Extract yolk with either one of the following methods:

2a. Dilute 1:10 of egg yolk in ddH₂O. Stir slowly at 4 °C for 6 hours. Precipitate lipids by centrifugation (10,000 g, 25 minutes, 4 °C). Transfer supernatant to a fresh tube.

2b. Dilute 1 part of egg yolk with 3 parts of 0.1 M sodium phosphate buffer (pH 7.5), and 1 part of 17.5% PEG in 0.1 M sodium phosphate buffer (pH 7.5). Stir slowly in room temperature for 20 minutes. Precipitate lipids by centrifugation (10,000 g, 25 minutes, room temperature). Filter the supernatant through a coarse filter paper.

3. Add potassium sulphate to 0.5 M, adjust the pH to 7.5, and filter through a 0.45 mm filter.

4. Equilibrate the IgY HiTrap column in 0.5 M potassium sulphate, 20 mM sodium phosphate buffer (pH 7.5).

5. Load the IgY extract onto the column.

6. Wash with 10 bed volumes of 0.5 M potassium sulphate, 20 mM sodium phosphate buffer (pH 7.5).

7. Elute in 10 bed volumes of 20 mM sodium phosphate (pH 7.5). Collect fractions.

8. Cleaning the column with 10 bed volumes of 20 mM sodium phosphate (pH 7.5) and 30% isopropanol.