Anti-S-protein antibody titers in serum or other biological fluids were determined using ELISA. Briefly, we coated 1 μg/ml spike protein (Sino Biological, PA, USA) onto ELISA plates (Corning, NY, USA) in PBS overnight at 4 °C or 2 hours at 37 °C. The plate was blocked with PBS +1 % BSA (Fisher Scientific, PA, USA) + 0.1 % Tween20TM (Sigma-Aldrich, MD, USA) for 2 hours at room temperature. After washing, we added the samples at different dilutions. We detected the antibodies by HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1 in 5,000; PA, USA), anti-mouse IgA (Bethyl Laboratories, 1 in 10,000; TX, USA), and detection antibody against mouse IgA (1 in 250) from the mouse total IgA ELISA kit from Invitrogen (CA, USA). The positive control (anti-S IgG) was obtained from Abeomics (CA, USA).
To estimate the fraction of the protein adsorbed onto the liposomes, trimeric S-protein (10 μl, 1 μg /μl) was gently mixed with NanoSTING (20 μg), and incubated at room temperature for 10 min. The mixture was centrifuged at 20,000 x g for 40 min, and the pellet was resuspend and washed in 25 μl PBS. All supernatant were collected and combined, and then the total volume was measured.
The total amount of S-protein, the fraction of S-protein sedimented on the NanoSTING, and the protein in the supernatant fraction were evaluated using a quantitative S-protein ELISA.
To estimate the fraction of the protein adsorbed onto the liposomes, trimeric S-protein (10 μl, 1 μg /μl) was gently mixed with NanoSTING (20 μg), and incubated at room temperature for 10 min. The mixture was centrifuged at 20,000 x g for 40 min, and the pellet was resuspend and washed in 25 μl PBS. All supernatant were collected and combined, and then the total volume was measured.
The total amount of S-protein, the fraction of S-protein sedimented on the NanoSTING, and the protein in the supernatant fraction were evaluated using a quantitative S-protein ELISA.