Salmonella typhimurium

For serum anti-commensal IgG analysis, colonic fecal contents were processed as described above. Bacteria were homogenized using BugBuster 10X protein extraction reagent (Novagen), centrifuged at 20,000 g for 10 min, and the supernatant recovered for a crude commensal bacterial antigen preparation. Protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Subsequently, 96-well Nunc ELISA plates (Thermo Fisher Scientific) were coated with 5 μg/mL commensal antigen preparation overnight at 4°C, washed extensively, and murine sera incubated in doubling dilutions for 4 h at room temperature. For small volumes of sera, samples were incubated at a 1:150 dilution. In the case of serum anti-flagellin IgG detection, 96-well Nunc plates were coated overnight with 200 ng/mL flagellin purified from Salmonella typhimurium (InvivoGen). Commensal antigen-specific IgG was detected using a goat anti-mouse IgG-horseradish peroxidase (HRP) conjugated antibody (Thermo Fisher Scientific, 1:10000 dilution), and TMB peroxidase substrate (BD biosciences). After 15-20 min, the reaction was quenched with 1 M Na₂SO₄ and the optical densities measured at 450 nm using a CLARIOstar spectrophotometer (BMG Labtech).

Flagellin immunization was carried out as previously described (Kobayashi et al., 2009 (link)). Briefly, C57BL/6 mice were hyperimmunized by two intraperitoneal (i.p.) injections with 10 μg of flagellin from Salmonella typhimurium (InvivoGen) in Incomplete Freund’s Adjuvant (IFA) (Sigma Aldrich) two weeks apart. Mice were culled at four weeks and total serum IgG, as well as control serum IgG from unimmunized mice, was purified using Pierce Protein G purification columns (Thermo Fisher Scientific), as per the manufacturer’s instructions.