Efficient and Specific Removal of Albumin from Human Serum Samples*

Laura F. Steel{ddagger},§, Michael G. Trotter{ddagger}, Pamela B. Nakajima, Taj S. Mattu||, Gregory Gonye** and Timothy Block{ddagger}

{ddagger} Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Jefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
|| Isis Innovation, Ewert House, Ewert Place, Summertown, Oxford OX2 7SG, United Kingdom
** Daniel Baugh Institute for Functional Genomics/Computational Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Patient serum or plasma is frequently monitored for biochemical markers of disease or physiological status. Many of the rapidly evolving technologies of proteome analysis are being used to find additional clinically informative protein markers. The unusually high abundance of albumin in serum can interfere with the resolution and sensitivity of many proteome profiling techniques. We have used monoclonal antibodies against human serum albumin (HSA) to develop an immunoaffinity resin that is effective in the removal of both full-length HSA and many of the HSA fragments present in serum. This resin shows markedly better performance than dye-based resins in terms of both the efficiency and specificity of albumin removal. Immunoglobulins are another class of highly abundant serum protein. When protein G resin is used together with our immunoaffinity resin, Ig proteins and HSA can be removed in a single step. This strategy could be extended to the removal of any protein for which specific antibodies or affinity reagents are available.


Serum is a rich source of biochemical products that can act as indicators of the physiological or clinical status of a patient. For instance, serum levels of hormones, cholesterol, small molecules, or enzymes and other proteins have all been assayed to provide information regarding conditions as diverse as pregnancy, cardio-vascular or nutritional status, viral disease, or cancer. It is not surprising then that many efforts to find new biomarkers for disease detection or progression have applied emerging analytical techniques to profile the protein content of human serum.

Serum presents some unique challenges to efforts to characterize its protein content by any single method (for review, see Ref. 1). Notably, different protein species are present in serum over a very wide range of abundance. A small number of highly abundant proteins such as albumin, immunoglobulins, {alpha}1-antitrypsin, transferrin, and haptoglobins are present in concentrations in the milligram to tens of milligrams per milliliter range and together account for as much as 80% of the total serum protein. A larger number of proteins, including many that are, or could be, diagnostically significant, are present at far lower concentrations. Removal of abundant serum proteins will help in the discovery and detection of less abundant proteins that may prove to be informative disease markers.

Over the last several decades, attempts have been made to remove albumin, the single most abundant serum protein, from serum (or plasma) and other body fluids, often by methods based on the high affinity of albumin for certain blue textile dyes (24). These methods can be effective in removing albumin, although they suffer from a lack of specificity as many serum proteins in addition to albumin will bind to the dye-based resins. Other methods reported include a proprietary polypeptide affinity matrix that removes albumin together with IgG, but is now apparently unavailable (5), and a method based on size separation in a centrifugal filtration device that was, perhaps predictably, unsuccessful (6).

In the work reported here, we have investigated the efficacy of an immunoaffinity resin for improving the specificity and efficiency of removing albumin from human serum samples. The use of immunoaffinity resins for the selective removal or purification of proteins from solution is well established. However, their application to the problem of albumin removal is not entirely straightforward, primarily due to the enormous load of this single protein species in serum and plasma. We show that by using high affinity antibodies heavily loaded on a beaded resin support, albumin and a large number of albumin fragments can be essentially quantitatively removed from human serum samples. Furthermore, when a protein G affinity resin is used together with the immunoaffinity resin, IgG and human serum albumin (HSA)1 are simultaneously removed from the samples. Similar methods could be applied to the removal of additional abundant proteins.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Monoclonal Antibody Production and Purification
Monoclonal antibodies were raised against HSA using standard techniques. Briefly, purified HSA (Sigma) was used to immunize BALB/c mice, and hybridomas were generated by fusion of spleen cells to SP2/0 myeloma cells. Hybridoma supernatants were tested for reactivity to HSA by enzyme-linked immunosorbent assay (ELISA) using the purified HSA preparation as a capture antigen. Hybridomas showing strong reactivity in these initial assays were retested for reactivity to each of the three major domains of HSA (7). Coding sequence from each of the major domains of HSA (indicated in Fig. 1) was amplified by reverse transcriptase-PCR using RNA isolated from HepG2 cells. These sequences were cloned into the vector pET28 (Novagen, Madison, WI) for expression as recombinant His6-tagged fusion proteins that were then purified on nickel-nitrilotriacetic acid resin (Qiagen, Valencia, CA) and used for testing the hybridoma supernatants by ELISA. Hybridomas showing strong reactivity against one of each of the three domains were single-cell cloned, retested by ELISA, and expanded for large scale production of monoclonal antibodies. Two of these antibodies, 12E8H, which is reactive against an epitope in domain II, and 2G4A, which is reactive against an epitope in domain I, have been used in the experiments described here.



View larger version (53K):
[in this window]
[in a new window]
 
FIG. 1. Amino acid sequence of HSA. The sequence of HSA (Swiss-Prot accession no. P02768) is represented showing the three major domains. Tryptic fragments that were detected and used in the identification of albumin-derived polypeptides by mass spectrometry are indicated in bold, underlined type.

 
Hybridoma cells were grown in CELLine 1000 flasks (Integra Biosciences, Ijamsville, MD) in serum-free BD Cell MAb medium (BD Biosciences, Mountain View, CA), and antibodies were purified from culture supernatants by affinity chromatography on HiTrap protein G columns (Amersham Biosciences). The eluted antibodies were dialyzed against phosphate-buffered saline and concentrated in Ultrafree-15 centrifugal filter units (Millipore, Bedford, MA).

Preparation of Antibody Affinity Resin
Antibodies were coupled to N-hydroxysuccinimide-activated Sepharose 4 Fast Flow resin (Amersham Biosciences) at a concentration of 15 mg antibody per milliliter resin, following procedures recommended by the manufacturer. After antibody coupling, the resin was washed first with 0.1 M NaCl, 0.1 M glycine, pH 2.8, then with 0.1 M NaCl, 0.1 M glycine, pH 9.0, and finally with 0.01 M Tris, pH 8.0. The resin was equilibrated with Tris-buffered saline/Tween-20 (TBS/T; 0.02 M Tris, 0.15 M NaCl, pH 7.6 with 0.1% Tween-20) prior to use or storage at 4 °C.

Removal of HSA from Serum Samples
Antibody Affinity Resin—Human serum was diluted into one column volume or less of TBS/T and applied to immunoaffinity resin that had been equilibrated in the same buffer. Proteins were allowed to bind while mixing gently for 30–60 min at room temperature. Unbound proteins were recovered from the resin by gravity flow in a standard column format (PolyPrep column; Bio-Rad) and then immediately reapplied to the column. This step was repeated so that unbound proteins were collected and immediately reapplied a total of four times. The column was then washed five times, each time with a single column volume of TBS/T, and all washes were combined as the unbound fraction of proteins. Bound proteins were eluted from the column with a total of five washes, each with a single column volume of 0.1 M NaCl, 0.1 M glycine, pH 2.8. Eluted fractions were neutralized by the addition of 0.05 volume of 1 M Tris, pH 8.0, and combined. Proteins from both the unbound and eluted fractions were concentrated by precipitation with 5 volumes of acetone.

SwellGel Blue—Albumin was removed from human serum samples using a SwellGel Blue Albumin Removal Kit (Pierce) according to the manufacturer’s instructions. Samples containing 650 µg of total protein (~260 µg albumin) were loaded onto a single removal disc, where each disc is reported to have a binding capacity of >2 mg of albumin.

Addition of Protein G Resin to Immunoaffinity Resin—For the removal of both HSA and IgG in a single column, ~0.1 column volume of protein G immobilized on Sepharose 4B Fast Flow (Sigma) was added to the immunoaffinity resin. Columns were washed, run, and regenerated exactly as for the immunoaffinity resin alone.

Two-dimensional Gel Electrophoresis (2DE)
2DE was carried out as described previously (8) with minor modifications. Acetone precipitated proteins were redissolved in sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM dithiothreitol, 5 mM tributylphosphine, and 0.4% ampholytes (Servalyt 3-10). Proteins were focused in an 18-cm pH 3-10NL immobilized pH gradient strip (Amersham Biosciences) after rehydration at 50 V for 14 h. Gel strips were equilibrated in 6 M urea, 2% SDS, 1.5% dithiothreitol, 30% glycerol, 50 mM Tris, pH 8.8, for 10 min followed by incubation in the same solution, but replacing dithiothreitol with 3% iodoacetamide, for an additional 10 min. The gel strips were placed on an 8–18% polyacrylamide gel for resolution in the second dimension. Gels were stained with silver (9) or colloidal Coomassie blue (10) and imaged with a FluorChem 16-bit charge-coupled device camera (Alpha Innotech, San Leandro, CA).

For immunoblotting, proteins were transferred to a polyvinylidene difluoride membrane and the blots were stained with SyproRuby protein blot stain (Bio-Rad). Blots were probed with goat anti-HSA (Fitzgerald Industries, Concord, MA) and developed using horseradish peroxidase-conjugated anti-goat antibody (Sigma) and SuperSignal West Pico detection reagents (Pierce).

Peptide Mass Fingerprint Analysis of Proteins
Protein spots excised from colloidal Coomassie blue-stained two-dimensional (2D) gels were destained, reduced, and alkylated and then digested with trypsin (11). Peptides were further treated by elution from ZipTip-C18 reversed phase pipette tips (Millipore). Recovered peptides were prepared for MALDI-TOF mass spectrometry by mixing with alpha-cyano-4-hydroxy cinnamic acid, 1% formic acid in 50% acetonitrile, and droplets were allowed to dry on the MALDI sample plate. Peptide mass maps were obtained using a Voyager DE (Applied Biosystems, Foster City, CA) MALDI-TOF mass spectrometer operated in positive ion reflectron mode. Proteins were identified from the peptide mass maps using MASCOT (Matrix Science, London, UK) to search the nonredundant protein data base (12).

Antibody Array
The following mouse monoclonal antibodies were used for capture: anti-HSA (Research Diagnostics, Flanders, NJ), anti-hepatitis B surface antigen (HBsAg; Fitzgerald Industries), anti-alphafetoprotein (AFP; U.S. Biological), and anti-hepatitis B e antigen (HBeAg; Fitzgerald Industries). Antibodies were diluted to a concentration of 0.5 mg/ml in PBS plus 0.02% Tween-20 and spotted onto Hydrogel slides (PerkinElmer Life Sciences) with a MicroGrid TAS (BioRobotics, Cambridge, UK) robotic spotter using MicroSpot 2500 pins. Slides were incubated in a humid chamber overnight and then washed briefly in TBS/T before blocking for 2 h in TBS/T plus 1% gelatin (Bio-Rad). Slides were rinsed briefly in TBS/T and then incubated with antigen solution (see below) for 2 h at room temperature. Slides were washed three times for 20 min in TBS/T and then incubated for 2 h at room temperature with antibodies that had been labeled with Cy3 fluorescent dye (Amersham Biosciences) according to manufacturer’s instructions. Antibodies used for detection were goat polyclonal anti-human AFP, anti-HBsAg, and anti-HSA (Fitzgerald Industries). Slides were again washed with TBS/T and then imaged using a ScanArray 5000XL scanner (PerkinElmer Life Sciences).

Antigen solution was prepared by adding purified AFP (Fitzgerald Industries) and HBsAg (Chemicon International, Temecula, CA) to human serum at a concentration of 1000 ng/ml each. The spiked serum samples were diluted into TBS/T and fractionated on immunoaffinity resin as described above. Proteins recovered in flow-through and eluted fractions were concentrated by acetone precipitation and resuspended in TBS/T. Unfractionated, spiked samples were similarly diluted, acetone precipitated, and redissolved in TBS/T. Antibody microarrays were incubated with the antigen solution at a concentration equivalent to a 1:10 dilution of the starting serum sample, so that AFP and HBsAg would each be present at a maximum of 100 ng/ml in any fraction.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Monoclonal Antibodies and HSA Immunoaffinity Resin
Monoclonal antibodies to human serum albumin are available from many commercial suppliers. However, albumin is present in human serum at concentrations in the range of 35 to 45 mg/ml and very large quantities of antibody are required for its quantitative removal. Our goal was to have a renewable source of high affinity monoclonal antibodies reactive against a number of different epitopes in the albumin molecule so that both full-length HSA and many HSA fragments could be efficiently removed. Therefore, we decided to develop our own source of monoclonal anti-HSA antibodies.

Hybridomas were prepared from the spleens of mice immunized with full-length HSA, as described in "Experimental Procedures." Supernatants from individual hybridoma clones were first tested for reactivity against full-length HSA by ELISA. In order to identify hybridomas producing antibodies reactive against different HSA epitopes, clones showing strong reactivity to the full-length molecule were further tested against recombinant His-tagged proteins representing each of the three major domains of HSA (see Fig. 1). Antibodies against epitopes in two different domains (I and II) were chosen for large scale preparation and purification. These two antibodies were covalently attached to Sepharose resin to prepare an immunoaffinity reagent, as described in "Experimental Procedures."

Efficiency and Specificity of HSA Removal from Serum
To test the efficiency of HSA removal from human serum, 10 µl of serum, containing ~700 µg of total protein and an estimated 400 µg of HSA, was incubated with 0.25 ml of the antibody affinity resin in a column format (see "Experimental Procedures"). Proteins that did not bind to the column (the flow-through fraction) and proteins specifically bound by the resin and then eluted (the eluted fraction) were prepared and examined by 2DE. The abundant HSA protein is evident as a large, poorly resolved and clearly overloaded spot when total serum proteins are displayed in these 2D gels (Fig. 2a). After separation of serum proteins on the affinity column, the flow-through fraction shows essentially no remaining HSA (Fig. 2b) while the HSA is recovered in the fraction eluted from the column (Fig. 2c).



View larger version (56K):
[in this window]
[in a new window]
 
FIG. 2. 2DE of total human serum proteins and proteins fractionated on anti-HSA immunoaffinity resin. Proteins were resolved by 2DE with separation by isoelectric focusing in pH 3–10NL immobilized pH gradient strips in the first dimension and 8–18% SDS-PAGE in the second dimension. Gels were stained with silver and are shown with the acidic end of the first dimension to the left. a, total human serum proteins with the positions of albumin, IgG heavy chains, and Ig light chains indicated. b, proteins in the flow-through fraction; and c, proteins eluted from the immunoaffinity column.

 
The capacity of the resin was tested by attempting removal of HSA from different volumes of serum with a constant volume of resin. As assessed by visual inspection of one-dimensional and 2D gels, the 0.25-ml column was able to remove HSA from volumes of serum up to ~12 µl, but became slightly overloaded with 15 µl, or ~1.05 mg of total serum protein and 600 µg of HSA (data not shown). No reduction in column capacity has been observed with re-use of the resin up to eight times (data not shown).

While HSA is by far the most abundant protein seen by 2DE of proteins that bind to and are eluted from the column, many other "spots" are present in the gel (Fig. 2c). A large number of these have been shown to be albumin or albumin-related fragments by two methods. First, eluted proteins separated by 2DE were transferred to polyvinylidene difluoride membranes for detection by immunoblotting using a polyclonal antibody against HSA. Almost all spots visualized by staining of the blot were also detected by reaction with the anti-HSA antibody, as shown in Fig. 3. This suggests that proteins (or protein fragments) that bind to the column are doing so by specific interactions with the antibody, rather than by nonspecific binding to the resin substrate or to HSA itself.



View larger version (52K):
[in this window]
[in a new window]
 
FIG. 3. Immunoblot analysis of proteins eluted from the anti-HSA immunoaffinity resin. Proteins eluted from an immunoaffinity column were resolved by 2DE, as shown in Fig. 2, and transferred to polyvinylidene difluoride membrane. After transfer, blots were stained with SyproRuby protein blot stain and then probed with polyclonal goat anti-HSA. a, stained blot; b, HSA detection.

 
Further identification of the numerous minor spots found in the eluted fraction was obtained by mass spectrometry. Individual spots excised from the gel were digested with trypsin and analyzed by MALDI-TOF peptide mass fingerprinting. Spots marked with numbers in Fig. 4 have been identified as albumin fragments in that peptides produced by trypsin digestion can be mapped to the albumin molecule over a region that is consistent with the size of the polypeptide in the excised spot. The tryptic fragments detected in this analysis are indicated in bold lettering in the albumin sequence shown in Fig. 1 and are also listed in Table I, together with their occurrence in each of the analyzed spots. In a small number of cases, for instance in the cluster of spots numbered 31, 33, 34, and 35, peptide mass matches were found over a region that exceeds the expected molecular mass of the fragment excised from the gel. This may be due to incomplete separation of adjacent spots in the gel, resulting in cross-contamination of the excised gel plugs. Identification by mass spectrometry was attempted on 23 spots, and 21 of those spots showed a tryptic mass fingerprint that could be matched to albumin (Table I). One spot was identified as {alpha}1-antitrypsin (AT in Fig. 4), and this is consistent with its migration in the 2D gel as compared with published data (Swiss 2D PAGE). The data obtained from one spot were insufficient for a protein identification.



View larger version (104K):
[in this window]
[in a new window]
 
FIG. 4. Proteins eluted from the anti-HSA column and identified by mass spectrometry. Proteins eluted after binding to an immunoaffinity column were resolved by 2DE, and the gel was stained with colloidal Coomassie blue. Protein spots were excised from the gel, digested with trypsin, and analyzed by MALDI-TOF peptide mass mapping. Spots identified as albumin or albumin fragments are indicated by numbered lines. The spot labeled AT is {alpha}1-antitrypsin. Approximate molecular weights are shown in kilodaltons to the left of the gel.

 

View this table:
[in this window]
[in a new window]
 
TABLE I Tryptic peptides used to identify albumin fragments by mass spectrometry

 
Very small amounts of some proteins that are most probably not albumin can sometimes be seen when 2D gel images of eluted proteins are strongly overexposed (data not shown). Based on their migration in the gel, these appear to be trace amounts of abundant proteins such as IgG, {alpha}1-antitrypsin, and haptoglobins.

Improvements in the Resolution and Detection of Serum Proteins in 2D Gels after HSA Removal
Small regions of the 2D gels shown in Fig. 2, a and b, are enlarged in Fig. 5 to demonstrate the improvement in the data available from 2D gels, even in the absence of significant overloading of the proteins that remain after albumin depletion. Many of the abundant proteins that were obscured by albumin are now visible (compare resolution in upper left of each image). Less abundant proteins that were lost in the confusion of albumin fragments become distinct and are now available for analysis (see spots indicated by arrows). Minor proteins that were previously hidden by comigration with albumin fragments or smears can now be resolved (see boxed region and three-dimensional depictions of that region below each image). It is evident that higher protein loads are now possible so that proteins of this and even somewhat lower abundance classes can be detected and analyzed without overwhelming the gel with albumin and its fragments. We expect that similar improvements will be realized using alternative fractionation technologies, such as liquid chromatography linked to mass spectrometry, for the analysis of serum proteins.



View larger version (81K):
[in this window]
[in a new window]
 
FIG. 5. Improved resolution and detection of proteins after HSA removal. A close-up view is shown of the gel region including and just below the position of the major albumin spot, as seen in Fig. 2. a, gel with total serum proteins; b, gel with HSA removed. A three-dimensional image of the boxed region is shown below each gel. Note the numerous low abundance spots that can now be visualized for analysis. Arrows in b point to spots that are obscured in the gel shown in a.

 
Albumin Removal using a Cibacron Blue-based Resin
Resins used to remove HSA from serum samples have been made from immobilized forms of blue dyes such as Cibacron Blue, which was shown in the early 1970s (2) to be effective in binding albumin in plasma samples. Since then, attempts have been made to modify the dye to improve the specificity of its binding to albumin, and one such product is marketed as SwellGel Blue (Pierce). Given the convenience and commercial availability of this, and similar, products, it was important to compare its performance with that of our immunoaffinity resin.

Ten microliters of serum, containing ~400 µg of HSA, was loaded onto SwellGel Blue resin with a capacity of >2 mg of HSA, as stated by the manufacturer. The column was processed according to the manufacturer’s instructions, and proteins in the flow-through and eluted fractions were analyzed by 2DE, as before. Results, shown in Fig. 6, indicate that a large portion of the HSA was not bound by the resin. Proteins that bound to and were eluted from the resin include albumin, many albumin fragments, and a large number of nonalbumin proteins. In particular, significant amounts of relatively abundant serum proteins such as Ig heavy and light chains, apolipoprotein A1, serotransferrin, haptoglobin, and {alpha}1-antitrypsin were bound by the column. One-dimensional gel analysis of several sequential wash and elution fractions suggested that this was not the result of incomplete washing during the initial binding steps, but instead represents proteins, in addition to HSA, that bind to the column under the recommended conditions (data not shown). We conclude that immunoaffinity reagents are far superior to the dye-based resin in terms of both the efficiency and specificity of albumin removal.



View larger version (63K):
[in this window]
[in a new window]
 
FIG. 6. 2DE of human serum proteins fractionated on SwellGel Blue. Human serum proteins were fractionated on SwellGel Blue and resolved by 2DE, as shown in Fig. 2. a, Proteins in the flow-through fraction; b, proteins eluted from SwellGel Blue resin. Some proteins can be identified as follows: 1, serotransferrin; 2, {alpha}1-antitrypsin; 3, IgG heavy chains; 4, apolipoprotein A1; 5, Ig light chains; 6, haptoglobins.

 
Potential Retention of Albumin-related and Albumin-binding Proteins by the Immunoaffinity Resin
While the immunoaffinity resin shows a high degree of specificity for binding to HSA, it was of interest to investigate whether proteins structurally similar to albumin, or proteins that can bind to albumin, are also removed by treatment of samples with the resin. Our interest in viral liver disease led us to examine the behavior of two such proteins, AFP and HBsAg. AFP is a fetal form of albumin with 40% amino acid sequence identity and a highly similar tertiary organization (13, 14). AFP is frequently monitored in patients chronically infected with hepatitis B or C virus, where serum levels greater than 20 ng/ml (usually 100–1000 ng/ml or higher) can be indicative of progression to hepatocellular carcinoma. Like albumin, AFP is folded into three major domains that are held together by disufide bonds at positions conserved between albumin and AFP (14). It was possible that conformation-dependent epitopes in AFP would be recognized by antibodies used to make the HSA immunoaffinity resin. HBsAg can be present at concentrations ranging from 1 µg/ml to over 1000 µg/ml in the serum of patients who are chronically infected with HBV (15). HBsAg has been shown to bind to some forms albumin (16), and therefore it could potentially be retained on the column through this interaction.

Human serum was spiked with purified AFP and HBsAg, each at a concentration of 1000 ng/ml serum. The sample was then treated to remove HSA using the immunoaffinity resin as described above. The total spiked sample (prior to HSA removal), the immunoaffinity column flow-through fraction, and proteins eluted after binding to the column were all assayed by an antibody microarray for the presence of HSA, AFP, and HBsAg. The array also contained antibody to HBeAg (anti-HBeAg) as a negative control. As shown in Fig. 7a, this method easily detected HSA, AFP, and HBsAg in the unfractionated, total sample. Both AFP and HBsAg are present in the flow-through fraction (Fig. 7b) but are undetectable above background in the eluted fraction (Fig. 7c). As expected, HSA is found in the eluted fraction, but is undetectable above background in the flow-through fraction (Fig. 7, b and c). These results provide further evidence of the high degree of specificity that is possible when using an immunoaffinity reagent for the removal of albumin from serum samples.



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 7. Antibody microarray analysis of AFP and HBsAg binding to HSA immunoaffinity resin. Capture antibodies were applied to three slides in 16 replicate spots, as labeled to the left. Serum spiked with AFP and HBsAg was fractionated on anti-HSA immunoaffinity resin, and the resulting flow-through and eluted fractions, as well as total protein prior to fractionation, were incubated on separate slides. Bound antigen was detected with Cy3-labeled antibodies. Slides were scanned at two different laser intensities to account for the very large amount of HSA relative to AFP and HBsAg. Low intensity scans are shown above the higher intensity scan of each slide, and the negative control Ab spots (anti-HBeAg) are the same spots in each of the two scans. a, slide incubated with total spiked sample prior to fractionation; b, slide incubated with flow-through fraction proteins; c, slide incubated with proteins in eluted fraction.

 
Although the overall recovery of proteins in the flow-through and eluted fractions from our immunoaffinity column is close to 100% (±10%) as determined by Bradford assays, we cannot rule out retention of individual low abundance proteins that are not evident in the 2D gel and antibody array analyses we have done to date. Preferential loss of low abundance, nonalbumin proteins would need to be checked on an individual basis if such a loss were suspected.

Simultaneous Removal of HSA and Immunoglobulin Chains
Removal of additional abundant proteins can be beneficial in the analysis of serum proteins, and therefore we attempted to remove albumin together with the IgG class of immunoglobulins. These proteins can account for ~20% of total serum proteins and are present at a concentration of ~8–16 mg/ml (17). To remove IgG, we took advantage of its well-known affinity for protein G. Protein G resin was added to the HSA immunoaffinity resin, and serum samples were processed as above. Fig. 8 shows a 2D gel of serum where both HSA and IgG have been removed. Very small amounts of IgG heavy chains remain in the flow-through fraction, and a more specific affinity reagent (e.g. anti-IgG antibody) might be required for their complete removal. Because protein G binds IgG, but has only a low affinity for other classes of human immunoglobulins, we expect that the light chain proteins that remain in the sample are derived primarily from these other immunoglobulin classes. Again, an affinity reagent with specificity for light chains (e.g. protein L or anti-light chain antibodies) could be used to achieve a more complete removal, if necessary. It was possible that protein G on the resin would interact with the densely loaded anti-HSA immunoaffinity resin when the two were used together in the same column, thereby reducing binding capacity. However, we saw no difference in binding capacity when the two resins were used together in the same column, or sequentially in separate columns (data not shown). Therefore, HSA and most IgG proteins are readily removed from serum samples in a single column.



View larger version (133K):
[in this window]
[in a new window]
 
FIG. 8. Simultaneous removal of albumin and IgG proteins. Human serum was fractionated on a column containing both anti-HSA and protein G affinity resins. The flow-through fraction was collected and resolved by 2DE, as shown in Fig. 2. The gel was stained with colloidal Coomassie blue.

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The rapidly evolving of techniques of proteome analysis are making protein profiling of both tissues and body fluids increasingly sensitive and informative. This has important clinical implications and is leading to new strategies for disease detection. Serum has been, and continues to be, a valuable source material for the monitoring of disease markers. However, the serum proteome is dominated by a small number of highly abundant proteins that can obscure detection of other potentially informative proteins. Here we describe a technique for removing albumin, by far the most abundant serum protein, in a way that is efficient and specific enough to be suitable for use with emerging proteome technologies. The method is easily adapted for the simultaneous removal of circulating IgG and could be applied to additional proteins as well.

Removal of albumin from serum samples is problematic because of its extremely high concentration. A high specificity and high capacity resin is required. While Cibacron Blue and related dyes have been shown to bind albumin with high affinity (2), they also bind many other abundant serum proteins (3). Dye-based resins that have been optimized for albumin binding are commercially available, but our results suggest that they still lack sufficient specificity. The need to compromise between specificity and capacity may account for the failure of the product we tested to perform well in either area.

Antibodies are an obvious choice as affinity reagents with the specificity required for selective removal of any single protein species. In the case of albumin removal, however, the quantity of antibody needed for essentially complete depletion of albumin, even from microliter quantities of plasma or serum, makes the use of antibodies problematic. To maximize the value of the antibodies used in our resin, we selected individual monoclonal antibodies that each react with high affinity to a unique epitope on the albumin molecule. This not only provides a renewable antibody source, but also is a reagent that can benefit from cooperative binding to different regions of the protein. By using antibodies reactive against different regions of the molecule, we are able to capture a large number of the albumin fragments that are present in serum samples. While our resin was effective when made with only one (not shown) or two of our antibodies, it is possible that further efficiencies could be achieved with additional antibodies.

Several modifications to our procedure that could improve its applicability in different circumstances are possible. For instance, the use of formats such as multi-well plates that are suitable for high throughput will be explored. The use of antibody fragments or single-chain antibodies derived from the anti-HSA monoclonals might be beneficial if they can be produced less expensively or loaded onto substrate at higher concentrations.

In the ideal, it would be best to use a nonproteinaceous surface or resin for the specific removal of proteins from any sample. This would eliminate the potential for contaminating the sample with protein leached from the resin and would likely be a more stable and less expensive product. To date, however, no affinity resin that can match the specificity and binding capacity of monoclonal antibodies has been discovered. Covalent binding of antibody to the resin has minimized the leaching problem, and the ability to reuse the resin helps to keep the cost reasonable for research purposes.

Our research involves the examination of proteins in human serum samples by 2DE and antibody microarrays, and both of these techniques benefit from the removal of the bulk of the albumin and IgG. Removal of highly abundant serum proteins improves the detection of less abundant proteins by 2DE, as well as the display of proteins that comigrate and are otherwise obscured by HSA or IgG. The removal of the large and poorly resolved spots created by these highly abundant proteins, and the numerous smaller spots that represent albumin fragments, also helps in computer-assisted gel analysis, particularly with spot delineation, quantitation, and gel-to-gel matching. We expect that many of the new technologies being developed for protein profiling will benefit in similar ways by the improvements in sensitivity and reduction of background that result from the removal of a small number of very abundant proteins from samples prior to analysis.


    ACKNOWLEDGMENTS
 
We thank Dr. Peter Lobel (University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School) for the use of his mass spectrometry facility and MaryAnn Communale for some coaching on the use of the mass spectrometer. We also acknowledge the skill and expertise of Drs. Michael Bodri and Janis Hammer (Delaware Valley College) in handling the mice.


    FOOTNOTES
 
Received, March 14, 2003, and in revised form, May 16, 2003.

Published, MCP Papers in Press, May 16, 2003, DOI 10.1074/mcp.M300026-MCP200

1 The abbreviations used are: HSA, human serum albumin; 2DE, two-dimensional gel electrophoresis; AFP, alpha fetoprotein; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; ELISA, enzyme-linked immunosorbent assay; 2D, two dimensional; TBS/T, Tris-buffered saline/Tween-20; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Back

* This work was supported by the National Cancer Institute, Early Detection Research Network, and by an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Jefferson Center for Biomedical Research, Doylestown, PA 18901. Tel.: 215-489-4946; Fax: 215-489-4920; E-mail: laura.steel{at}jefferson.edu


    REFERENCES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anderson, N. L., and Anderson, N. G. (2002) The human plasma proteome: History, character, and diagnostic prospects. Mol. Cell. Proteomics. 1, 845 –867[Abstract/Free Full Text]

  2. Travis, J., and Pannell, R. (1973) Selective removal of albumin from plasma by affinity chromatography. Clin. Chim. Acta 49, 49 –52[Medline]

  3. Gianazza, E., and Arnaud, P. (1982) A general method for fractionation of plasma proteins: Dye-ligand affinity chromatography on immobilized Cibacron Blue F3-GA. Biochem. J. 201, 129 –136[Medline]

  4. Raymackers, J., Daniels, A., DeBrabandere, V., Missiaen, C., Dauwe, M., Verhaert, P., Vanmecheien, E., and Meheus, L. (2000) Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry, nanoliquid chromatography-electrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry. Electrophoresis 21, 2266 –2283[CrossRef][Medline]

  5. Lollo, B. A., Harvey, S., Liao, J., Stevens, A. C., Wagenknecht, R., Sayen, R., Whaley, J., and Sajjadi, F. G. (1999) Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClear. Electrophoresis 20, 854 –859[CrossRef][Medline]

  6. Georgiou, H. M., Rice, G. E., and Baker, M. S. (2001) Proteomic analysis of human plasma: Failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins. Proteomics 1, 1503 –1506[CrossRef][Medline]

  7. Brown, J. R. (1976) Structural origins of mammalian albumin. Fed. Proc. 35, 2141 –2144[Medline]

  8. Steel, L. F., Shumpert, D., Trotter, M., Seeholzer, S. H., Evans, A. A., London, W. T., Dwek, R., and Block, T. M. (2003) A strategy for the comparative analysis of serum proteins for the discovery of biomarkers for hepatocellular carcinoma. Proteomics 3,601 –609[CrossRef][Medline]

  9. Rabilloud, T. (1999) in 2-D Proteome Analysis Protocols (Link, A. J., ed.) pp.297 –305, Humana Press, Totowa

  10. Rabilloud, T., and Charmont, S. (2000) in Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Rabilloud, T., ed.) pp.107 –126, Springer, Berlin

  11. Spodik, B., Seeholzer, S. H., and Coleman, T. R. (2002) in Protein-Protein Interactions (Coleman, E. A., ed.) pp.355 –374, Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  12. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551 –3567[CrossRef][Medline]

  13. Morinaga, T., Sakai, M., Wegmann, T. G., and Tamaoki, T. (1983) Primary structures of human alpha-fetoprotein and its mRNA. Proc. Natl. Acad. Sci. U. S. A. 80, 4604 –4608[Abstract]

  14. Min, X., and Carter, D. C. (1992) Atomic structure and chemistry of human serum albumin. Nature 358, 209 –215[CrossRef][Medline]

  15. Evans, A. A., O’Connell, A. P., Pugh, J. C., Manson, W. S., Shen, F., Chen, G.-C., Lin, W.-Y., Dia, A., M’Boup, S., Drame, B., and London, W. T. (1998) Geographic variation in viral load amon hepatitis B carriers with differing risks of hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev. 7, 559 –565[Abstract]

  16. Krone, B., Lenz, A., Heermann, K. H., Seifer, M., Lu, X. Y., and Gerlich, W. H. (1990) Interaction between hepatitis B surface proteins and monomeric human serum albumin. Hepatology 11, 1050 –1056[Medline]

  17. Harlow, E., and Lane, D. (1999) Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor