SARS-CoV-2 Spike Protein ELISA Protocol

The binding antibody titers using ELISA were assessed according to methods previously published [20 (link)]. Briefly, 96-well half-area ELISA plates (Corning (Tewksbury, MA, USA)) were coated with the recombinant Washington strain (WA-1) spike protein (expressed as a prefusion-stabilized S-2P construct) [21 (link)] at 2 μg/mL in PBS, followed by overnight incubation at 4 °C. The next day, the plates were washed three times with PBS-T (PBS containing 0.05% Tween 20) and blocked with 5% milk (Oxoid skim milk powder, Thermo Scientific, Waltham, MA, USA) in PBS (w/v) for 1 h at room temperature. Serially diluted plasma samples in duplicate were added to the plates and incubated for 2 h at room temperature. The plates were washed three times, and a goat anti-monkey IgG–horseradish peroxidase secondary antibody (Nordic-MUBio (Susteren, the Netherlands)) was added for 1 h at room temperature. The plates were washed three times again, and 1-Step Ultra TMB-ELISA substrate (Thermo Fisher Scientific, Waltham, MA, USA) was added for 5 min. The reaction was stopped with 1 M H₂SO₄, and the absorbance was measured at 450 nm with background correction at 570 nm. The data were analyzed using Prism v9.4.1 using a 4-parameter logistic curve fit.