Journal: Viruses
Article Title: The Glycan Ectodomain of SARS-CoV-2 Spike Protein Modulates Cytokine Production and Expression of CD206 Mannose Receptor in PBMC Cultures of Pre-COVID-19 Healthy Subjects
doi: 10.3390/v16040497
Figure Lengend Snippet: Spike (S) proteins bind Con A mainly through mannan constituents. ( A ) Binding of S-in (top) or S-hu (bottom) protein (0.75 µg/mL) coated on a microplate and HRP-Con A (0.0125 µg/mL), measured by optical density (O.D.) using an in-house ELISA assay. Scalar concentrations of saccharides were incubated together with HRP-Con A directly on the S protein-coated microplates. Data are reported as mean ± sd of two independent experiments run in duplicate. ( B ) Binding of S-in, S-hu, S1 and S2 proteins (all at 0.75 µg/mL) coated on a microplate and HRP-Con A (0.025 µg/mL) measured by optical density (O.D.) through an in-house ELISA assay. Mannan (50 µg/mL) was incubated together with HRP-Con A directly on the spike-coated microplates. Data are reported as mean ± sd of two independent experiments run in duplicate. Statistical analysis: one-way ANOVA with Tukey’s multiple comparisons post hoc test. ** p < 0.01; **** p < 0.0001. In panel ( A ), the asterisks indicate the differences with respect to the control condition Con A without saccharides. In panel ( B ), only differences in O.D. values between binding of Con A to each specific S protein in the absence or presence of mannan, and between binding of Con A and S-hu protein versus those of Con A and S1 or S2 subunits, are shown.
Article Snippet: Plates were blocked for 1 h at 37 °C with 3% BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS-T. After the BSA solution was removed, in a first set of experiments, the wells of plates coated with S-in or S-hu were incubated for 1 h at 37 °C with 0.0125 μg/mL of HRP-conjugated Con A (Sigma L-6397) in PBS-T containing 3% BSA, alone or in combination with scalar concentrations of mannan ( S. cerevisiae , Sigma M3640); dextran 1000 (Fluca 31416); alfa-methyl-mannoside (Sigma-Aldrich, REF. M6882); N-acetil-glucosamine (Sigma-Aldrich, REF. A8625).
Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation
