Goat anti chicken iga horseradish peroxidase conjugate

The level of antibodies against C.jejuni protein in intestinal secretions and sera of chicken was quantified by ELISA.
Ninety-six-well plates (Nunc, Thermo Scientific) were coated overnight at 4 °C with whole-cell C. jejuni proteins (20 μg/well), washed three times with PBS containing 0.02 % Tween 20 (Sigma-Aldrich), blocked for 1 h at 37 °C with PBS containing 2 % bovine serum albumin (Sigma-Aldrich), washed as described previously, and incubated for 1.5 h at room temperature with either diluted sera (1:300) or intestinal secretion samples (1:10). The plates were developed with 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), using goat anti-chicken IgA horseradish peroxidase conjugate (Thermo Scientific, Rockford, IL) or rabbit anti-chicken IgY (whole molecule)-peroxidase (Sigma-Aldrich) (dilution 1:2000), respectively. The plates were incubated with the substrate for 25 min at room temperature and then the colorimetric reaction was stopped by adding 2-M H₂SO₄ (Sigma-Aldrich). Absorbance was measured at 490 nm using an ELISA reader (Tekan). Each sample was analyzed in duplicate.

The 6xHis-tagged rCjaAD protein purified as described above was also used as a coating antigen. The levels of antibody against rCjaAD protein in chicken intestinal secretions were quantified by ELISA. Briefly, 96-well Maxisorp plates (Nunc, Rochester, NY, USA) were coated with purified rCjaAD protein (5 μg per well) in PBS and incubated overnight at 4°C. Then, plates were blocked for 1 h at 37°C with PBS containing 0.1% Tween 20 (Sigma–Aldrich) and 1% bovine serum albumin (BSA), washed three times with PBS containing 0.1% Tween 20 (Sigma–Aldrich) and incubated for 1 h at room temperature with the intestinal secretion samples (1:10). Goat anti-chicken IgA horseradish peroxidase conjugate (Thermo Fisher, Scientific) was employed to detect chicken IgA that bound to Campylobacter antigens. The plates were developed with 3,3′,5,5′-tetramethylbenzidine (Sigma–Aldrich), according to the manufacturer’s directions. The reaction was stopped with 3M H₂SO₄ and optical density was determined at A 490 using an ELISA reader (Tekan). Each sample was analyzed in triplicate.