Chicken iga elisa kit

Blood samples were collected from the brachial vein on day 7 post S. Enteritidis challenge in Vacutainer tubes containing sodium heparin to determine the serum component (Lan et al., 2005 (link)). Approximately 8 mL of blood was collected from all the birds, using an 18-gauge needle. Blood sera was isolated by centrifuging the tubes at 3,000 × g for 10 min at 4°C. The separated serum, was transferred into individual vials and stored at -20°C for biochemical analysis. Whole blood samples were used to determine the WBC and RBC count. The whole blood and serum analysis was carried out at the Diagnostic Services, Faculty of Medicine, University of Montreal.
The serum immunoglobulin (IgA) were measured using a Chicken IgA ELISA kit (Bethyl Laboratories, Inc., Cedarlane, Hornby, ON, Canada). Serum IgA present in the sample was captured by anti-chicken IgA antibody pre-absorbed on the surface of microtiter wells. The quantity of IgA analyte present in the sample was proportional to the absorbance at 450 nm.

Ileal mucosal scrapings were collected by removing the mid-ileum, rinsing the tissue with PBS, and scraping the mucosal layer from the underlying connective tissue using a microscope slide. The homogenate was centrifuged at 3000 rpm at 4 °C for 10 min, and the supernatant was collected for subsequent measurements. Ileum mucosa samples were used to measure various biomarkers of oxidative stress, including levels of glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and secretory immunoglobulin A (sIgA). GPX and SOD activities were measured by using EnzyChrom superoxide dismutase assay kit (ESOD-100) and EnzyChrom glutathione peroxidase assay kit (EGPX-100), respectively, as per the manufacturer’s instructions. Both kits were bought from BioAssay Systems (Hayward, CA, USA). CAT was analyzed using an OxiSelect catalase activity assay kit (Cell Biolabs, Inc., San Diego, CA, USA). MDA was measured using an OxiSelect thiobarbituric acid reactive substances (TBARS) assay kit (Cell Biolabs, Inc., San Diego, CA, USA). sIgA was determined using a chicken IgA ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA). Bradford reagent (Sigma-Aldrich, St. Louis, MO, USA) was used to quantify the protein concentration of the ileal mucosa samples.