Ortho phenyl diamine

ELISA plates (Costar, Corning Life Sciences, Lowell, MA, USA) were coated with So (1 μg in 100 μL of 50 mM Carbonate-bicarbonate buffer pH 9.6, in each well), incubated overnight at 4 ^oC and washed twice in 0.05 % PBS Tween-20 (PBS-T). Plates were then blocked with 200 μL of 5 % skim milk in 0.05 % PBS-T and incubated for 2 h at 37 ^oC. Next, the plates were washed once with PBS-T and 50 μL of diluted serum (1:50 in 1 % skim milk/PBS-T) was added to each well. Plates were then incubated for 1 h at 37 °C and washed five times with PBS-T. Next, wells were filled with 50 μL/well of HRP conjugated rabbit Ig antimouse IgG (Sigma-Aldrich, St Louis, MO, USA) at a dilution of 1:10,000 in 1 % skim milk/PBS-T to assess total IgG. To evaluate IgG1, IgG2a, IgG2b and IgG3, wells were filled with 50 μL/well of HRP conjugated rabbit Ig antimouse IgG1, IgG2a, IgG2b or IgG3 (Zymed, San Francisco, CA, USA), respectively, each diluted at 1:8.000 in 1 % skim milk/PBS-T. All plates were then incubated for 45 min at 37 ^oC. Each plate was washed five times in PBS-T and 50 μL/well of Citrate Phosphate Buffer pH 5.1, ortho-phenyl-diamine (Sigma, St Louis, MO, USA) and 30 % H₂O₂] were added and left for 15 min at room temperature in a dark chamber. Reactions were stopped with 25 μL/well of 4 N H₂SO₄ and Optical Density (OD) was measured at 490 nm using an ELISA Plate Reader (BIORAD, Hercules, CA, USA).

Colon fragments (100 mg for each mouse) were homogenized in 1 mL of PBS buffer containing 0.05% tween-20 (Vetec, Rio de Janeiro, Brazil), 0.1 mM phenylmethylsulfonyl fluorid (MP Biomedicals, Solon, Ohio, USA), 0.1 mM benzethonium chloride (Sigma-Aldrich), 10 mM EDTA (ethylenediaminetetraacetic acid) (Synth, Brazil) and 20 KIU aprotinin A (Sigma-Aldrich). Tissues mixtures were centrifuged (3.000× g, 10 min) and supernatants were collected for ELISA immunoassays using DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA). Plates (Nunc^®, Sigma-Aldrich) were coated with purified monoclonal antibodies anti IL-10, IL-1β, IL-12p70, IL-17, IFN-γ, TGF-β, TNF-α and IL-6, overnight at 4 °C. Plates (Nunc-Immuno Plates, MaxiSorp) were washed by TBS (Tris-buffered saline) and supernatant from homogenized colon tissues were added. Plates then were incubated overnight at 4 °C. After plates washing, biotinylated monoclonal antibodies against different cytokines were added to coated plates and incubated for 2 h at room temperature. The revelation was performed by adding 100 µl/well of a citrate buffer containing Orthophenyldiamine (Sigma-Aldrich) (1 mg/mL) and 0.04% (v/v) H₂O₂. Then, 2N H₂SO₄ solution was added to stop the reaction. The absorbance was measured at 492 nm using an ELISA reader (Bio-Rad, Philadelphia, PA, USA).