The concentration of SIgA and IL-6 in saliva was measured with the SIgA and IL-6 Quantikine ELISA (DB-100; R&D Systems, Inc., Minneapolis, MN, USA) following the protocol of the manufacturer. The samples were incubated in duplicate on microtiter plates coated with anti-SIgA monoclonal antibody (cat. no. 11137A12) or anti-IL-6 (cat. no. 11134A09) antibodies (both R&D Systems, Inc.) for 2 h at room temperature. The plates were then washed with the washing buffer of the ELISA kit to remove the unbound antibodies. Subsequent to the incubation at 25°C for 60 min of the conjugate solution, horseradish peroxidase-conjugated reagent, and the biotin solution of the ELISA kit was added. Color development was stopped subsequent to 20–25 min, depending on the assay (11 (link)). The ELISA assay results were read using a VersaMax microplate spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA) set at 450 nm.