Purification of IgYs

Purification of IgY from egg yolks was performed according to the protocol described by Hatta et al. (1990), with some modifications. Egg yolks showing higher ELISA values for one individual hen in Groups 2–5 at 6–12 weeks post-administration and egg yolks of one individual in Group 1 (control group) at 6–12 weeks post-administration were pooled together. These egg yolk samples were stored at −30 °C for more than two weeks to denature the egg yolk lipoprotein and then used for IgY purification. Purified IgY was pulverized after freeze-drying and stored at −30°C. The purity of the purified IgYs was determined using Laemmli’s sodium dodecyl sulfate-polyacrylamide gel electrophoresis method on a 5–20% polyacrylamide gel (c-PAJEL, ATTO, Japan). The gel was stained and photographed using Lumino Graph II (ATTO). The purity of each IgY antibody was determined as the ratio of heavy and light chains to the total protein fraction in the lane.