Ovalbumin from chicken egg white

Serum samples were analyzed for anti-ovalbumin IgG₁ antibodies 14 days after ip injection at 3 weeks (primary) and 9 weeks (secondary) by ELISA as previously described in detail (45 (link)). Briefly, 96 well microplates were coated with ovalbumin from chicken egg white (Sigma) before non-specific binding sites were blocked with 2% bovine serum albumin (BSA) (Sigma) in PBS-tween 20. After washing, serial dilutions of serum samples and reference standard were added to the plates. Reference standard was porcine serum obtained following hyperimmunization with ovalbumin. Bound anti-soya IgG₁ antibodies were detected using isotype-specific monoclonal antibodies (clone K139 3C8, Biorad) followed by HRP-conjugated goat anti-mouse as above, and relative concentrations of antibody were determined by interpolation of samples onto the reference standards.
In order to compare changes in serum antibody generated by weaning and by injection of novel proteins in outbred animals, in which the starting levels differ, results are expressed as the ratio of antibody after manipulation to that before manipulation (the –fold change in antibody following exposure to OVA).

Ovalbumin from chicken egg white (Ova) (Sigma-Aldrich Canada Ltd, Oakville, ON; A5503) was labeled with Cyanine-5 (Cy5) reactive dye (Ambion/ThermoFisher Scientific, Burlington, ON, Canada; 5831G). The following formula was used to determine the amount of Cy5 needed for labeling: 8 × molecular weight (MW) of Cy5 x (amount of Ova)/ MW of Ova. Each Cy5 tube was re-suspended in 100 μl of dimethyl sulfoxide (DMSO; Sigma-Aldrich; D2650). A 1:10 ratio of Cy5 dye to protein and 0.3 M sodium carbonate buffer (Sigma-Aldrich) were incubated overnight at 4 °C with nutation and then placed on top of a 3 K Amicon centrifugal filter (ThermoFisher Scientific) before centrifugation at 16,000 × g for 10 min. After centrifugation, filters were inverted, and samples were washed 4 times with distilled water. The filters were then placed in new microcentrifuge tubes and then centrifuged at 1000 × g for 2 min to dispense the Cy5-labeled Ova.