10% FBS and 0.05% Tween20 in PBS. Serum were serially diluted in blocking buffer and added to the plates. Plates were incubated for 90 min at room temperature and then washed 3 times with 0.05% Tween-20 in PBS. Goat anti-human IgG-HRP (Jackson ImmunoResearch, 1:2,500) was diluted in blocking buffer before adding to wells and incubating for 60 min at room temperature. Plates were washed 3 times with 0.05% Tween-20 in PBS, and then washed 3 times with PBS before the addition of peroxidase substrate (SigmaFAST o-Phenylenediamine dihydrochloride, Sigma-Aldrich). Reactions were stopped by the addition of 1 M HCl. Optical density measurements were taken at 490 nm. The half-maximal binding dilution for each serum or plasma sample was calculated using nonlinear regression. The limit of detection was defined as 1:30.

Ninety six-well microtiter plates (Thermo Fisher) were coated with 50 µL recombinant protein at a concentration of 2 µg/mL in coating buffer (KPL) overnight at 4 °C. The next day, 220 µL blocking solution (phosphate-buffered saline (PBS; Gibco) supplemented with 0.1% Tween-20 (T-PBS; Fisher Scientific), 0.5% milk powder (AmericanBio), and 3% goat serum (Life Technologies)) were added to all wells of the microtiter plates and incubated for 1 h at room temperature. mAbs were diluted to a starting concentration of 10 µg/mL, serially diluted 1:3, and incubated for 2 h at room temperature. The microtiter plates were washed three times with T-PBS and 50 µL anti-mouse IgG (whole molecule) peroxidase antibody (produced in rabbit; Sigma, #A9044) diluted 1:3000 in blocking solution was added to all wells and incubated for 1 h at room temperature. The microtiter 96-well plates were washed four times with T-PBS and were developed with 100 µL/well SigmaFast o-phenylenediamine dihydrochloride (Sigma). After 10 min the reaction was stopped with 50 µL 3 M hydrochloric acid (Thermo Fisher) and the plates were read at 490 nm with a microtiter plate reader (BioTek). The data were analyzed in Microsoft Excel and GraphPad Prism. The cutoff value was defined as the average of all blank wells plus three times the standard deviation of the blank wells and the area under curve values were calculated.