Goat anti mouse igg horseradish peroxidase hrp conjugated antibody

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).

The strain pPG-SD-S1/Δupp ATCC 393 was inoculated into MRS broth (1:100) and incubated for 12 h at 37 °C. Next, the culture was centrifuged at 4 °C, and the pellet and supernatant obtained were sonicated. Further, proteins in the sonicated supernatant and pellet were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Milford, MA, USA). The membranes were incubated with mouse S1 monoclonal antibody (stored in our laboratory) as the primary antibody for 1 h at 37 °C and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5000) (Thermo Scientific, Durham, NC, USA) as the secondary antibody for 1 h at 37 °C. The results were observed using a chemiluminescent substrate reagent (Solarbio, Beijing, China) according to the manufacturer’s instructions.