SARS-CoV-2 Antigen-Based Serology Assay

As an antigen, we used recombinant proteins derived from the SARS-CoV-2 (S1 domain, RBD domain; Receptor Binding Domain, N, M, and E), and purification was performed as previously described [18 (link)]. Microtiter plates (Sigma-Aldrich, St. Louis, MO, USA) were coated with 100 µL/well of each antigen (S1 domain, RBD domain, N, M, and E) to a final concentration of 0.1 µg/mL in a coating buffer (50 mM Na₂CO₃/NaHCO₃, pH 9.6). The plates were incubated for one hour at 37 °C and then blocked for 40 min at 37 °C with 200 µL/well of 5% skimmed milk diluted in phosphate-buffered saline (PBS)-Tween 20 (0.05%). For the primary antibody, 100 µL/well of serum samples (1:50 diluted in PBS) or breast milk (direct) by duplicate were incubated for one hour at 37 °C. Finally, plates were incubated with 100 µL/well of monoclonal anti-human IgA (Sigma-Aldrich; 1:500 dilution) and IgG (Sigma-Aldrich; 1:1500 dilution, St. Louis, MO, USA) coupled to horseradish peroxidase (HRP) for one hour at 37 °C. After every step, the plates were washed thrice with 200 μL/well of PBS-Tween 20 0.05% for 5 min. The enzymatic reaction was developed using o-phenylenediamine dihydrochloride (Sigma-Aldrich) and stopped using 2 N H₂SO₄. The optical density (OD) was measured at 492 nm using a microplate reader. Samples with OD > 0.300 were considered positive for both antibodies.