Quantification of Secretory IgA in Intestinal Fluids

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.