2.3. Spike-ACE2 binding kit assay

To test antibody specificity to spike S1, a commercially available COVID-19 Spike-ACE2 binding in vitro kit was used (RayBiotech, Peachtree Corners, GA; Code: CoV-SACE2-1). Manufacturer instructions were followed for the reagents and sample preparation. 100 μL of each sample were added to each well in triplicate and incubated overnight at 4 °C with shaking. The solution was discarded the following day and washed 4 times in 1× wash solution. 100 μL of 1× horseradish peroxidase (HRP) conjugated IgG was added to each well for 1 h at room temperature with shaking. Samples were washed three times with 1× wash solution. 100 μL of TMB one-step substrate reagent was added to each well, incubated for 30 min at room temperature with shaking in the dark. 50 μL of stop solution were added to each well and the plate was immediately read at 450 nm in a plate reader. As a final step, the content of each well was lysed using 100 μL of 1 M sodium hydroxide and collected into a disposable plastic culture tube (12 × 75 mm). Wells were washed three times with PBS and each wash was added to the corresponding a disposable plastic culture tube (12 × 75 mm). Tubes were then counted on a gamma counter to detect radioactivity.