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IgY Purification from Chicken Egg Yolks

Once you've immunized your hens for IgY antibody production and collected immune eggs, the next step is to purify the IgY from the egg yolks.

Unlike serum, which contains a number of immunoglobulin classes – IgG, IgM, IgD, IgA and IgE, yolk contains only one class of antibody – IgY. However, the egg yolk is a complex mixture of water (50%) lipids (32-35%) and proteins (16%)1 and for most experimental purposes, it is advisable to, at least, partially purify the IgY protein, by removing the bulk of the lipids and lipoproteins. Proteins residing within the yolk are of 4 types: lipovitellins, phosphorous-containing lipoproteins (40%), apovitellenins, containing less phosphorous but more lipid (37.3%), phosvitin, a phosphoprotein (13.4%) and the livetins (9.3%), of which IgY is part1.

Removal of the yolk lipids and lipoproteins leaves a water soluble fraction, containing IgY along with other proteins, which crudely could be compared to an animal serum, in terms of useability in immunoassays. A number of methods which remove most of the lipids/lipoproteins have been described (reviewed in Refs. 1, 2):

  • Polyethylene Glycol precipitation3
  • Dextran Sulphate Precipitation4
  • Organic Solvent Lipid Solubilization5
  • Natural Gums, Xantham/Carageenan Precipitation6
  • Lipid Dilution/Ultrafiltration7

Following the delipidation of your egg yolk, the almost lipid-free solution can now be treated in a number of ways to concentrate/purify the IgY fraction:

  • Polyethylene Glycol Precipitation3
  • Sodium Sulphate Precipitation4
  • Ultrafiltration/Ammonium Sulphate Precipitation7
  • Preparative Electrophoresis8

There are a number of IgY Purification Kits commercially available that use one or more of the methods described above (Gallus Immunotech also provides an IgY purification service.):

Manufacturer IgY Purification Kit Name IgY Yield
(mg IgY /gram yolk)		 IgY Purity
(% Purity by SDS PAGE)		 Cost**
(per mg IgY purified)
Gallus Immunotech IgY Eggspress Purification Kit 4-9 85-90 a
Pierce Biotechnology/ Thermoscientific Eggcellent IgY Purification Kit 4-9 85-90 b
Promega Eggstract IgY Purification System 2-5 75-90* d
Agro-Bio EggsPure 2-5 90 c
GE Healthcare HiTrap IgY Purification Information not available Information not available Information not available
Affiland IgY Purification Kit Information not available 98 Information not available
*75% for 2-step protocol, 90% purity for 3-step protocol
a = cheapest, d = most expensive

References:

  1. Kovacs-Nolan, J. and Y. Mine, 2004. Avian egg antibodies: basic and potential applications. Avian and Poultry Biology Reviews 15 (1), 2004, 25-46.
  2. Akita, EM and S. Nakai, 1993. Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. Coli strain. J. Immunol. Methods, 160 :207-14.
  3. Polson, A., von Wechmar, M.B. and Fazakerley, G. 1980. Antibodies to proteins from yolk of immunized hens. Immunol. Commun., 9 :495-514.
  4. Jensenius, J.C., Anderson, I., Hau, J., Crone, M. and Kock, C. 1981. Eggs: conveniently packaged antibodies. Method for puri®cation of yolk IgG. J. Immunol. Methods, 46, 63-8.
  5. Bade, H. and Stegemann, H., 1984. Rapid method of extraction of antibodies from hen egg yolk. J. Immunol. Methods, 71, 421-6.
  6. Hatta, H., Kim, M. and Yamamoto, T.,1990. A novel isolation method for hen egg yolk antibody IgY\\\'. Agric. Biol. Chem., 54 : 2531-5.
  7. Akita, E.M. and Nakai, S. 1992. Immunoglobulins from egg yolk: Isolation and purification. J. Food Sci., 57, 629-34.
  8. Gee, Sarah C., Irene M. Bate, Theresa M. Thomas and Dennis B. Rylatt. 2003. The purification of IgY from chicken egg yolk by preparative electrophoresis. Protein Expr Purif 30:151-5.

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