Purification of Hyperimmune IgY from Egg Yolk

The hyperimmune egg yolk generated in our recent study (9 (link)) was used for IgY purification following a published protocol (10 (link)) with slight modifications. Briefly, one volume of egg yolk was diluted with four volumes of PBS and then mixed with 3.5% (w/v) of polyethylene glycol 6000 (PEG 6000, Fisher Scientific, MA, USA) for lipid removal. After 20 min of incubation at room temperature, the mixture was centrifuged at 5,000g for 20 min. Subsequently, the supernatant was added with 12% (w/v) of PEG 6000 for IgY precipitation and the mixture was incubated at room temperature for 10 min. Following centrifugation at 5,000g for 20 min, the precipitate was dissolved in PBS and added with 12% (w/v) of PEG 6000 again for another round of IgY precipitation. After centrifugation, the precipitate was dissolved in a small volume of PBS, added with equal volume of precooled (−20°C) ethanol, and centrifuged at 5,000g and 4°C for 20 min to remove PEG 6000. Lastly, the precipitate containing a high proportion of IgY was dissolved in a small volume of PBS and the solution was dialyzed against PBS. Initial egg yolk, the supernatant and precipitate fractions obtained from the centrifugation of 12% PEG 6000 mixture, and the final dialyzed IgY extract were subjected to SDS-PAGE analysis.