9018 elisa plate

The total IL-6 levels in rats’ CSF were assessed using the rat IL-6 ELISA kit (Elabscience, E-EL-R0015). Briefly, a Corning Costar 9018 ELISA plate was coated with capture antibody and incubated overnight at 4 °C. After blocking the coated wells with Blocking Buffer for 1 h at room temperature, CSF samples were added following a 100-fold dilution. Detection of total IL-6 was achieved using an HRP-conjugated anti-rat IL-6 monoclonal antibody. Then, Streptavidin-HRP monoclonal antibody was applied as a second-step reagent.

The titre level of IgG total from blood sera of day 0, 21, 33 and 45 was determined using eBioscience Mouse IgG total ready-SET-Go (eBioscience, Austria). Briefly, Corning Costar 9018 ELISA plate was coated with 100 μl/well of antigen ACERL in coating buffer, seal and incubate overnight at 4 °C. The wells were aspirated and washed twice with 250 μl/well wash buffer (1× PBS, 0.05% Tween-20) and the wells blocked with 200 μl of blocking buffer at room temperature for 2 h. The wells were aspirated and washed three times with wash buffer and 100 μl reconstituted standard and sample in assay buffer was added in triplicate into well and incubated for 2 h at room temperature. The wells were aspirated and washed three times with wash buffer and 100 μl/well of diluted detection antibody was added to all wells for 1 h at RT. Then, the wells were aspirated again and washed three times with wash buffer and 100 μl/well of substrate solution was added to each well and incubated at RT for approximately 15 min and before 100 μl of Stop Solution was added to each well and the plate read at 450 nm. The plate was read using the Bio-Plex Suspension Array System model Bio-Plex 100 System (BioRad, USA) and analysed using the Bio-Plex Manager 4.0 (BioRad, USA) software.

Murine TNF‐ɑ or Murine IL‐10 Ready set go enzyme‐linked immunosorbent assay (ELISA) kits were used (eBioscience). Protocol was performed as per manufacturer's instructions. In brief, Costar 9018 ELISA plates were coated with 100 µL/well of capture antibody diluted in Coating Buffer overnight at 4°C followed by blocking with ELISASPOT diluent. Small intestinal samples were homogenized in Cell Lytic MT buffer (Sigma) using Lysing matrix E beads (MP Biomedicals) and homogenates cleared by centrifugation at 10 000 rpm for 15 minutes. After quantification using BCA kit (Pierce), equivalent amounts of test samples or standard material were applied to wells in duplicate and incubated overnight at 4°C. Detection Antibody, Streptavidin‐HRP and substrate were then applied, and plates were read at 450 nm with 570 nm correction.