Peroxidase streptavidin goat anti mouse iga hrp

Levels of IgA were determined in the intestinal lavage. Small intestine was rinsed with 10 ml of cold PBS. Intestinal lavages were centrifuged at 12,000 g for 20 min at 4°C, and levels of IgA in the supernatants were determined by ELISA. Briefly, 96-well plates were coated with Ig goat anti-mouse unlabeled antibody in coating buffer (pH 9.6) overnight at 4°C. Wells were washed and blocked with 200 µl PBS containing 0.25% casein for 1 h at room temperature. Samples were added to the plates and incubated for 1 h at 37°C. The plates were then washed, peroxidase-streptavidin goat anti-mouse IgA-HRP (Southern Biotechnology, Birmingham, AL, USA) diluted 1:10,000 was added, and the plates were incubated for 1 h at 37°C. Color was developed at room temperature with 100 μl/well of orthophenylenediamine (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and 0.04% H₂O₂ substrate in sodium citrate buffer. The reaction was interrupted by the addition of 20 μl/well of 2 N H₂SO₄. Absorbance was measured at 492 nm using an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).

The levels of sIgA were determined by ELISA. Briefly, 96-well plates (NUNC) were coated with Ig goat anti-mouse UNLB antibody in coating buffer (pH 9.6) overnight at 4°C. The wells were washed and blocked with 200 μl of PBS containing 0.25% casein for 1 h at room temperature. Samples were added to the plates and incubated for 1 h at 37°C. The plates were then washed, peroxidase-streptavidin goat anti-mouse IgA-HRP (Southern Biotechnology) diluted 1:10000 was added, and the plates were incubated for 1 h at 37°C. Colour was developed at room temperature with 100 μl/well of orthophenylenediamine (1 mg/ml) (SIGMA) and 0.04% H₂O₂ substrate in sodium citrate buffer. The reaction was interrupted by the addition of 20 μl/well of 2 N H₂SO₄. The absorbance was measured at 492 nm using an ELISA microplate reader (Bio-Rad).