SARS-CoV-2 Spike Protein Antibody Assay

Interleukin (IL)-4, IL-10, and interferon (IFN)-γ in the serum of immunized mice were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). Standard curves were generated for each cytokine. Values were measured at an optical density of 450 nm using an ELISA plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA). Antigen-specific immunoglobulin (Ig)G from serum or IgA from fecal samples was measured using ELISA based on our previous study (Hwang et al., 2023 (link)). Briefly, each well of a 96-well plate was pre-coated with E. coli-expressed recombinant SARS-CoV-2 spike S1 antigens (1 µg per well) overnight at 4°C. The antigen-coated wells were blocked with bovine serum albumin for 1 h at 37°C. After blocking, immunized mouse serum (1:100 dilution) or fecal extract (1:10 dilution) was added to the wells and incubated at 37°C for 1 h. HRP-conjugated goat anti-mouse IgG or IgA antibody (1:1000; Invitrogen, Waltham, MA, USA) was added to the wells and incubated for 1 h at 37°C. The antigen-specific antibody was detected by adding 3, 3’, 5, 5’- tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped by adding 0.5 N H₂SO₄. Optical density (450 nm) was measured using an ELISA plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA).