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.

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

“…For instance, in proteomics, pretreating of serum samples with IgY to specifically neutralize highly abundant serum components was found to improve downstream analyses [155]. In another study the identification of underrepresented serum proteins and disease marker candidate discovery was simplified when specific IgY was used as a blocking reagent [156,157]. This approach was facilitated by the general characteristics of IgY such as the ease of production and the low incidence of cross-reactivity.…”

High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery

“…Another method employed for the removal of multiple high‐abundance proteins with the aim of enriching low‐molecular‐weight species (peptides/proteins) was centrifugal ultrafiltration 31–34. A significant advance in the area of high‐abundance protein depletion and plasma proteome characterization was achieved by applying antibodies generated against several of the most abundant plasma proteins 35–43.…”

IgY ‑ turning the page toward passive immunization in COVID-19 infection (Review)

“…As already mentioned, IgY purification is currently achieved by conventional precipitation methods with ammonium sulphate or PEG and by chromatographic procedures. Sequential precipitation with 31% ammonium sulfate and 12% polyethylene glycol (PEG) produces IgY antibodies with above 95% purity without any loss in immunoreactivity (55).…”