Sars cov 2 spike s1 his recombinant protein

10,12-pentacosadiynoic acid (PCDA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). PVDF transfer membranes (0.45 µm) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Artificial saliva with mucin (Pickering Laboratories) was purchased from Fisher Scientific (Waltham, MA, USA). SARS-CoV-2 Spike S1 Antibody, SARS-CoV-2 Spike S1-His Recombinant Protein, and MERS-CoV Spike S1 Protein were purchased from Sino Biological (Beijing, China). All other compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA).

RBD protein (Abcam, recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein RBD [Active] 50 µg, #ab273065, Cambridge, UK) or S1 protein (Sino Biologicals, SARS-CoV-2 Spike S1-His Recombinant Protein [HPLC-verified], #40591-V08H) was diluted with a coating solution (0.1 mM NaHCO₃, pH 9.6) to a concentration of 2 µg/mL. Microtiter plates (430341, Nunc C96 Maxisorp; Thermo Fisher Scientific, Waltham, MA, USA) were coated for 2 h at 4 °C with each diluted antigen. Subsequently, 200 µL/well of blocking solution was added and incubated overnight at 37 °C or for 2 h or four times. A 1000-fold dilution of mouse serum or BAL fluid was used as the primary antibodies and added to the plates (100 μL/well) and incubated overnight at 4 °C. Peroxidase-labeled anti-mouse IgG (Sigma-Aldrich, anti-mouse IgG [whole molecule] produced in goat-affinity isolated, buffered aqueous solution, #A4416, St. Louis, MO, USA) and anti-mouse IgA (Abcam, Waltham, MA, USA, goat anti-mouse IgA alpha chain [HRP], #ab97235) were applied at 1:60,000 for 1 h at 37 °C. After six washes, the plates were incubated with 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (A3219; Sigma-Aldrich) at room temperature for 30 min. Next, 0.5 M H₂SO₄ at 100 µL was added per well, and the optical density (OD) was measured at 450 nm using a microplate reader (MTP-880Lab; Corona Electric, Hitachinaka, Japan).