2.3. Treatment of Saliva with Receptor-Destroying Enzyme (RDE)

Where specified, saliva was treated with RDE to remove sialic acid. One volume of saliva in 8 volumes of Ca-Mg saline (0.24 mM CaCl₂, 0.8 mM MgCl₂, 20 mM boric acid, 0.14 mM sodium tetraborate, 0.15 M NaCl) was treated with one volume of Vibrio cholerae RDE (Sigma, MO, USA) for 30 min at 37 °C. The final concentration of RDE was 50 mU/mL. RDE was then inactivated by incubation at 56 °C for 1 h. Samples were transferred to 1000 MW cut-off Spectrapor membrane tubing and dialyzed against 50 mM NH₄HCO₃ at 4 °C overnight. Following freeze-drying, samples were reconstituted to their original volumes with PBS and stored at −20 °C.