Biotin conjugated anti mouse iga antibody

Levels of secretory IgA were determined by enzyme linked immunosorbent assay (ELISA) in small bowel intestinal fluids. Microtitre plates (Nunc-Immuno Plates, MaxiSorp) were coated with goat anti-mouse antibody (Southern Biotechnology, Birmingham, AL, USA) in carbonate-bicarbonate buffer (0.1 M Na2CO3/NaHCO3—pH 9.6) for 18 h at 4 °C. Wells were washed with washing solution (saline 0.9% plus 0.05% tween 20) and blocked with 200 µL of 0.05% casein in PBS for 1 h at room temperature. Intestinal fluids previously centrifuged at 432g for 20 min were added to the plate and diluted in PBS-0.25% casein (two times until dilution 1:80). After incubation of 1 h at room temperature, plate was washed and biotin conjugated anti-mouse IgA antibody (Southern Biotechnology) in PBS-0.25% casein (1:10.000) was added to the wells. After incubation of 1 h at 37 °C, peroxidase-streptavidin goat anti-mouse IgA (Southern Biotechnology, Birmingham, AL, USA) was added; plate was incubated for 1 h more and, then, coated with 100 µL/well of orthophenylenediamine (OPD) (1 mg/mL) (Sigma, St. Louis, MO, USA) and 0.04% H₂O₂ substrates. Color was developed at room temperature and reaction was stopped by the addition of 20 µL/well of 2 N H₂SO₄. Absorbance was measured at 492 nm using a Bio-Rad Model 450 Microplate Reader. Results were expressed as concentration (µg/mL), according to the standard curve.

The quantification of total sIgA and Salmonella-specific sIgA in fecal extracts has been described (31 (link), 32 (link)). MaxiSorp 96-well plates (Nunc, Roskilde, Denmark) were coated with goat anti-mouse Ig H + L [IgA, IgG, and IgM] antibody (Southern Biotech, Birmingham, AL, USA) and incubated overnight at 4°C for total IgA. For detection of anti-Salmonella IgA, plates were coated with heat-killed Salmonella (1 × 10⁶ CFUs/ml) and incubated overnight at room temperature. IgA was detected using biotin-conjugated anti-mouse IgA antibody (Southern Biotech) followed by streptavidin-HRP. Color was developed by using TMB substrate solution and plate was read with the TECAN microplate reader (Maennedorf, Switzerland) at λ = 450 nm.

Levels of secretory IgA (sIgA) were determined by enzyme-linked immunosorbent assay (ELISA) in small bowel intestinal fluids (Carvalho et al., 2017a (link)). Microtiter plates (Nunc-Immuno Plates, MaxiSorp) were coated with anti-IgA antibodies (Southern Biotechnology, Birmingham, AL, United States) for 18 h at 4°C. The plates were washed with saline (NaCl 0.9%) added with Tween 20 (0.05%) and blocked with 200 μl PBS-casein (0.05%) for 1 h at room temperature. Intestinal fluid samples were diluted in PBS-casein (0.25%) and then added to the plate. After incubation for 1 h at room temperature, the wells were washed and biotin-conjugated anti-mouse IgA antibody (Southern Biotechnology) diluted in PBS-casein (0.25%) (1: 10,000). The plates were incubated for 1 h at 37°C and anti-IgA conjugated to streptavidin peroxidases (1:10,000) were added (Southern Biotechnology). After 1 h of incubation, 100 μl of orthophenylenediamine (OPD) (Sigma, St. Louis, MO, United States) and H₂O₂ (0.04%) were added to each well. Plates were kept away from light until the coloration developed. The reaction was stopped by addition of 2 N H₂SO₄. Reading was performed on a plate reader (Bio-Rad Model 450 Microplate Reader) at 492 nm absorbance. The results were measured in concentration of sIgA (μg) per ml of intestinal fluid, according to the standard curve.