Protein Depletion Using IgY from Chickens Immunised with Human Protein Cocktails

Abstract

Given that proteomic analysis of complex protein mixtures may be restricted by the presence of highly abundant proteins, sample preparation to remove abundant proteins is essential for the analysis of low abundance proteins. Chickens are effective producers of antibodies (IgY) against mammalian proteins, able to produce large quantities of antibodies that can be recovered by simple non-intrusive extraction of egg yolk. The extraction procedure described uses a modification of the water dilution method (WD) to deplete lipids and lipoproteins followed by sequential precipitation with 31% ammonium sulphate and 12% poly ethylene glycol (PEG) producing IgY antibodies with greater than 95% purity and no loss in immunoreactivity. In the present study, various cocktails of the 12 most abundant human plasma proteins were used as immunogens to produce IgY antibodies. The anti-cocktail IgY antibodies were effectively used to sequentially and selectively immunodeplete abundant proteins from plasma. Also, affinity depletion (e.g., Affi-Gel Blue) was combined with immunodepletion to sequentially deplete abundant proteins from both plasma and urine. The current approach described allows the end user to mix and match sets of IgY cocktails to deplete tailored sets of targeted proteins dependent on their end use application.

Keywords:

• Chicken antibodies
• Extraction
• Human proteins
• Immunodepletion
• Protein cocktails
• Proteomics

ACKNOWLEDGMENTS

The authors wish to thank Dr. Tracey Edgell for critical reading of the manuscript.

Notes

The data is representative of six eggs. Values are mean ± SD (n = 3). Protein determination was performed by BCA protein assay as mentioned in the text. Recovery was calculated as a percentage of total protein yield of purified IgY/total protein. The purity of IgY was determined by visual inspection of SDS-PAGE gels.