Human sera

A 96-well microplate was coated with sEOD1PM (25 μg/mL) in 0.1 M carbonate buffer (pH 9.6) by incubation at 4 °C overnight. The plate was washed with PBST and blocked with 0.5% sodium caseinate in PBST at 37 °C for 60 min. The plate was washed with PBST and incubated with human sera (Sigma-Aldrich) or saliva at 37 °C for 60 min. The plate was then washed with PBST and incubated with peroxidase-conjugated anti-human IgG, IgM, or IgA (1:2000; Sigma-Aldrich) in PBST containing 0.5% sodium caseinate and developed with a TMB substrate system (KPL, Gaithersburg, MD, USA). Color development was stopped with 1 N phosphoric acid, and optical density was measured at 450 nm with the absorbance reader MTP-450 (CORONA Electric, Ibaraki, Japan).
For testing the specificity of the anti-sEOD1PM antibody, an ELISA plate was coated with sEOD1PM (25 µg/mL in carbonate buffer) and blocked with 0.5% sodium caseinate in PBST before use. human sera or pooled human saliva were mixed with serially diluted competitive soluble antigen, namely sEOD1PM, CSBG, OVA, AgHWE, ASBG, or dextran, and then applied to the ELISA plate. The amount of plate-bound Ig was determined using peroxidase-conjugated anti-human IgG or IgA (1:2000).

Bacterial cells (∼3.5 × 10⁵ CFU) from a mid-log culture were treated with 100 μg/ml of purified RAD2-DpK2. Human sera (Sigma-Aldrich catalog no. S7023) were prewarmed to either 37°C (active) or 50°C (heat inactivated) for 30 min and then added to enzyme-treated cells (80% final concentration) and incubated at 37°C for 3 h. Viable bacterial counts were determined before and after incubation with serum.