RBD-specific IgG Antibody ELISA Protocol

RBD-specific IgG antibodies were determined using Enzyme-Linked Immunosorbent Assay (ELISA) as previously described (6 (link), 15 (link), 16 (link)). To determine IgG subclasses antibodies (IgG1, IgG2 IgG3 and IgG4), 100 µL of serum samples were added to the plate which were initially diluted to 1:10 and then 3-fold, 8 serial dilution was performed. Plates were subsequently incubated at 37°C for an hour. Mouse anti-human IgG1 (1:3000), IgG2 (1:4000), IgG3 (1:3000) and IgG4 (1:3000) Fc specific HRP (Southern Biotech) were added to the plates and incubated for one hour at room temperature. Subsequently, Ortho- phenylenediamine in 0.1 M sodium citrate buffer (100 µL; pH 4.5) and 30% hydrogen peroxide were added to the plate. The reactions were stopped after 20 minutes by adding 1 M H₂SO₄ (25 µL) and endpoint titers were determined as the reciprocal interpolated dilutions of the samples at 492 nm that were 0.4 above the background.