WO2008058325A1 - Immunoaffinity compositions for immunodepletion of proteins - Google Patents

Abstract

The present invention relates generally to the use of affinity compositions to clarify or modify a sample with respect to its complex constituency. More particularly, the present invention enables the treatment of a heterogeneous, complex sample to enrich the sample for particular molecules or groups of molecules. The clarified or modified samples of the present invention are useful inter alia in proteomic studies, diagnostics and in detection of selected low abundance, prognostic biomarkers and in therapeutic agent discovery regimes. The affinity compositions also have use for industrial applications. Affinity compositions are useful, therefore, in the removal of selected molecules from a sample. Affinity compositions in the form of a kit also form part of the present invention.

Description

IMMUNOAFFINITY COMPOSITIONS FOR IMMUNODEPLETION OF PROTEINS

FIELD

The present invention relates generally to the use of affinity compositions to clarify or modify a sample with respect to its complex constituency. More particularly, the present invention enables the treatment of a heterogeneous, complex sample to enrich the sample for particular molecules or groups of molecules. The clarified or modified samples of the present invention are useful inter alia in proteomic studies, diagnostics and in detection of selected low abundance, prognostic biomarkers and in therapeutic agent discovery regimes. The affinity compositions also have use for industrial applications. Affinity compositions are useful, therefore, in the removal of selected molecules from a sample. Affinity compositions in the form of a kit also form part of the present invention.

BACKGROUND

Bibliographic details of references provided in the subject specification are listed at the end of the specification.

Reference to any prior art is not, and should not be taken as an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Proteomic analysis seeks to define the function and relative expression profiles of subsets of proteins encoded by a given genome at a given time in a given cellular location. Proteomics separates, identifies and characterizes the proteins expressed, retained, secreted or released by a cell or tissue in order to establish their function(s) and their potential relationship to the onset, type, stage and progression of diseases, as well as response to therapy and/or relapse. Proteomics may be used to compare tissue samples from diseased and healthy people, in order to identify proteins whose expression is changed in disease. Proteins which are significantly altered in their expression, location or post-translational modifications (PTM) in patients with a disease, compared to those in a group of healthy individuals, may represent protein targets for drug or discovery of biological markers and for endpoint and/or surrogate biomarkers. One application of proteomics is in the search for biological markers of disease onset, progression and treatment in elements of the blood, such as serum or plasma.

Serum and/or plasma proteins are useful diagnostic tools, and alteration of the expression of some serum/plasma proteins is an early sign of an altered physiology, which may be indicative of disease. In routine diagnostic laboratories, identification of specific low abundant disease-associated proteins in serum/plasma relies heavily on time-consuming and expensive radiolabeled or enzyme-linked immunoassay methods (RIA or ELISA) which only have the ability to evaluate a single protein component at a time. Due to the heterogenous nature of most physiological disorders, it is generally considered that no single marker is likely to be sufficiently predictive of disease, so that there is a need for more than one candidate biomarker to enhance already available diagnostic or prognostic tests. It has been suggested that a panel of multiple diagnostic/prognostic markers in serum/plasma can be identified by utilizing proteomic approaches which have the capacity to profile multiple biomarkers (Daly and Ozols, Cancer Cell 7:111-112, 2002).

One tool used in proteomic methods for protein separation and analysis of proteins is two- dimensional gel electrophoresis (2DE). Following separation by 2DE, proteins are characterized and identified, usually by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting or other forms of advanced/hybrid mass spectrometry (e.g. MS/MS), coupled to protein and genomic database searching.

Unfortunately, the analysis by 2DE and other multidimensional chromatographic technologies of proteins in samples of biological fluids such as serum and plasma is very difficult. This is because of the limited amount of protein able to be resolved, and the great variation in the concentration of proteins in many samples. This variation in concentration is frequently referred to as "dynamic range". These factors result in data obtained by 2DE and other chromatographic media from complex samples, such as unfractionated serum and plasma being dominated by the presence of proteins which are of high abundance in blood, for example human serum albumin, immunoglobulin G (IgG), haptoglobin, fibrinogen, transferrin, oci-antiptyrpsin, α₂-macroglobulin, IgA and IgM. Of these, six (albumin, IgG, IgA, IgM, macroglobulin and fibrinogen) constitute 85-90% of the protein mass in blood serum. Proteins with a concentration higher than lmg/mL are generally considered to be of high abundance, and such proteins may represent 2-60% of the total protein present.

Thus, the application of current proteomic technologies is limited by the presence of high abundance "housekeeping" proteins like albumin and immunoglobulins, which constitute approximately 60-97% of the total serum protein (Georgiou et al, Proteomics i:1503-1506, 2001). Such proteins hinder the detection of hundreds of low abundance proteins, some of which might potentially be relevant to a particular disease state. Moreover, the widely spread pattern of albumin and immunoglobulin in chromatographic separations such as the 2-D gel can also obscure proteins with a similar pi and molecular weight. Theoretically, by removing albumin and immunoglobulin, which together constitute 60-97% of the total serum protein, 3-5-fold more protein can be analyzed. If proteomic technologies are to be used routinely for diagnostic purposes, a rapid, inexpensive and simple method is required to remove the high abundant proteins.

In particular, the presence of these abundant proteins severely limits the utility of methods used in wide scale analysis of proteins present in complex mixtures of proteins, such as single dimension electrophoresis (IDE), 2DE, multi-dimensional liquid chromatography and MS. These methods are often used in the investigation of low-abundance proteins such as cytokines, signal transduction proteins, hormonal mediators and cancer biomarkers.

Current load limits for gel electrophoresis restrict the ability to visualize and identify putative clinically-relevant low abundance biomarker proteins. Rare proteins may be difficult if not impossible to detect. Similar, although less extreme, dynamic range problems are experienced with 2DE, multidimensional LC and other chromatographic separative analyses of other types of biological samples, such as urine, tissue extracts, milk, and cell lysates.

Similar problems arise in fermentation fluids and even industrial fluids such as paint and complex chemical solutions.

There is a need to deplete fluids being tested of high abundant molecules or particular types or families or groups of molecules so that other targeted molecules can be assayed for bioactivity or biomarker or component profiles or used in diagnostic protocols and quality control.

One approach is immunodepletion of high abundance proteins using avian-derived antibodies (Ig Y antibodies) selectively raised to specific proteins (see International Patent Application No. PCT/AU2003/001075 [WO 2004/019009] which is incorporated herein by reference in its entirety).

Another approach for immunodepletion is described in US Patent Application No. 09/977,358 (US Publication No. 20020127739). Specific antibodies that have affinity to protein A and protein G are raised against one specific abundant protein at a time and are combined in appropriate ratios in a matrix to deplete the abundant proteins. A similar approach is described in International Patent Application No. PCT/US2004/022786 [WO2005/049653].

Despite these methodologies, there is a need to more efficiently and selectively deplete high abundant molecules or other groups of molecules from complex samples. SUMMARY

Tliroughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The present invention contemplates the use of an affinity composition to modify or clarify a sample with respect to molecules of a particular type, group, family or level of abundance. More particularly, the present invention enables the selective depletion of certain target molecules by affinity depletion such as immunodepletion using immune sera or a fraction or component thereof generated against cocktails of antigens comprising at least two of the target molecules to be depleted. Hence, the present invention contemplates the clarification or modification of complex samples to remove selected molecules such as high abundance molecules, selected proteins or other molecules or therapeutic agents. The present invention is directed, therefore, to affinity compositions comprising ligands to two or more selected molecules and the use thereof to remove or reduce the level of the selected molecules from a sample after passage of the sample through or over the affinity composition. In a particular embodiment, the affinity composition comprises at least two species of antibodies each having specificity for a separate target molecule. Reference to "species" in this context relates to specificity and not class of antibody.

In an embodiment, the target molecules are proteins. However, the present invention extends to non-proteinaceous target molecules including lipids, phospholipids, polysaccharides, nucleic acids, organisms and viruses. The present invention extends to biological samples including body fluids as well as industrial or chemical samples. The present invention enables removal of any targeted interfering molecules or agents. An example of an "interfering molecule" includes a high abundance agent which may mask the presence of a low abundance or rare target molecule.

Hence, in one embodiment, the present invention contemplates a method for generating a sample enriched for a first population of molecules, the method comprising subjecting the sample to affinity-mediated depletion of a second population of molecules using immune sera or a fraction or component thereof from an avian species generated by immunization of the avian species by two or more members of the second population of molecules.

In an embodiment, the first and/or second population of molecules is/are proteins which include peptides and polypeptides. In another embodiment, the first and/or second populations of molecules is/are interring agents in a biological, chemical or industrial sample. The affinity-mediated depletion is generally referred to in this context as immunodepletion.

Hence, in one embodiment the present invention provides a method for generating a sample enriched for a first population of protein, the method comprising subjecting the sample to immunodepletion of a second population protein using immune sera or a fraction or component thereof from an avian species generated by immunization of the avian species by two or more members of the second population of protein.

Whilst the preferred mode of molecule depletion is immunodepletion, other forms of affinity depletion may also occur in combination with the immunodepletion.

In a particular embodiment, the present invention provides a method for clarifying/enriching a sample, the method comprising subjecting the sample to immunodepletion of at least two target molecules potentially present in the sample fluid using avian-derived antibodies generated following the immunization of an avian species with a cocktail comprising at least two molecules and collecting antibodies from egg yolk; and optionally further clarifying the immunodepleted sample by affinity depletion using ligands of said at least two selected molecules.

In an even more particular embodiment, the present invention is directed to a method for clarifying/enriching a sample, the method comprising subjecting the sample to immunodepletion of at least two target proteins potentially present in the sample using avian-derived antibodies generated following the immunization of an avian species with a cocktail comprising the at least two proteins and collecting antibodies from egg yolk; and optionally further clarifying the immunodepleted sample by affinity depletion using ligands of the at least two selected proteins.

The sample includes a biological fluid.

The biological fluid may be whole blood, serum, plasma, milk, lymph, cerebrospinal fluid, urine, amniotic fluid, lavage fluid, cervicovaginal fluid, uterine fluid, seminal fluid or tissue fluid. The biological fluid may also be conditioned medium (CM) from a cell or tissue culture or may be a tissue or cell extract from any species of plants, animals or microbes. A "microbe" may be a prokaryote or eukaryotic cell. The biological sample may also be used for natural product screening (e.g. coral, water, silt etc.). The sample may also be any chemical or industrial sample or an environmental sample. In fact, any sample having high abundance and low abundance including rare molecules is contemplated herein.

The enriched sample may be used as a source of bioactive molecules, as a source to determine bioprofile markers of disease or a state of wellbeing or health and/or to monitor treatment protocols or to assess, for example, markers in an industrial sample. The enriched sample may also be useful in diagnostic protocols for lower abundant molecules.

The level of enrichment of low abundance molecules or level of immunodepletion with optional affinity depletion is readily tested using one-dimensional gel electrophoresin (1 DE), two-dimensional gel electrophoresis (2DE), capillary electrophoresis (CE), mass spectrometry (MS) including advanced and hybrid forms thereof (e.g. MS/MS), high- pressure liquid chromatography (HPLC), gas-chromatography (GC), multi-dimensional liquid chromatography (MDLC) or liquid chromatography/mass spectrometry (LC/MS). These separation techniques may also be used in combination with immunodepletion.

The level of enrichment or depletion can be monitored by chromatographic separation before and after affinity depletion.

Kits comprising particular avian-derived antibodies and/or fractions of sample and/or reagents for chromatographic separation techniques also form part of the present invention. In particular, the present invention provides affinity compositions comprising sera, and/or egg yolk or a fraction or component thereof comprising at least two immunoglobulins each having specificity for differential target molecules.

Abbreviations used in accordance with the present invention are defined in Table 1.

Table 3 Abbreviations

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

Figure 1 is a photographic representation of a ID gel illustrating the purity of a representative IgY fraction.

Figure 2a through e are photographic representations of imrnunoblot detection of antigens using IgY preparations derived from immunizations A to E.

Figure 3 is a photographic representation illustrating specific depletion and enrichment of plasma proteins following SDS-PAGE. NP, neat plasma; AGB, Affigel Blue depleted plasma; AGB/IgY, Affigel Blue and IgY depleted plasma; MW, molecular weight markers.

Figure 4 is a photographic representation illustrating specific depletion and enrichment of plasma proteins following 2D gel electrophoresis. See Figure 1 for legend for abbreviations.

Figure 5 is a graphical representation illustrating specific depletion and enrichment of plasma proteins following HPLC.

Figures 6a and b are graphical representations of mass spectra and identification score illustrating the enrichment and identification of low abundance proteins in a HPLC fraction of neat and depleted plasma.

Figure 7 is a photographic representation illustrating the specific depletion and enrichment of urine proteins following SDS-PAGE. NLJ, neat urine, AGB, Affigel Blue depleted urine, AGB/IgY, Affigel Blue and IgY depleted urine, MW, molecular weight markers. Figure 8 is photographic representation illustrating specific depletion and enrichment of urine proteins following 2D gel electrophoresis. See Figure 5 for legend abbreviations.

Figure 9 is a graphical representation of specific depletion and enrichment of urine proteins and peptides following HPLC.

Figures 10a through d are graphical representations of mass spectra illustrating specific depletion and enrichment of polypeptide peaks from HPLC fractions of neat and depleted urine.

Figures 11a through d are graphical representations mass spectra illustrating specific depletion and enrichment of polypeptides peaks from neat and depleted urine following magnetic bead separation.

Figure 12 is a comparative photographical representation of 2D gels illustrating specific and non-specific depletion of plasma proteins using IgY or dye affinity coupled with IgY technology.

Figure 13 is a photographical representation a ID gel illustrating the re-usability of IgY columns.

DETAILED DESCRIPTION

Unless otherwise indicated, the subject invention is not limited to specific assay steps, reagents, antibodies, manufacturing methods, diagnostic regimes, or the like as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be necessarily limiting.

The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a single biomarker as well as two or more biomarkers; reference to "an assay" includes a single assay, as well as two or more assays; reference to "the invention" includes a single and multiple aspects of an invention; and so forth.

Abbreviations used herein are defined in Table 1.

All scientific citations, patents, patent applications and manufacturer's technical specifications referred to hereinafter are incorporated herein by reference in their entirety.

For the purposes of this specification, "affinity depletion" means the removal of components from a complex mixture based upon chemical or immunological characteristics by specific agents. hi one aspect, the affinity depletion is by immunodepletion. Hence, the present invention encompasses affinity compositions comprising immunoglobulins specific for at least two target molecules immobilized to a solid support such that upon contact between the target molecule and the immobilized immunoglobulin, an immuiiocomplex forms comprising the target molecule and immunoglobulin. The at least two immunoglobulins are generated or located in avian sera immunized by at least two molecules to which the immunoglobulin have specificity.

The term "affinity support" refers to a matrix or support to which specific agents are bound or coupled and which is used to deplete components from a complex mixture. Hence, the affinity composition of the present invention comprises at least two immunoglobulins, or a sera or IgY fraction immobilized to an affinity support. The immunoglobulins are, therefore, affinity reagents.

The term "affinity reagents" is used to define biological substances or macromolecules that can specifically bind to targets in a biological sample through affinity recognition and attractive forces between reagents and targets. Affinity recognition, resembling the relationship between lock and key, is highly specific for the target and usually has a dissociation constant below 10^"8 M (Winzor, J. Chromatogr. 1037(1-2) :351-367, 2004; Chaiken, J Chromatogr. 376:11-32, 1986). The affinity reagents can include IgY antibodies, proteins, peptides, affibodies, minibodies, aptamers, nucleotides, polymers and others. However, in an embodiment, the affinity reagents comprise at least two immunoglobulins of differing specificities to two target molecules.

A "solid support" is the material to which the affinity reagents are attached through a linkage and can mediate the affinity reagents to separate bound targets from those nonspecific targets. A "solid support" is similar to an "affinity support". The solid support generally comprises surface materials and a core or base. The surface materials are the active chemical or biological materials that can link the solid support to the affinity reagents. These materials comprise hydrazide, active chemicals, polystyrene, receptor, protein A/G, biotin avidin, strepavidin, macromolecules and others. The core or base is coated with the surface materials and linked to affinity reagents via surface materials. The core or base can be the materials that help or mediate the separation of that affinity reagent-target complex. Examples of the core or base include microbeads, nanobeads, microtiter wells, flat supports, acrylamide/azlactone copolymer, polystyrenedivinylbenzene, polystyrene, agarose, paramagnetic, magnetic and others.

The term "separation devices" includes the forces, attractions, apparatus, or processes that mediate the separation of the affinity reagent-target bound solid support form mixture of targets or materials. Examples of the separation devices include gravity, centrifugation, liquid chromatography, magnetic force, multiple tubes or wells, microfluidic and others. Hence, separation devices are employed to assist passage of a sample along or through an affinity support in order to facilitate affinity depletion of target molecules.

The term "target molecules" is used to define the molecules in the sample to be removed. For example, the target molecules may be proteins such as albumin or immunoglobulins or other high abundance molecule. In accordance with the present invention, at least two target molecules are selected at a time for depletion. Furthermore, in a diagnostic aspect, the diagnostic target may be a low abundance or rare molecule. Hence, high abundance molecules are removed or their level depleted to facilitate identification of the low abundance molecule.

"hnmunodepletion" means the use of immunoglobulins raised against specific components of a complex mixture to remove those components from the mixture. The terms "antibody" and "immunoglobulin" may be used interchangeably throughout the subject specification.

"Immunoaffmity" refers to the association between an immunoglobulin and its corresponding antigen or epitope.

The terms "high abundance protein", "high abundance molecule" or "highly abundant protein" refer to a protein or molecule which is present at the highest or higher concentration relative to other proteins or molecules in the sample. Similarly, a low abundance molecule includes a rare molecule which is present in a lower concentration relative to other molecules.

The term "IgY" is used to denote the avian equivalent of IgG and in particular an immunoglobulin isolated from egg yolk. It is significantly different in its chemical and physical properties from IgG. In particular, in addition to having different amino acid composition and sequence, IgY has a much higher electrophoretic mobility, a much lower isoelectric pH and a higher molecular weight than IgG, and has substantially different chemical stability. Under certain conditions IgY requires stabilization by non-ionic surfactants, whereas IgG is stable in the absence of surfactants. Ionic detergents can inhibit the reaction of IgG with some antigens, but these agent have little effect on the ability of IgY to bind antigens. IgY is monomelic in 0.15 M NaCl (low salt conditions), and is dimeric in 1.5 M NaCl (high salt conditions), while IgG is monomeric at both low and high salt conditions. The properties of IgY are described in detail in US Patent No. 4,550,019. The structural differences between the two molecules mean that the hinge region which is present in IgG between the Fab pieces is absent in IgY, and hence IgG is slightly less suitable than IgY for use in solid-phase extraction procedures.

The yolk of eggs laid by immunized avian species is an abundant source of polyclonal antibodies (pAb). Specific antibodies produced in avian species offer several important advantages over antibodies produced in mammals.

Due to the phylogenetic distance between birds and mammals, there is greater probability of producing a higher percentage of specific antibody against mammalian antigens by antigens by immunizing avian species than by immunizing other animals. Highly conserved mammalian proteins sometimes fail to elicit a humoral immune response in animals, such as rabbits, which are traditionally used for generating polyclonal antibody.

Since avian IgY does not cross-react with mammalian IgG, and does not bind bacterial or mammalian Fc receptors, non-specific binding is reduced, and the need for cross-species immunoabsorption is also eliminated.

The sample may be any complex mixture of high and low abundance molecules. The sample may be, for example, a biological, industrial, chemical or environmental sample. Examples of industrial samples include paint or polymer formulations, industrial waste preparation and the like.

The term "biological sample" includes a biological fluid, such as whole blood, serum, plasma, milk, lymph, lavage fluid, cerebrospinal fluid, amniotic fluid, cervicovaginal fluid, uterine fluid or seminal fluid. The biological sample may also be conditioned medium

(CM) from a cell or tissue culture, or may be a tissue or cell extract, especially an extract of a highly vascularized tissue.

The biological sample may be obtained from any mammalian species, including humans, companion animals such as dogs and cats, domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids and ungulates.

The biological sample may also be obtained from plants or microbes. Reference to a

"microbe" includes prokaryotic and eukaryotic cells. In one embodiment, the sample is obtained from a human. Hence, the present invention extends to clarification and/or enrichment of samples useful, for example, in natural product screening. It also extends to clarification and/or enrichment of non-biological samples such as industrial, chemical and environmental samples.

In relation to a biological sample, the mammal may be of either sex, may be of any age, and may be either healthy or suffering from any kind of pathological condition, including but not limited to infections, cancers, or chronic degenerative conditions. In other words, the method of the invention is applicable to any situation where it is desired to perform analysis in order to detect a low abundance molecule, or to identify whether there is a change in the pattern of expression of such a molecule in a mammal. This is useful in the screening of low abundant bioactive molecules, proteomic studies and diagnostic assays for target molecules which are present in low concentrations.

The present invention provides means for modifying a complex sample (e.g. biological fluid) to remove selected molecules or groups or families of molecules. Hence, by "modifying" in this context includes clarifying and/or enriching the biological fluids to reduce the level of selected species of molecules. The selected species may be high abundance proteins, molecules or agents introduced as part of a therapeutic regime or any other naturally occurring or introduced species. Hence, the molecules to be removed or reduced may be proteinaceous in nature including peptides, polypeptides or proteins, nucleic acids, lipids including cholesterol, phospholipids, polysaccharides or complexes comprising same, cells and viruses. Particular molecules targeted for removal or depletion are proteins. Reference to "proteins" includes peptides and polypeptides. However, the present invention extends to the removal or depletion of any interfering molecule or agent from any type of sample.

The selected removal or depletion of targeted molecules has a corollary of enriching the biological sample for other molecules such as low abundance molecules. Accordingly, the present invention relates to the selective depletion of target molecules to enable enrichment of a second group, family or species of molecules.

One aspect of the present invention is the use of at least two target molecules to generate avian-derived immunoglobulins having specificity to one or other of the target molecules which are then used to immunodeplete the target molecules from the sample. Further affinity depletion or other form of purification may also occur. Thus, at least two target molecules may be referred to as a "cocktail" of target molecules or a cocktail of antigens. The present invention extends, therefore, to affinity compositions comprising at least two immunoglobulins each specific for a target molecule.

The present invention also provides cocktails or compositions comprising at least two target molecules for use in immunizing an avian species to generate IgY antibodies.

The present invention contemplates therefore a method for clarifying/enriching a sample, the method comprising subjecting the sample to immunodepletion of at least two target molecules potentially present in the sample using avian-derived antibodies generated following the immunization of an avian species with a cocktail comprising the at least two molecules and collecting antibodies from egg yolk; and optionally further clarifying the immunodepleted sample by affinity depletion using ligands of said at least two selected molecules.

As indicated above, an example of target molecules is protein (including peptides and polypeptides). Hence, another aspect of the present invention provides a method for clarifying a sample, the method comprising subjecting the sample to immunodepletion of at least two target proteins potentially present in the sample using avian-derived antibodies generated following the immunization of an avian species with a cocktail comprising the at least two proteins and collecting antibodies from egg yolk; and optionally further clarifying the immunodepleted sample by affinity depletion using ligands of said at least two selected proteins.

Examples of targeted proteins for immunodepletion including albumin, microglobulin, immunoglobulin, transferrin, antitrypsin, glycoprotein and/or an apolipoprotein protein.

The present invention further contemplates a method for enriching a sample for low abundance proteins or otherwise reducing the level of targeted proteins, the method comprising subjecting the sample to immunodepletion using sera or antibody fraction isolated from egg yolk of an avian species immunized with at least two proteins selected from the list consisting of albumin, macroglobulin, immunoglobulin, fibrinogen, haptoglobulin, transferrin, antitrypsin, glycoprotein and an apolipoprotein ; and optionally further clarifying the immunodepleted sample by affinity depletion using a ligand to two or more of the above or other plasma/serum proteins.

The sample may undergo multiple rounds of immunodepletion with different combinations of antibodies. In addition, the antibodies may not necessarily be purified antibodies but a mixture of IgY antibodies which include specific antibodies to the target proteins. Polyclonal IgY antibodies are particularly useful. The antibodies may also be re-used. Hence, where the antibodies are immobilized to a solid support in a clarification device, the clarification device may be re-used any number of times such as from twice or over 15 times (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 re-uses).

The present invention contemplates a method for generating a modified sample, the method comprising subjecting a non-modified sample to one or more rounds of immunodepletion using IgY antibodies which comprise at least two species of antibodies each specific for a protein selected from the list consisting of albumin, macroglobulin immunoglobulins, fibrinogen, haptoglobin, transferrin, antitrypsin, glycoprotein and apolipoproteins; and optionally subjecting the immunodepleted modified sample to one or more rounds of affinity depletion using at least two ligands each specific for at least one of the above proteins.

Another aspect of the invention contemplates methods for combining individual sets of IgY antibodies which comprise at least two species of antibodies each specific for a selected protein of interest. In this way, immunodepletion of multiple selected proteins can be achieved simultaneously rather than by sequential steps using a combination of different antibody sets.

A further aspect of the invention relates to methods for generating immunodepletion kits with varying selectivities by combining different sets of IgY antibodies each of which comprises at least two species of antibodies each specific for a selected protein. In this way, application-specific kits can be provided for immunodepletion of various sets of high abundance proteins peculiar to a particular biological fluid, tissue or cell. For example, an immunodepletion kit for human plasma or serum might combine multiple sets of IgY antibodies which comprise at least two species of antibodies each specific for a protein selected from the list consisting of albumin, macroglobulin immunoglobulins, fibrinogen, haptoglobin, transferrin, antitrypsin, glycoprotein and apolipoproteins. Antibody sets directed against major tissue-specific proteins would be included to generate immunodepletion kits with tailored application to extracts derived from heart, lung, kidney or other tissue extracts or biological samples. As indicated above, the kits may comprise a clarification device comprising immobilized antibodies (e.g. IgY antibodies). The clarification device (i.e. the device which immunodepletes targeted molecules from a sample) may be a single use device (i.e. discarded after a single use) or may be multiple use device (i.e. re-usable for any number of times such as from 2 to 15 times).

Another aspect of the invention is the production of custom combinations of IgY affinity columns/matrices to parallel the relative abundance of complicating proteins (i.e. mole for mole), also that for specific applications, i.e. 2D gels. For LC- MS/MS one can selectively generate immunodepletion media to selectively remove interfering proteins that complicate that particular technology. Thus the high abundant proteins that complicate ID & 2D gel s can be removed, whereas with some of the LCMS/MS based methods other proteins can be depleted. The advantage would be that with judicious selection of IgY antibodies someone skilled in the art can create "custom (bespoke) affinity purification media" to suit specific applications. Furthermore, if someone skilled in the art is looking at CSF, urine, lung lavage, serum, plasma, etc., then the relative ratio of dominating proteins changes, again, one can generate specific columns/purification strategies to accommodate this. Therefore someone skilled in the art has the capacity to provide specific depletion columns for specific applications.

Notwithstanding that the present invention is particularly useful for targeting the listed proteins, it is clear that any two or more proteins or non-pro teinaceous molecules may be selectively removed from the sample. Hence, a "modified" sample is a fluid in which at least one protein or other molecule has been removed or depleted or where the fluid has undergone at least one purification or clarification step. In addition, the sample may be a biological fluid or a non-biological fluid. A non-biological fluid includes an industrial, chemical and environmental sample.

The extent to which a sample has been modified, clarified, immunodepleted and/or enriched for particular molecule types or fractions can be readily determined using a range of chromatographic or spectrometric techniques including one or two dimensional gel electrophoresis, capillary electrophoresis, mass spectrometry, high-pressure liquid chromatography, gas chromatography, liquid chromatography and/or liquid and mass chromatography. Alternatively, the modified or clarified sample may be subject to further purification using any one or more of the above techniques.

Although the order of clarification steps is preferably affinity depletion followed by immunodepletion, the present invention extends to either order. However, non- immunological-based affinity depletion is an optional step. With respect to affinity depletion, this may be accomplished in any number of ways including affinity matrix chromatography wherein the two or more specific ligands or generic ligands or dyes are immobilized to a solid support. The affinity depletion step comprises, in one embodiment, the use of a clarification device comprising immobilized antibodies. As indicated above, the clarification device may be a single or multiple use device.

Typically, the support is a dye affinity chromatography resin, in which a solid support is coupled to a dye such as a chlorotriazine compound, including but not limited to Cibacron blue F3GA affinity supports such as Affi-gel Blue (Bio-Rad Laboratories), or Blue

Sepharose (Amersham Biosciences). Other dye-ligands which could also alternatively be employed to remove abundant blood proteins include Procion Red HE3B, Reactive Blue

MRB, Reactive Green H4G, Reactive Green HE4BD, Reactive Green HE4BD, React4ive Yellow M8G, and Reactive Brown M4R, all of which can be coupled to supports such as

Sepharose 4B and 6B. Dyes suitable for use in affinity chromatography are discussed in a review by Scawen, Anal. Proc. 25:143-144, 1991. Alternatively, the support may be coupled to a protein such as Protein A, Protein G or Protein A/G fusions. Affinity chromatography techniques are well known in the art and are reviewed in Hage, Clinical Chemistry 45:593-615, 1999 and Larsson, Methods Enzymol 104:212-223, 1987.

The affinity depletion step may involve the use of magnetic beads such as agarose (Dynabead M-280) as a solid phase matrix support for an affinity ligand for the magnetic separation of the targeted at least two molecules from the biological fluid.

Similarly, any solid-phase support which can be coupled to an immunoglobulin to form an affinity support may be used for immunodepletion, these include but are not limited to agarose gels such as Sepharose 4B or Sepharose 6B (Pharmacia), cross-linked agarose, or acrylamide-based and cellulose-based beads.

The antibodies used in immunodepletion may be polyclonal antibodies raised against whole serum or plasma, or against any fraction of these complex proteinaceous mixtures or against purified or semi-purified proteins and may suitably be raised using an immunization schedule comprising multiple booster injections. The antibodies may also be raised to any combination of target molecules such as in an industrial sample. The antibodies may be raised in any convenient avian species. Where the antibodies are avian antibodies, these may be raised in any convenient species of bird, but most conveniently will be raised in a poultry species such as a chicken, turkey, duck or goose.

Most particularly, the avian antibody is a chicken antibody. In one embodiment the antibody is chicken IgY from egg yolk. The targeted molecule for immunodepletion may be conjugated to a carrier protein if necessary in order to increase immunogenicity.

The antibodies, may be produced and purified using any conventional method or modification thereof. Suitable methods for preparation of IgY are disclosed in US Patent Nos. 5,367,054; 5,420,253; 4,550,019 and 4,056,737.

The present invention further provides a composition for immunodepletion of a high abundance molecule from a biological sample, comprising antibody preparations directed against targeted molecules in a biological sample coupled to an affinity support. In one embodiment, the antibody is an avian polyclonal antibody.

The present invention provides a device for the rapid processing of samples comprising a generally cylindrical chamber having an opening at either end, in which each opening is adapted to interact with a receptacle, in which the sample can be transferred from one receptacle to the other via the chamber, and in which the chamber has transversely disposed within it multiple layers of an affinity support having a high affinity for two or more targeted molecules in a biological sample, separated by a layer of an affinity support coupled to two or more antibodies or sera or fraction of sera comprising same directed against the targeted molecules. The device may be a single use or multiple use device.

The targeted molecules are, for example, albumin and immunoglobulins and the antibody is avian IgY. However, the target molecules may be any defined molecule or family or group of molecules in any sample.

The term "interact" means that the chamber fits to the receptacle such that minimal fluid can escape when fluid is passed from one receptacle to another via the chamber. The plane of each layer of the support is generally perpendicular to the axis of the chamber. In use, the chamber is connected at one end to a receptacle containing a fluid sample, and at the other end to an empty receptacle, and the sample is passed a number of times from one receptacle to the other through the chamber.

In one embodiment the receptacles are hypodermic syringes and the chamber is a Luer- type cartridge. More particularly, both the chamber and the receptacles are made of plastics. In a second embodiment the chamber is adapted to be coupled directly to a separation or analytical apparatus such as an HPLC or LC/MS.

The present invention also provides a kit for removal of targeted molecules from a sample, comprising an affinity support with affinity for targeted molecules in a sample and optionally, the kit comprises a second or further affinity supports each coupled to at least two antibodies directed against other targeted molecules.

The invention is further described by way of reference to the following non-limiting examples and drawings. In the Examples, the following materials and methods are employed:

Antigen cocktails

Cocktails of two or more protein antigens were prepared for use in immunizing chickens. The cocktails are conveniently referred to as Cocktails A through E and are defined below:

Cocktail A:

Human Serum Albumin (66kD) - 500 μg per injection IgG (170 kD) - 500 μg per injection

Cocktail B:

IgM (900 kD) - 500 μg per injection IgA (250 kD) - 500 μg per injection

Cocktail C: α2-Macroglobulin (725 kD) - 500 μg per injection

Fibrinogen (341 kD) - 500 μg per injection

Cocktail D:

Haptoglobin (86 kD) - 500 μg per injection

Transferrin (80 kD) — 500 μg per injection al -Antitrypsin (52 kD) - 500 μg per injection

Cocktail E: cd -Acid glycoprotein (44 kD) - 250 μg per injection

Apolipoprotein AI (28 kD) — 250 μg per injection

Apolipoprotein All (17 kD) - 250 μg per injection

Cocktail F:

Human Serum Albumin (66kD) - 500 μg per injection

Immunizations

The following protocol was adopted for immunizing the chickens with Cocktails A through F.

Two chickens were used for each of the five antigen cocktails.

IgY extraction

IgY purification was conducted using acid water extraction followed by PEG precipitation.

In general terms, ten eggs were opened and yolk separated and retained from the egg white. The yolk sac was rinsed in distilled water and then dried on clean dry paper towel. The yolk sac was suspended over a beaker and with a Pasteur pipette, the egg sac was punctured and yolk collected in the beaker.

One volume of yolk was suspended in nine volumes of 3 mM HCl and the pH adjusted to pH 5.5 with 10% v/v acetic acid. The suspension was stirred overnight in a cold room and then centrifuged for 30 minutes at 2000 g. Solid ammonium sulfate is added to 31% and the suspension stirred for 1 hour in the cold room. The suspension was then centrifuged for 30 minutes at 2000 g.

The pellet was washed with 30 ml 31% w/v ammonium sulfate solution for 30 minutes and centrifuged again for another 30 minutes at 2000 g. The resulting pellet was dissolved in 50 ml 20 mM phosphate buffer and one volume of IgY extract combined with PEG 6000 (24% w/v) [one volume] and stirred for 1 hour in a cold room. The mixture was then centrifuged again at 2000 g for 30 minutes and the pellet re-dissolved in 50 ml 20 mM phosphate buffer. The protein solution was stored 10 ml aliquots at -2O⁰C.

IgY analysis

The quality of the IgY extract was determined using a BCA assay of crude yolk and the IgY preparation followed by gel electrophoresis, liquid chromatography, magnetic bead separation and mass spectrometry (see Figure 1).

Anti-Cocktail A IgY antibodies

Cocktail A comprises human serum albumin (66 kD) at 500 μg and IgG (170 kD) at 500 μg. Immunization and IgY purification resulted in 935 mg of protein from 82 g of egg yolk at a concentration of 18.7 mg protein/ml in 50 ml final volume. Purity of the preparation was over 90%.

Specificity

Antibody specificity of the IgY fraction generated following immunizations with the different cocktails were determined by Western Blot. In general, the respective cocktails gave strong signals to their respective purified antigens as well as with human serum and plasma samples (see Figure 2). EXAMPLE 1

Depletion of plasma proteins

SDS-PAGE was performed using 4-12% Bis-Tris gels on a Novex NuPAGE system. 25 μg of neat protein (NP), Affigel Blue (AGB) and Affigel Blue and IgY (AGB/IgY) depleted plasma were analyzed per sample. The results are shown in Figure 3. Proteins were visualized with colloidal coomassie and gels imaged using a BioRad densitometer.

Band 2 (human serum albumin) is depleted after AGB treatment. Bands 1 and 3 (macro globulin and immunoglobulin light chain, respectively) were depleted upon IgY treatment. Bands 4, 5, 6, and 7 were enriched during the depletion process, while bands 8 and 9 appear only in the AGB/IgY-depleted plasma samples. Proteins enriched were identified from 2D gels as transferrin, antitrypsin, haptoglobin, apolipoprotein Al and A2, transthyretlirin, and alpha 2-glycoprotein.

This Example illustrates specific depletion of plasma proteins using affinity immunodepletion. Consequently, less abundant proteins are enriched and can be detected using SDS-PAGE.

EXAMPLE 2 Selective depletion of plasma proteins

2D gel electrophoresis was performed with a No vex NuPAGE system using 3-10 NL strips in the first dimension and 4-12% Bis-Tris gels in the second dimension. The equivalent of 50 μg of NP, AGB- and AGB/IgY-depleted plasma was analyzed. See Example 1 for abbreviations. Proteins were visualized with SyproRuby. The stained gels were imaged using a BioRad FX laser scanner. The results are shown in Figure 4.

AGB depleted plasma removed human serum albumin (spot 1) and enriched immunoglobulins (2 and 3), transferrin (4), antitrypsin (5), and haptoglobin (6). IgY treatment most notably depleted the immunoglobulins and further enriched proteins 4 to 6 and ceruloplasmin (7), apolipoproteins (8, 10), and transthyrethrin (9) as identified by peptide mass fingerprint.

This Example illustrates specific depletion of plasma proteins using the invention. Consequently, less abundant proteins are enriched and can be detected and identified using 2D gel electrophoresis and MALDI-ToF peptide mass fingerprinting.

EXAMPLE 3 Selective depletion and enrichment of plasma proteins

The equivalent of 120 μg neat or AGB/IgY-depleted plasma was fractionated on an Agilent HPLC using a Phenomenex Jupiter C18 column (3OθA, 2.0 x 150 mm, particle size 5μm). See Example 1 for abbreviations. Elution carried out using a linear gradient of 0-70% Buffer B over 35 mins after an initial 10 min wash at a flow rate of 0.3 ml/min, Buffer A consisted of 0.1% v/v TFA pH 2 and Buffer B 0.08% v/v TFA, pH 2 and separation monitored by absorption at 214 nm. The results are shown in Figure 5.

The chromatogram indicates significant depletion of the major peak at a retention time of 37 min from neat plasma in comparison to AGB/IgY-depleted plasma, which was identified by peptide mass fingerprinting (mascot score 135) as human serum albumin from the neat plasma fraction. This depletion was accompanied by increased intensities of peaks with retention times of 36 min (identified as transferrin, mascot score 240), 39 min (identified as transthyrethrin, mascot score 87), 42 min (identified as mixture of alpha 1- antitrypsin and Apo Al, mascot score 228), and 43 min (identified as Apo Al, mascot score 123). The RP-HPLC chromatogram of the AGB/IgY depleted plasma also showed some unique peaks occurring at retention times 30, 31, 34 and 45 min, which did not yield significant peptide mass fingerprints or were not analyzed.

This Example illustrates specific depletion of major plasma proteins. This allows the selective enrichment of less abundant proteins to be detected using RP-HPLC and identified by mass spectrometry tools. As such, the method enables the visualization and identification of multiple protein/peptide species that could not be previously detected by RP-HPLC. EXAMPLE 4 Detection and identification of low abundance proteins in plasma

Mass spectra of tryptic digests of fraction 43 (retention time 43 mins) from above chromatogram (see Figure 5) were generated on a Bruker autoflex ToF/ToF operated in reflectron mode, acquiring a mass range from 700 to 5,000 Da. Samples were analyzed in HCCA matrix with a total of 500 laser shots summed per sample. The results are shown in Figures 6a and 6b.

As an example, only the peptide mass fingerprint of the AGB/IgY-depleted plasma yielded a significant result (mascot score 228) and was identified that a mixture of alpha- 1- antitrypsin and Apolipoprotein AL Peaklists of fraction 43 from neat plasma or AGB- depleted plasma did not yield significant peptide mass fingerprint results.

This Example illustrate that using affinity immunodepletion, low abundant proteins can be enriched to detectable levels in depleted samples.

EXAMPLE 5 Depletion of urine proteins

SDS-PAGE was performed using 4-12% Bis-Tris gels on a Novex NuPAGE system. The equivalent of 25 μg of neat, AGB or AGB/IgY-depleted urine from a healthy male was analyzed. Proteins were visualized with colloidal Coomassie stain and gels were imaged using a flatbed scanner. The results are shown in Figure 7.

Band 3, presumably human serum albumin, was depleted from neat urine using AGB treatment. Bands 1, 2, A, and 5 were depleted with IgY treatment. These samples were not further analyzed, it is presumed that some of the IgY-depleted protein bands are heavy and light chains of immunoglobulins. Enrichment of specific protein/polypeptide species upon

AGB/IgY treatment was not detected using this technology. Other strategies for sample analysis such as liquid chromatography (LC) or magnetic bead separation coupled with MS may alternatively be used for urine samples due to their high peptide/protein ratio (see also Figure 9).

The Example, illustrates specific depletion of major urine proteins.

EXAMPLE 6

Selective depletion of urine proteins

2D gel electrophoresis was performed on a Novex NuPAGE system using 3-10 NL strips in the first dimension and 4-12% Bis-Tris gels in the second dimension. The equivalent of 50 μg of neat, AGB- and AGB/IgY-depleted urine from a healthy male was analyzed. Proteins were visualized with SyproRuby and imaged using a BioRad FX laser scanner. The results are shown in Figure 8.

Human Serum Albumin (spot 1) was not detected following AGB depletion. Immunoglobulins, heavy and light chains, (spot 2) were depleted using AGB, upon which spots 4, 8, and 9 appeared and spots 3, 5, 6, and 7 were enriched. The identities of these enriched proteins were not determined.

This Example illustrates specific depletion of major urine protein. Consequently, some less abundant proteins are enriched and can be detected.

EXAMPLE 7 Selective depletion and enrichment of urine proteins and polypeptides

The equivalent of 50 μg of neat and AGB/IgY-depleted urine was fractionated on an Agilent HPLC using a Phenomenex Jupiter Cl 8 column (300A, 2.0 x 150 mm, particle size 5μm). Elution was carried out using a linear gradient of 0-100% Buffer B over 75 mins after an initial 5 min wash at a flow rate of 0.3 ml/min, Buffer A consisted of 0.1% TFA pH 2 and Buffer B 0.08% TFA, pH 2 and separation was monitored by absorption at 214 nm. The results are shown in Figure 9.

The chromatogram indicates (partial) depletion of major peaks at retention times of 33 to 38 min from neat urine. The depletion is accompanied by an increase in relative intensities of peaks with lower retention times (20 to 30 min), indicating a possible an enrichment of peptides. The composition of some of these fractions was further investigated with MALDI-TOF mass spectrometry (see Figure 8 below).

This Example illustrates the depletion of some major urine proteins using immunodepletion. Consequently, the less abundant peptides/polypeptides are enriched to enable detection and identification using mass spectrometry tools.

EXAMPLE 8 Selection enrichment and detection of polypeptides in urine fractions

Please refer to Figures 10a through 1Od.

Mass spectra of fractions 25 (retention time 25 mins) from the chromatogram (of Figure 9) were generated on a Bruker autoflex ToF/ToF operated in linear mode, acquiring a mass range from 700 to 10,000 Da. Samples were analyzed in HCCA matrix with a total of 500 laser shots summed per sample.

The obtained mass spectra indicate a loss of 1 peak (m/z 3724.641) coupled with the emergence of 4 new peaks (m/z 952.104, 1140.350, 1623.934 and 2483.519) in AGB/IgY- depleted urine fraction when compared to the neat urine (Figure 10a).

Mass spectra of fractions 30 (retention time 30 mins) from the chromatogram (of Figure 9) were generated on a Bruker autoflex ToF/ToF operated in linear mode, acquiring a mass range from 700 to 10,000 Da. Samples were analyzed in HCCA matrix with a total of 500 laser shots summed per sample.

The obtained mass spectra indicate loss of 7 peaks (m/z 1318.511, 1880.576, 2010.690, 2067.898, 3324.612, 3437.738, and 3518.213) coupled with the emergence of 8 new peaks (m/z 897.229, 1287.6, 1553.888, 1965.856, 1986.976, 2174.988, 2526.854, 3364.122) in the AGB/IgY- depleted urine fraction when compared to the neat urine fraction (Figure 10b).

Mass spectra of fractions 36 from the HPLC fractionations (of Figure 9) were generated on a Bruker autoflex ToF/ToF operated in linear mode, acquiring a mass range from 700 to 10,000 Da. Samples were analyzed in HCCA matrix with a total of 500 laser shots summed per sample. The obtained mass spectra indicate a loss of 10 peaks (m/z 1347.649, 1596.931, 1734.911, 1743.871, 2091.044, 2277.777, 2294.378, 2390.839, 2407.371, and 2826.951) coupled with the emergence of three new peaks (m/z 1623.137, 1641.332, and 2475.253) in the AGB/IgY-depleted urine fraction when compared to the neat urine fraction (Figure 10c).

Mass spectra of fractions 37 (retention time 37 mins) from the chromatogram (of Figure 9) were generated on a Bruker autoflex To/ToF operated in linear mode, acquiring a mass range from 700 to 10,000 Da. Samples were analyzed in HCCA matrix with a total of 500 laser shots summed per sample.

The obtained mass spectra indicate a loss of 17 peaks (m/z 1045.386, 1174.553, 1243.706, 1506.652, 1562.881, 1614.817, 1743.96, 1933.868, 1967.008, 1983.123, 2004.013, 2339.198, 2444.254, 3636.287, 3776.401, 3855.521, and 4093.403) coupled with the emergence of 4 new peaks (m/z 2578.527, 2651.511, 3124.951, 3182.121, and 3444.214) in the AGB/IgY-depleted urine fraction when compared to the neat urine fraction (Figure 1Od).

This Example illustrates that polypeptide peaks are removed from specific fractions of neat urine, which coincides with the appearance of previously non-detected polypeptides in depleted urine samples. These peaks could be characterized further using mass spectrometry tools.

EXAMPLE 9

Selective depletion and enrichment of polypeptides in urine preparations

Please refer to Figures 1 Ia through 1 Id.

Neat and AGB/IgY depleted urine samples were further enriched using Bruker Clinprot magnetic bead HIC 8 chemistry at 0.8 and 1 mg/ml, according to manufacturer's instructions. The resulting samples were analyzed in HCCA matrix. Mass spectra were generated on a Bruker autoflex ToF/ToF operated in linear mode, acquiring a mass range from 1,000 to 20,000 Da using a total of 1,000 laser shots summed per sample.

The obtained mass spectra indicate a loss of 6 peaks (m/z 2186.738, 2430.312, 2784.061, 4361.905, and 4747.74) coupled with the emergence of 1 new peak (m/z 9814.446) in AGB/IgY-depleted urine when compared to a neat urine sample (Figure 1 Ia).

Neat and AGB/IgY depleted urine samples were further enriched using Bruker Clinprot magnetic bead IMAC-Cu chemistry at 0.8 and 1 mg/ml according to manufacturer's instructions. The resulting samples were analyzed in HCCA matrix. Mass spectra were generated on a Bruker autoflex ToF/ToF operated in linear mode, acquiring a mass range from 1,000 to 20,000 Da using a total of 1,000 laser shots summed per sample.

The obtained mass spectra indicate a loss of 2 peaks (m/z 1835.179 and 2316.055) coupled with the emergence of 5 new peaks (m/z 1448.526, 1748.005, 1818.268, 1884.423, and 5423.932) in AGB/IgY-depleted urine when compared to a neat urine sample. The identities of these peaks were not further investigated (Figure 1 Ib).

Neat and AGB/IgY depleted urine samples were further enriched using Bruker Clinprot magnetic bead HIC 8 chemistry at 4.8 and 5.1 mg/ml according to manufacturer's instructions. The resulting samples were analyzed in HCCA matrix. Mass spectra were generated on a Bruker autoflex ToF/ToF operated in linear mode, acquiring a mass range from 1,000 to 20,000 Da using a total of 1,000 laser shots summed per sample. The obtained mass spectra indicate a loss of 5 peaks (m/z 1711.117, 6108.365, 8009.201, 8386.747, and 11708.727) coupled with the emergence of 14 new peaks (m/z 2103.749, 3017.964, 3177.826, 3378.526, 3516.668, 3707.311, 4020.803, 4247.398, 4415.325, 5021.711, 5508.458, 5690.493, 10749.171, and 15647.550) in AGB/IgY-depleted urine when compared to a neat urine sample. The identities of these peaks were not further investigated (Figure l ie).

Neat and AGB/IgY depleted urine samples were further enriched using Bruker Clinprot magnetic bead IMAC-Cu chemistry at 4.8 and 5.1 mg/ml according to manufacturer's instructions. The resulting samples were analyzed in HCCA matrix. Mass spectra were generated on a Bruker autoflex TOF/TOF operated in linear mode, acquiring a mass range from 1,000 to 20,000 Da using a total of 1,000 laser shots summed per sample.

The obtained mass spectra indicate a loss of 18 peaks (m/z 1775.999, 1984.695, 2223.218, 3570.219, 3838.763, 3965.705, 4150.555, 4703.405, 4790.568, 4888.704, 4976.557, 5577.153, 6169.663, 7187.852, 7553.560, 8764.786, 9062.022, and 9206.085) coupled with the emergence of 6 new peaks (m/z 1886.242, 2242.721, 2383.436, 3202.695, 3285.102, and 5688.44) in AGB/IgY-depleted urine when compared to a neat urine sample (Figure lid).

This Example illustrates that polypeptide peaks are removed from neat urine, which coincides with appearance of previously non-detected polypeptides in the depleted samples. These peaks could be characterized further using mass spectrometry tools. EXAMPLE 10

Non-specific and specific depletion of plasma proteins

2D gel electrophoresis was performed with a Novex NuPAGE system using 3-10 NL strips in the first dimension and 4-12% Bis-Tris gels in the second dimension. The equivalent of

50 μg of neat, IgY (cocktail 1) and IgY (cocktail 2)-depleted plasma were analyzed per sample. Proteins were visualized with SyproRuby. The stained gels were imaged using a

BioRad FX laser scanner. The top panel of gels is identical to those in Figure 2, which consist of neat plasma, AGB-depleted plasma and AGB-IgY depleted plasma and is included in this figure for comparison. The bottom panel of gels represents IgY cocktail depletion (cocktail 1 and 2) without AGB treatment. See Figure 12.

Affinity depletion of neat plasma with IgY cocktail 1 removed human serum albumin (spot 1) and enriched immunoglobulins (2 and 3), transferrin (4), antitrypsin (5), and haptoglobin (6). hi comparison to the AGB treatment, the IgY cocktail 1 enabled detection of ceruloplasmin (7), and the additional spots 11 and 12, which have not been characterized. Affinity depletion with IgY cocktails 1 and 2 removed human serum albumin further than cocktail 1 and AGB/IgY treatment. It also depleted the immunoglobulins (2 and 3). Proteins 4 to 6 and ceruloplasmin (7), apolipoproteins (8), and transthyrethrin (9) were further enriched by treatment with both IgY cocktails. In comparison to the AGB/IgY treatment, IgY cocktail combination 1 and 2 enabled detection of additional spots 11 to 15, which have not been characterized.

This Example illustrates specific depletion of plasma proteins using the invention. The results presented in figure 12 also illustrate that the combination of IgY cocktails 1 and 2 improves specific enrichment of plasma proteins when compared to depletion with AGB and IgY affinity depletion. EXAMPLE 11

Re-usability of IgY columns

To test re-usability and reproducibility of IgY columns, three 200 μg aliquots of AGB- depleted plasma were processed using the same IgY cocktail column. 39 μl of resulting flowthrough (FT) and eluent (E) fractions were analyzed bylD SDS-PAGE using 4-12% Bis-Tris gels on a Novex NuPAGE system. Proteins were visualized with colloidal Coomassie stain and gels imaged using a BioRad densitometer. The results are shown in Figure 13.

Both, flowthrough and eluent fractions of the three replicate treatments show identical protein patterns indicating that the depletion method provides reproducible results after column re-use.

Summary of Examples

The data presented illustrate targeted proteins or polypeptides can be depleted from a variety of biological fluids using the IgY columns. This selective depletion allows enrichment visualization and identification of less abundant and previously non-detectable proteins or polypeptides. The data presented also illustrate that the invention is compatible with gel electrophoresis, liquid chromatography, magnetic bead separation, and mass spectrometry.

This is the first report of applying IgY-based depletion with magnetic bead separation for subsequent MALDI-ToF/ToF peptide profiling of human plasma and urine samples.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

Chsdkea. JChromatogr. 376:11-32, 1986 Daly and Ozols, Cancer Cell 1:111-112, 2002 Georgiou et al, Proteomics 7:1503-1506, 2001 Hage, Clinical Chemistry 45:593-615, 1999 Larsson, Methods Enzymol 104:212-223, 1987 Scawen, Anal. Proc. 28Λ43-H4, 1991 US Patent No. 4,550,019 US Patent No. 4,056,737 US Patent No. 5,367,054 US Patent No. 5,420,253 US Patent Application No. 09/977,358 Winzor, J Chromatogr. 1037(l-2):351-367, 2004 WO 2004/019009

Claims

CLAIMS:

1. A method for generating a sample enriched for a first population of molecules said method comprising subjecting said sample to affinity-mediated depletion of a second population of molecules using immune sera or a fraction or component thereof from an avian species generated by immunization of the avian species by two or more members of said second population of molecules.

2. The method of Claim 1 wherein the first and second populations of molecules are proteins.

3. The method of Claim 1 or 2 further comprises one or more additional affinity depletion steps of the enriched sample.

4. The method of Claim 3 wherein the affinity depletion is immunodepletion.

5. The method of any one of Claims 1 to 4 wherein the avian immune sera comprises egg yolk antibodies (IgY).

6. The method of any one of Claims 1 to 5 wherein the sample is a biological fluid selected from the list comprising blood, serum, plasma, milk, lymph, cerebrospinal fluid, urine, amniotic fluid, lavage fluid, cervicovaginal fluid, uterine fluid, seminal fluid or tissue fluid.

7. The method of any one of Claims 1 to 5 wherein the sample is conditioned medium or a cell extract from a plant, animal or microbe.

8. The method of any one of Claims 1 to 5 wherein the sample is an industrial or chemical sample.

9. The method of any of Claims 1 to 8 wherein the second population of molecules is selected from two or more of albumin, microglobulin, immunoglobulins, fibrinogen, haptoglobin, transferrin, antitrypsin, glycoprotein and/or apoliproteins.

10. The method of any one of Claims 1 to 8 wherein the affinity depletion step comprises a device which can be re-used.

11. A method for clarifying/enriching a sample said method comprising subjecting said sample to immunodepletion of at least two target molecules potentially present in said sample using avian-derived antibodies generated following the immunization of an avian species with a cocktail comprising the at least two molecules and collecting antibodies from egg yolk; and optionally further clarifying the immunodepleted sample by affinity depletion using ligands of said at least two selected molecules.

12. A method for enriching a sample for low abundance proteins or otherwise reducing the level of targeted proteins said method comprising subjecting said sample to immunodepletion using sera or antibody fraction isolated from egg yolk of an avian species immunized with at least two proteins selected from the list consisting of albumin, macroglobulin, immunoglobulins, fibrinogen, haptoglobin, transferrin, antitrypsin, glycoprotein and apolipoproteins ; and optionally further clarifying the immunodepleted sample by affinity depletion using a ligand to two or more of the above or other plasma/serum proteins.

13. The method of any one of Claims 1 to 12 wherein the avian species used to generate immune sera is a chicken.

14. A composition for immunodepletion of a high abundance molecule from a sample, comprising antibodies preparation directed against targeted molecules in a biological sample coupled to an affinity support.

15. The composition of Claim 14 wherein the antibody is an avian polyclonal antibody.

16. The composition of Claim 15 wherein the avian polyclonal antibody is a chicken IgY antibody.

17. A device for the rapid processing of biological samples comprising a generally cylindrical chamber having an opening at either end, in which each opening is adapted to interact with a receptacle, in which the sample can be transferred from one receptacle to the other via the chamber, and in which the chamber has transversely disposed within it multiple layers of an affinity support having a high affinity for two or more targeted molecules in a biological sample, separated by a layer of an affinity support coupled to two or more antibodies or sera or fraction of sera comprising same directed against the targeted molecules.

18. The device of Claim 17 which is re-usable.

19. A kit for removal of targeted molecules from a sample, comprising an affinity support with affinity for targeted molecules in a sample and optionally, the kit comprises a second or further affinity supports each coupled to at least two antibodies directed against other targeted molecules.