Preparation of Egg Yolk IgY Fraction

5 M EDTA, disodium salt, pH 7.5 E saturated ammonium sulfate solution (see Protocol 4.3) solid ammonium sulfate 

Separate yolks of chicken eggs from egg white and discard egg white. Wash the yolks carefully with water to remove adhering egg white. Suspend the yolks in 5 vol. Soln. A by vigorous stirring. Precipitate lipids and lipoproteins by addition of 6 ml Soln. B and 15 ml Soln. C per 100 ml yolk suspension. Stir at RT for 30-60 min and spin at 5000 x g for 10 min. Wash the pellet with a small volume of Soln. A (about 20 ml per yolk) and centrifuge again. Combine the supernatants and filter through a paper filter. Add Soln. D to the clear filtrate to a final concentration of 30 mM EDTA. 

Add 24.3 g solid ammonium sulfate per 100 ml EDTA-filtrate (yields a 40% saturation) and stir at 4 °C for 30 min. Collect the precipitate by centrifugation. Wash the pellet once with 30% saturated ammonium sulfate (3 vol. Soln. E -i- 7 vol. ddH20), spin down again and dissolve the precipitated IgY in a small volume of Soln. A (about 1/10 of the original yolk volume). Dialyze against TBS and determine the IgY content spectrophotometrically. 

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