U.S. patent application number 09/017284 was filed with the patent office on 2001-12-13 for removal of abundant interfering proteins from a liquid sample using a collapsible affinity matrix.
This patent application is currently assigned to FISH AND RICHARDSON P.C.. Invention is credited to STEVENS, ANTHONY C..
Application Number | 20010051380 09/017284 |
Document ID | / |
Family ID | 21781755 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051380 |
Kind Code |
A1 |
STEVENS, ANTHONY C. |
December 13, 2001 |
REMOVAL OF ABUNDANT INTERFERING PROTEINS FROM A LIQUID SAMPLE USING
A COLLAPSIBLE AFFINITY MATRIX
Abstract
A method for specifically immunoprecipitating albumin from a
serum sample, using a "collapsible affinity matrix." Also provided
is a method for the co-removal of immunoglobulin using a
"collapsible affinity matrix." Removal of the highly abundant serum
proteins, albumin and immunoglobulin, thereby improves the
fractionation of the remaining serum proteins. Due to the
collapsible nature of the matrix, less protein is trapped in the
void space. Through specific removal of the abundant serum proteins
by the collapsible affinity matrix and application of a two
dimensional gel electrophoresis method, HiCap 2-D PAGE, the
concentrations of a large number of low abundant serum proteins are
estimated simultaneously, allowing the identification of several
disease-related proteins in a relatively short period of time.
Inventors: |
STEVENS, ANTHONY C.; (SAN
DIEGO, CA) |
Correspondence
Address: |
FISH & RICHARDSON, PC
4350 LA JOLLA VILLAGE DRIVE
SUITE 500
SAN DIEGO
CA
92122
US
|
Assignee: |
FISH AND RICHARDSON P.C.
|
Family ID: |
21781755 |
Appl. No.: |
09/017284 |
Filed: |
February 2, 1998 |
Current U.S.
Class: |
436/516 |
Current CPC
Class: |
C07K 16/065 20130101;
C07K 16/18 20130101; C07K 1/32 20130101 |
Class at
Publication: |
436/516 |
International
Class: |
G01N 033/536; G01N
033/561 |
Claims
What is claimed:
1. A method of preparing a liquid sample for fractionation,
comprising: a) contacting the sample with a polypeptide affinity
reagent having specific binding affinity for an abundant
macromolecule in the sample, the polypeptide affinity reagent
comprising a first member of a high affinity binding pair system,
to form a macromolecule-polypeptide affinity reagent complex; and
b) contacting the macromolecule-polypeptide affinity reagent
complex with the second member of the high affinity binding pair
system to form a collapsible affinity matrix.
2. The method of claim 1, wherein the sample is selected from a
group consisting of plasma or serum.
3. The method of claim 1, wherein the abundant macromolecule is
serum albumin.
4. The method of claim 1, wherein the abundant macromolecule is
immunoglobulin.
5. The method of claim 1, wherein the method is performed multiple
times, simultaneously or sequentially, on a single serum sample to
remove multiple abundant molecules.
6. The method of claim 5, wherein the multiple abundant molecules
are serum albumin and immunoglobulin.
7. The method of claim 1, wherein the polypeptide affinity reagent
is anti-serum albumin antibody.
8. The method of claim 7, wherein the anti-serum albumin antibody
is monoclonal.
9. The method of claim 8, wherein the monoclonal anti-serum albumin
antibody has the binding specificity of HSA2126NX.012, ATCC
accession No. HB12464.
10. The method of claim 8, wherein the monoclonal anti-serum
albumin antibody is HSA2126NX.012, having ATCC accession No.
HB12464.
11. The method of claim 1, farther comprising, following step b),
the step of fractionating the liquid sample.
12. The method of claim 11, wherein the fractionating is by two
dimensional electrophoresis.
13. The method of claim 12, wherein the two dimensional
electrophoresis is the HiCap method.
14. A serum substantially and specifically depleted in serum
albumin and immunoglobulin.
15. A monoclonal antibody capable of immunoprecipitating serum
albumin from serum.
16. The monoclonal antibody of claim 15, wherein the monoclonal
antibody has the binding specificity of HSA2126NX.012, ATCC
accession No. HB12464.
17. The monoclonal antibody of claim 15, wherein the monoclonal
antibody is HSA2126NX.012, having ATCC accession No. HB 12464.
18. A host cell that expresses monoclonal antibody,
HSA2126NX.012.
19. A kit, comprising two or more containers, wherein: a) the first
container contains a monoclonal antibody capable of
immunoprecipitating serum albumin from serum, to which is bound a
first member of a high affinity binding pair; and b) the second
container contains a second member of the high affinity binding
pair.
20. The kit of claim 19, wherein the first container contains a
monoclonal antibody having the binding specificity of
HSA2126NX.012, to which is bound the first member of the high
affinity binding pair.
21. The kit of claim 19, wherein the first container contains
HSA2126NX.012, to which is bound the first member of the high
affinity binding pair.
22. The kit of claim 19, further comprising a third container,
wherein the third container contains protein A, to which is bound
the first member of the high affinity binding pair.
23. The kit of claim 19, wherein the second container contains
avidin.
24. The kit of claim 19, wherein the second container contains
streptavidin.
Description
BACKGROUND OF THE INVENTION
[0001] Since the development of high resolution two-dimensional
(2-D) electrophoresis by O'Farrell, the technique has been applied
to mapping the protein composition of human serum and of various
tissues. 2-D electrophoresis consists of isoelectric focusing
electrophoresis (IEF) in the first dimension and SDS polyacrylamide
gel electrophoresis [SDS-PAGE] in the second dimension. Current
interest in using 2-D electrophoresis to identify disease related
proteins is exemplified by the existence of databases dedicated to
2-D polypeptide maps of serum and tissue samples of different
disease states.
[0002] Although 2-D electrophoresis is considered to be the most
powerful separation technique for resolving highly complex protein
mixtures, the method has limitations. Most of these limitations are
related to sample composition, such as high concentrations of salt
and protein. The advent of immobilized pH gradient (IPG) strips has
greatly minimized these limitations. Even when using immobilized pH
gradient strips, however, suggested sample loadings of human serum
are on the same order of magnitude (1-5 .mu.L) as that used with
the "classical" O'Farrell technique for analytical 2-D
electrophoresis.
[0003] The limitation of human serum sample volume is due to the
protein distribution and not necessarily the total protein,
although total protein is a significant limitation with the
O'Farrell technique. A single protein, albumin (HSA), makes up
approximately 50% of the total human serum protein. This protein
can distort the gel image of a 2-D protein map when large sample
volumes are used. The limitation in sample volume ultimately limits
the number of proteins that can be detected by 2-D
electrophoresis.
[0004] The distortion in the gel image is particularly evident in
the area of the albumin (molecular weight [MW] 66,000, pI 4.9)
where vertical and horizontal streaking masks a large portion of
the protein map. In addition, a group of abundant serum proteins,
immunoglobulin (Ig), contributes approximately 20% to total human
serum protein. Vertical and horizontal streaking also masks the
portion of the protein map in the area of the gel image where Ig
light and heavy chains are located. Furthermore, the presence of
the abundant HSA and Ig alters the pI of the isoelectric focusing
electrophoresis gel in these proteins, impeding effective
resolution and detection of many other protein spots. To improve
2-D electrophoresis human serum maps, in both quality of image and
the number of detectable proteins, human serum albumin must be
specifically removed.
[0005] There are currently several methods for removing albumin
from serum, such as adsorption to activated carbon particles,
binding to Cibacron-blue dye coupled to Sepharose beads, and the
use of anti-albumin polyclonal antibodies. Removal of serum albumin
using carbon or the Cibacron-blue Sepharose is relatively
inexpensive, but these methods suffer from a lack of specificity.
The Cibacron-blue dye binds many proteins other than albumin, such
as interferon, lipoproteins, blood coagulation factors, kinases,
dehydrogenases and most enzymes requiring adenyl-containing
cofactors. Also, because of the microporous nature of the Sepharose
beads, additional proteins are trapped in the dead volume of the
rigid matrix.
SUMMARY OF THE INVENTION
[0006] The present invention provides a method for removing
interfering macromolecules from a liquid sample before protein
fractionation. This method involves contacting the liquid sample
with a polypeptide affinity reagent that has specificity for an
abundant macromolecule in the sample, and is one member of a high
affinity binding pair system. A macromolecule-polypeptide affinity
reagent complex is formed, that is then contacted with the other
member of a high affinity binding pair system to form a
"collapsible affinity matrix." The collapsible affinity matrix is
specific for the abundant macromolecule and, when centrifuged,
contains very little dead volume that would otherwise trap
additional sample macromolecules. In one embodiment, the invention
provides a method for specifically removing macromolecules from a
sample using biotinylated adsorptive proteins. In a specific
embodiment, a biotinylated anti-HSA antibody, in conjunction with
avidin and human serum, forms a collapsible affinity matrix,
containing albumin. The combination of biotinylated protein A,
avidin, and human serum, followed by contact with biotinylated
anti-HSA and avidin allows simultaneous co-precipitation of albumin
and immunoglobulin (Ig). The practice of the method of the
invention can thereby provide serum samples substantially depleted
of albumin and immunoglobulin.
[0007] The invention also provides a monoclonal antibody
(HSA2126NX.012) that can specifically immunoprecipitate albumin
from serum. The invention further provides a kit useful for
specifically removing abundant macromolecules from a sample using
biotinylated adsorptive proteins.
[0008] This unique method for removing albumin and immunoglobulin
from serum permits the full potential of the powerful protein
fractionation technique of high resolution 2-D electrophoresis to
be attained, by making possible visualization of low abundant serum
proteins, as well as those proteins that would normally be obscured
by the serum albumin and immunoglobulin. This advantage allows for
identification and characterization of a variety of novel markers
that may have diagnostic or therapeutic utility. For example, the
discovery of novel biochemical serum markers for the diagnosis of
various disease states such as osteoporosis, arthritis, cancer or
cardiovascular disease can aid immensely in the management of these
conditions.
[0009] The removal of high abundant macromolecules from a liquid
sample followed by a high resolution 2-D electrophoresis allows for
visualization of low abundant sample proteins that might not be
visualized with limits in total protein load. When the high
resolution 2-D electrophoresis includes in-gel sample rehydration
of immobilized pH gradient strips, followed by isoelectric focusing
in the first dimension and SDS-PAGE in the second dimension, this
method is called "High Capacity Two-Dimensional Polyacrylamide Gel
Electrophoresis" ("HiCap 2-D PAGE"). HiCap 2-D PAGE permits
relatively high amounts of low abundant proteins to be loaded
following the removal of albumin and immunoglobulin. HiCap 2-D PAGE
also permits the use of large sample load due to in-gel sample
rehydration. The combination of abundant serum protein removal by
the collapsible affinity matrix and HiCap 2-D PAGE produces highly
reproducible maps of low abundance serum proteins in human
serum.
DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a 2-D PAGE of 5 .mu.L human serum, where the serum
is A) untreated; B) treated with anti-HSA monoclonal HSA2126NX.012;
and C) treated with Cibacron-blue.
[0011] FIG. 2 is a 2-D PAGE of 250 .mu.g total protein from human
serum, where the serum is A) untreated; B) treated with anti-HSA
monoclonal HSA2126NX.012; and C) treated with Cibacron-blue.
[0012] FIG. 3 is a 2-D PAGE polypeptide map of 100 .mu.L human
serum treated for the removal of HSA and Ig. The 100 .mu.L human
serum was treated (albumin and Ig removed) and analyzed according
to the methods described in EXAMPLES III and IV. The entire map (pH
3.5-8.0) is a composite of three separate 2-D gels spanning three
different pH regions (3.5-5.0, 4.5-6.5 and 6.0-8.0). When 100 .mu.L
of treated human serum is analyzed, approximately 4000 polypeptide
spots can be detected. The greatest number of protein spots
previously reported to be detected in serum was 2500.
[0013] FIG. 4 is a duplicate of 2-D PAGE gels showing gel-to-gel
spatial reproducibility. Two identical 100 .mu.L human serum
samples were treated and analyzed according to the methods
described in EXAMPLES III and IV. Within the region of interest,
there were 212 spots detected in gel 1, as shown in FIG. 4A, and
232 spots detected in gel 2, as shown in FIG. 4B.
[0014] FIG. 5 is a bar graph showing gel-to-gel spatial
reproducibility. FIG. 5 shows normalized intensity of 18 paired
spots from gel 1 (see, FIG. 4A) and gel 2 (see, FIG. 4B). FIG. 5
represents the reproducibility of HiCap 2-D PAGE from a
quantitative point of view. Two identical 100 .mu.L human serum
samples were treated and analyzed using HiCap 2-D PAGE according to
the methods described in EXAMPLES III and IV. Eighteen paired spots
were randomly chosen and the individual normalized densities (NOD)
between the two gels were compared. The average variation in NOD
between the duplicate gels was about 25%.
[0015] FIG. 6 is a summary of the preliminary results from analysis
of patient samples. FIG. 6A shows spot ID 118 concentration (2-D
PAGE) and NTx concentration (commercial assay) in the serum of a
Paget's disease patient over time. FIG. 6B shows how the spot ID
118 concentration and NTx concentration correlate with one
another.
[0016] FIG. 7 is a duplicate of 2-D PAGE gels showing that more
polypeptides are present in a sample prepared using the collapsible
affinity matrix than using an immobilized matrix (a rigid
streptavidin-Sepharose matrix, Ultralink Immobilized Streptavidin
on 3M Emphage Biosupport Medium; Pierce). FIG. 7A is the
collapsible affinity matrix sample. FIG. 7B is an immobilized
matrix sample.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides a method for removing
interfering macromolecules from a liquid sample before protein
fractionation. The liquid sample is contacted with a polypeptide
affinity reagent having specificity for an abundant macromolecule
in the sample. The polypeptide affinity reagent is one member of a
high affinity binding pair system, and contacting the sample with
the polypeptide affinity reagent forms a macromolecule-polypeptide
affinity reagent complex. Then, the macromolecule-polypeptide
affinity reagent complex is contacted with a second member of a
high affinity binding pair system to form a "collapsible affinity
matrix." The collapsible affinity matrix is a stable aggregation of
the macromolecule-polypeptide affinity reagent complexes.
[0018] In one embodiment, the invention provides a method for
specifically removing macromolecules from a liquid sample using
biotinylated adsorptive proteins and a second member of the high
affinity binding pair system, such as avidin, streptavidin, or
NeutrAvidin.TM.. For example, albumin can be specifically removed
from a serum sample using biotinylated anti-human serum albumin
monoclonal antibody, with avidin, streptavidin, or NeutrAvidin.TM..
Alternatively, human serum albumin and immunoglobulin can both be
removed from a serum sample using biotinylated anti-human serum
albumin monoclonal antibody and biotinylated protein A, with
avidin, streptavidin, or NeutrAvidin.TM.. The invention thus
provides a serum sample substantially depleted in albumin and
immunoglobulin.
[0019] The invention further provides a kit useful in the practice
of the methods of the invention. The kit has two or more
containers. A first container contains a monoclonal antibody that
can immunoprecipitate albumin from serum, to which is bound a first
member of a high affinity binding pair, for example biotin. A
second container contains a second member of a high affinity
binding pair, for example, avidin, streptavidin, or
NeutrAvidin.TM..
[0020] Liquid Sample
[0021] The invention provides a method for preparing a liquid
sample for fractionation. As used herein, the term "sample"
includes material derived from a mammalian subject, e.g., human. As
well as non-mammalian animals. Such samples include but are not
limited to hair, skin samples, tissue samples, cultured cells,
cultured cell media, and biological fluids. The term "tissue"
refers to a mass of connected cells (e.g., CNS tissue, neural
tissue, or eye tissue) derived from an animal or human subject, and
includes the connecting material and the liquid material in
association with the cells. As used herein, the term "liquid
sample" refers to liquid material derived from a human, animal, or
the cells derived therefrom. Such liquid samples include but are
not limited to blood, plasma, serum, serum derivatives, bile,
phlegm, saliva, sweat, amniotic fluid, and cerebrospinal fluid
(CSF), such as lumbar or ventricular CSF. As used herein, the term
"liquid sample" also includes solutions containing an isolated
macromolecule, media into which the macromolecule has been
secreted, and media containing cells that produce a macromolecule
of interest. For example, a liquid sample may be a protein sample
that is to be resolved by SDS-PAGE and transferred to
nitrocellulose for Western immunoblot analysis. The quantity of
sample required for the protein fractionation can be determined by
one skilled in the art by standard laboratory techniques. The
optimal quantity of sample may be determined by serial
dilution.
[0022] Polypeptide Affinity Reagent
[0023] The invention provides a method for preparing a liquid
sample for fractionation, by contacting the liquid sample with a
polypeptide affinity reagent. As used herein, the terms
"polypeptide affinity reagent" refers to a polypeptide that
specifically binds to a macromolecule of interest in a liquid
sample to be fractionated. "Specifically binds" means the
adsorptive protein recognizes and binds a specified macromolecule,
but does not substantially recognize and bind other molecules in a
sample, e.g., a liquid biological sample, that naturally includes a
variety of macromolecules. The principle is to contact the liquid
sample with reagents having specific affinity for a particular
component. These reagents have narrow specificities for particular
sets of macromolecules.
[0024] Antibodies represent the main class of polypeptide affinity
reagents that are immunoreactive or bind to epitopes of
macromolecules. The term "epitope" refers to any antigenic
determinant on an antigen to which an antibody binds. Epitopes
usually are chemically active surface groupings of molecules such
as amino acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics.
[0025] As used herein, the term "antibody" includes intact antibody
molecules as well as fragments thereof, such as Fab, Fab',
F(ab').sub.2, Fv, and single chain antibody that can bind the
epitope. These antibody fragments retain some ability selectively
to bind with corresponding antigen or receptor. Particularly useful
antibodies include polyclonal and monoclonal antibodies, chimeric
antibodies, single chain antibodies and the like, having the
ability to bind with high immunospecificity to abundant
macromolecules. These antibodies can be unlabeled or suitably
labeled.
[0026] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, for example, Green et al.
("Production of Polyclonal Antisera", in Immunochemical Protocols,
Manson, ed., Humana Press, 1992, pages 1-5) and Colligan et al.
(Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in: Current Protocols in Immunology, section 2. 4. 1,
1992), incorporated herein by reference.
[0027] The preparation of monoclonal antibodies likewise is
conventional. Monoclonal antibodies can be produced using methods
well known in the art. See, Kohler et al. (Nature 256: 495, 1975);
Current Protocols in Molecular Biology (Ausubel et al., ed., 1989);
and Harlow and Lane (Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York, current edition), incorporated herein
by reference. Briefly, monoclonal antibodies can be obtained by
injecting mice with an antigenic composition, verifying the
presence of antibody production by removing a serum sample,
removing the spleen to obtain B lymphocytes, fusing the B
lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones that produce antibodies to
the antigen, and isolating the antibodies from the hybridoma
cultures. Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography. In EXAMPLE I, HSA2126NX.012 cell
culture supernatant was run over a Protein A Sepharose column.
[0028] Methods of in vitro and in vivo multiplication of monoclonal
antibodies are well known to those skilled in the art.
Multiplication in vitro may be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640
medium, optionally replenished by a mammalian serum such as fetal
calf serum or trace elements and growth-sustaining supplements such
as normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. In Example I, monoclonal antibody HSA2126NX.012 was
produced from hybridoma (ATCC accession No. HB12464) cultures grown
in medium that is serum free, contains no albumin and is low in
total protein content. Production in vitro provides relatively pure
antibody preparations and allows scale-up to yield large amounts of
the desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. In Example I, for larger scale production, an
artificial capillary system was used, where well-established
bioreactor culture yields 1-3 mg antibody per mL of supernatant.
Multiplication in vivo may be carried out by injecting cell clones
into mammals histocompatible with the parent cells, e.g., syngeneic
mice, to cause growth of antibody-producing tumors. Optionally, the
animals are primed with a hydrocarbon, especially oils such as
pristane (tetramethylpentadecane) prior to injection. After one to
three weeks, the desired monoclonal antibody is recovered from the
body fluid of the animal.
[0029] If desired, polyclonal or monoclonal antibodies can be
further purified, for example, by binding to and elution from a
matrix to which the polypeptide or a peptide to which the
antibodies were raised is bound. A purified antibody may be
obtained, for example, by affinity chromatography using
recombinantly-produced protein or conserved motif peptides and
standard techniques. Those of skill in the art will know of various
techniques common in the immunology arts for purification or
concentration of polyclonal antibodies, as well as monoclonal
antibodies. See, e.g., Colligan, et al. (Unit 9, Current Protocols
in Immunology, Wiley Interscience, 1997).
[0030] As used herein, the term "albumin-specific monoclonal
antibodies" refers to monoclonal antibodies that specifically bind
to serum albumin. "Specifically binds to albumin" means the
monoclonal antibody recognizes and binds to serum albumin, but does
not substantially recognize and bind other molecules in a sample,
e.g., serum, that naturally includes serum albumin. The invention
provides a monoclonal antibody that can immunoprecipitate serum
albumin from serum. This means that the monoclonal antibody
recognizes an epitope on the HSA molecule that is not blocked by
the numerous serum proteins that bind to HSA in serum. Thus, a
monoclonal antibody, specific for HSA, can be used in an improved
method for the removal of albumin from human serum. 2-D
electrophoresis of human serum treated in this way is therefore
improved in both the quality of image produced and the number of
proteins detected. In a specific embodiment, the monoclonal
antibody is HSA2126NX.012. A method for making monoclonal antibody
HSA2126NX.012 is provided in EXAMPLE I.
[0031] Although polyclonal antibodies against HSA provide
specificity, there is the inherent variability in antibody
population that occurs during separate immunization schedules that
can lead to reproducibility problems. Additionally, the supply of
polyclonal antibody containing serum is limited by the health and
finite lifespan of the producing animal. Considering the large
quantities of anti-HSA antibody required for the treatment of
serum, the use of polyclonal antibodies is possible but not
preferred.
[0032] Other polypeptide affinity reagents include protein A and
protein G. As used herein, "protein A" is a protein of MW 42,000
from the bacterium Staphylococcus aureus that binds to IgG from a
wide range of species, including human, rabbit, donkey, pig, and
guinea-pig. Protein A is commonly used as a secondary reagent in
immunological and biological techniques, as described by Goding (J.
Immunol. Meth. 20. 241-253, 1978), and is commercially available.
In EXAMPLE II, recombinant protein A was obtained from Scripps
Laboratories. As used herein, "protein G" is a monomeric protein
(MW 63,000) from human group G streptococcus. Protein G possess two
or three antibody-binding sites and binds IgG from a wide range of
species. Compared to protein A, protein G binds with a higher
affinity to rat, mouse and goat IgG, as described by Bjrk et al. (J
Immunol. 133, 969-974, 1984).
[0033] Other polypeptide affinity reagents include lectins, which
specifically bind sugars (saccharides). The definition adopted by
the Nomenclature Committee of the International Union of
Biochemistry states that "a lectin is a sugar-binding protein of
non-immune origin that agglutinates cells or precipitates
glycoconjugates." This definition provides positive and easily
testable properties for identifying possible lectins. The
sugar-binding property is the predominant feature of lectins and is
responsible for their biological actions and their value in
biological experimental techniques. Sugar-binding, in conjunction
with the related agglutination action, serves to identify lectins
in tissue extracts and facilitates their subsequent isolation.
Although binding to red blood cells has traditionally been the way
of distinguishing lectins, a few lectins do not agglutinate red
blood cells. Thus, although the original definition of lectin
specified the agglutination of red blood cells, the term now
incorporates those proteins that agglutinate other cells, as well
as some proteins that are not known at present to agglutinate any
cells at all, but do bind sugars and have stretches of amino-acid
sequence in their polypeptide subunits that are similar to those of
more characteristic lectins. Individual lectins are usually named
after the organism, in most cases a plant, from which they were
obtained. Examples of individual lectins include wheat-germ
agglutinin, concanavalin A from the jack-bean, and pea, lentil, and
potato lectins.
[0034] Lectins are distinguished from the numerous immunoproteins
and enzymes that may also bind sugars, although some lectins may
have glycosidase activity.
[0035] Lectins usually consist of two or four identical polypeptide
subunits. When differences between the subunits are found, however,
they can be quite marked. There is usually one sugar-binding site
per subunit and these sugar-binding sites are normally for the same
sugar, are all of the same type, and do not interact with each
other. Lectins composed of different subunits can be found in
different forms (isolectins) arising from various combinations of
the monomers in the complete dimer or tetramer. Subunits may differ
in their amino acid sequences and, if the lectin is a glycoprotein,
the subunits may also differ in the nature and linkages of the
sugars in the attached oligosaccharide side chains.
[0036] The properties of lectins make them extremely important
components of many techniques in cell biology and biochemistry.
They are used extensively, for example, in the procedures for
glycoprotein isolation, as described by Lis & Sharon ("Lectins
as molecules and tools." Annu. Rev. Biochem. 55: 35-67, 1986).
[0037] Other polypeptide affinity reagents include DNA-binding
proteins. As used herein, the term "DNA-binding proteins" refers to
proteins that bind to DNA, including gene regulatory proteins,
enzymes involved in DNA replication, recombination, repair,
transcription, and degradation, and proteins involved in
maintaining chromosome structure. They can be divided into two
large groups: (1) Those that have some sequence-specific or
secondary structure-specific requirement for DNA-binding, and (2)
those that bind DNA nonspecifically. Examples of sequence-specific
DNA-binding include homeodomain proteins; proteins involved in
protein-nucleic acid interactions during recombination; restriction
enzymes; and transcription factors. Examples of
sequence-nonspecific DNA-binding include chromatin; proteins
involved in DNA repair and DNA replication; and nucleases.
[0038] The method of the invention can be performed multiple times
on a single liquid sample to remove multiple abundant molecules.
For example, in EXAMPLES III and VI, the multiple abundant
molecules removed from serum are albumin and immunoglobulin.
[0039] Abundant Macromolecules
[0040] The invention provides a method for preparing a liquid
sample for fractionation, by contacting the liquid sample with a
polypeptide affinity reagent having specificity for an abundant
macromolecule in the sample. As used herein, a "macromolecule" is a
molecule with a molecular weight in excess of 1,000 kilodaltons
(kDa). Examples of macromolecules include polynucleotides,
polypeptides, and polysaccharides. Examples also include
glycoproteins, in which saccharide (sugar) moieties are covalently
bound to polypeptides, and nucleoproteins, which are complexes of
polynucleotide and polypeptide. The terms "albumin" and "serum
albumin" refer to the most abundant of the serum proteins. In one
embodiment, the serum albumin is human serum albumin (HSA). In
another embodiment, described in EXAMPLE XI, serum albumin is
monkey serum albumin (MSA).
[0041] An "abundant macromolecule" is a macromolecule present in a
sample in such quantity that the presence of the macromolecule
interferes with an aspect of the analysis of the sample. For
example, a single protein, serum albumin, makes up over 50% of the
total protein in human serum. This can have deleterious effects on
2-D protein maps prepared with large sample volumes, by distorting
the gel image. The distortion in the gel image is particularly
evident in the area of the albumin where vertical and horizontal
streaking can mask a large portion of the protein map. The
limitation in sample volume ultimately limits the number of other
proteins that can be detected by 2-D electrophoresis.
[0042] High Affinity Binding Pair System
[0043] The invention provides a method for preparing a liquid
sample for fractionation, by contacting the liquid sample with a
polypeptide affinity reagent having specificity for an abundant
macromolecule in the sample. The polypeptide affinity reagent is
one member of a high affinity pair system. As used herein, a "high
affinity binding pair system" is a pair of reagents where a first
member of the high affinity binding pair system binds to the second
member of the high affinity binding pair system with a functional
affinity (or avidity) sufficiently strong to allow stable
aggregation of the macromolecule-polypeptide affinity reagent
complexes in the liquid sample under physiological conditions over
the length of time that the method of the invention is practiced. A
high affinity binding pair system typically exhibits an affinity
between the first and second members of the high affinity binding
pair of at least about K.about.10.sup.-10. Specifically excluded
from the definition of high affinity binding pair systems are
antibody-second antibody systems and antibody systems that comprise
complement, protein A, protein G, or Fc receptors.
[0044] In one embodiment, the high affinity binding pair system is
the avidin and biotin system. Avidin binds to biotin almost
irreversibly, with a dissociation constant of K.about.10.sup.-15 M.
As used herein, "avidin" is a tetrameric glycoprotein from egg
white that binds to biotin. In another embodiment, the high
affinity binding pair system is streptavidin and biotin. As used
herein, "streptavidin" is a tetrameric protein from the prokaryote
Streptomyces avidinii that, like avidin, binds to biotin. In
another embodiment, the high affinity binding pair system is
NeutrAvidin.TM. and biotin. NeutrAvidin.TM. is an avidin protein
that has been processed to remove the carbohydrate and lower its
isoelectric point. The methods used to deglycosylate the avidin
retain both its specific binding and its complement of
amine-conjugation sites.
[0045] The polypeptide affinity reagent is linked to a first member
of a high affinity binding pair. The polypeptide affinity reagent
may be linked either directly to a first member of the high
affinity binding pair (i.e., the polypeptide affinity reagent and
the first member of the high affinity binding pair constitute the
same polypeptide) or covalently bound to a first member of the high
affinity binding pair. Biotin can be covalently linked to proteins;
the proteins can then be cross-linked using avidin, streptavidin,
or NeutrAvidin.TM.. As used herein, the term "biotinylation" refers
to the methods by which biotin can be linked covalently to
polypeptides for use as a label. Use of this technique is well
known in the art for localization of biotinylated primary reagents
such as antibodies, lectins or cDNA, and localization of proteins
that have been applied to living cells before processing, such as
endocytosed ligands. Biotinylation is commonly used as an
alternative method to radioactivity for labeling polypeptide.
EXAMPLE II provides a description of one method of both the
biotinylation of antibody and the biotinylation of protein A.
[0046] In another embodiment, the high affinity binding pair system
is a hapten, such as dinitrophenol, pyridoxal, or fluorescein, and
a specific anti-hapten antibody.
[0047] Collapsible Affinity Matrix
[0048] The polypeptide affinity reagent is one member of a high
affinity binding pair system, and contacting the sample with the
polypeptide affinity reagent forms a macromolecule-polypeptide
affinity reagent complex. Then, the macromolecule-polypeptide
affinity reagent complex is contacted with a second member of a
high affinity binding pair system to form a collapsible affinity
matrix. As used herein, a "collapsible affinity matrix" is a stable
aggregation of the macromolecule-polypeptide affinity reagent
complexes in the liquid sample. A collapsible affinity matrix is
stable under physiological conditions over the length of time that
the method of the invention is practiced. In EXAMPLE III, a
collapsible affinity matrix specific for HSA and Ig was prepared by
precipitating biotinylated anti-HSA bound to HSA and biotinylated
protein A bound to Ig (i.e, macromolecule-polypeptide affinity
reagent complexes) with avidin, the second member of the
avidin/biotin high affinity binding pair system.
[0049] An advantage of the collapsible affinity matrix for protein
removal is that when the matrix is pelleted by centrifugation, a
low void volume pellet is formed. The collapsible nature of this
novel collapsible affinity matrix is therefore superior to existing
methods that use coated Sepharose beads. The collapsible affinity
matrix contains less "dead" space and therefore traps less low
abundant and potentially interesting protein, due to the low-volume
void space, than does a rigid, microporous immobilized matrix.
[0050] The collapsible affinity matrix can be removed from the
liquid sample by means known to those of skill in the art. For
example, the collapsible affinity matrix can be removed by
centrifugation. For another example, the collapsible affinity
matrix can be removed by filtration.
[0051] Serum Substantially Depleted in Serum Albumin and
Immunoglobulins
[0052] The invention thus provides a serum substantially depleted
in albumin and immunoglobulins. As used herein, the term
"substantially depleted" means that the serum sample, after the
collapsible affinity matrix is removed, contains less than 50% of
the total protein of control serum that has not been treated with
the method of the invention. In EXAMPLE V, serum samples treated
with the collapsible affinity matrix contained less total protein
than on average than before treatment.
[0053] Serum Specifically Depleted in Serum Albumin and
Immunoglobulins
[0054] The invention thus provides a serum specifically depleted in
albumin and immunoglobulins. As used herein, the term "specifically
depleted" means that the serum sample, after the collapsible
affinity matrix is removed, contains a substantial amount of
identifiable low abundant protein than control serum that has not
been treated with the method of the invention. In EXAMPLE VI,
treating human serum with the monoclonal HSA2126NX.012 by the
collapsible affinity matrix method resulted in the visualization of
20% more 2-D PAGE protein spots when compared to treatment with
Cibacron-blue. Cibacron-blue Sepharose treatment of serum (by the
immobilized affinity matrix method) quantitatively removes the
serum glycoprotein HC gp-39, as determined by immunoassay, while
treatment with the HSA2126NX.012 monoclonal results in>75%
recovery of this glycoprotein. In EXAMPLE XIII, more polypeptides
remained in the collapsible affinity matrix sample compared to the
immobilized matrix sample. Analysis of the most clearly resolved
area of the gels calculated 164 polypeptide spots for the
collapsible affinity matrix versus 108 polypeptide spots for the
immobilized matrix. Due to the microporous nature of the rigid
Sepharose beads, that possess an inherent dead volume, low abundant
proteins of interest are trapped. By contrast, the collapsible, low
void-volume, affinity matrix does not trap low abundant proteins of
interest.
[0055] The removal of albumin and immunoglobulin, the two most
abundant proteins in serum, allows one to load a higher percentage
of novel polypeptides that are relevant to a variety of disease
states yet are low in abundance and are not detectable using
untreated serum and the existing limitations of 2-D gel
electrophoresis techniques. The more specific removal of serum
albumin is accompanied by the less removal of other proteins,
either by trapping or nonspecific binding. The removal of serum
albumin and immunoglobulin is particularly important for
identification of proteins that have isoelectric points or
molecular weights that are similar to these proteins. In the case
of an untreated serum sample, potentially novel proteins with
diagnostic or therapeutic potential are masked by the overwhelming
amount of HSA or Ig on the 2-D gels. The "overloading" of serum
albumin and immunoglobulin can also effect the focusing and running
of nearby proteins and can cause undesired distortion of the 2-D
protein pattern.
[0056] Fractionation
[0057] The invention provides a method for preparing a liquid
sample for protein fractionation. As used herein, the term "protein
fractionation" refers to an analytical technique used to separate
molecules. Several of the methods of fractionation well-known to
those of skill in the art include chromatography, electrophoresis,
and isoelectric focussing.
[0058] Chromatography is an analytical technique used to separate
molecules based on how they tend to cling to or dissolve in various
solids, liquids and gases. Many chromatographic methods are known
to those of skill in the art. Gel filtration chromatography is used
most often to separate proteins by running the solution containing
the proteins through a column filled with porous carbohydrate gel
beads that traps or slows down smaller molecules but allows larger
molecules to slide past. Paper chromatography and thin-layer
chromatography separate molecules by taking advantage of their
differing solubilities in a mix of solvents. The material to be
separated is applied to a special piece of material, and the edge
material is put in the solvent mix. The material to be separated
travels through the chromatographic material by capillary action
and the solvent carries the different molecules at different rates.
Ion exchange chromatography is a technique of analytical chemistry
used to separate and purify a biological molecule from a mixture,
based on the attraction of the charge of the molecule of interest.
The mixture, present in a buffer having one ionic property is
passed through a column containing a resin of polymers that have
fixed charged groups attached to the stationary substance. The
molecule of interest stays within the column while much of the rest
of the mixture continues through to the end. Then, a buffer having
a different ionic property is flushed through the column to detach
the molecule from the resin and separate the molecule from the
portion of the mixture that has a different charge. Affinity
chromatography is a technique of analytical chemistry used to
separate and purify a biological molecule from a mixture, based on
the attraction of the molecule of interest to a particular ligand
that has been previously attached to a solid, inert substance. The
mixture is passed through a column containing the ligand attached
to the stationary substance, so that the molecule of interest stays
within the column while the rest of the mixture continues through
to the end. Then, a different chemical is flushed through the
column to detach the molecule from the ligand and separate the
molecule from the rest of the mixture. High-performance liquid
chromatography (HPLC) is a type of column chromatography that uses
a combination of several separation techniques to separate
substances at higher resolution. Extremely sharp peaks on the
elution profile can be produced with high-performance liquid
chromatography.
[0059] Electrophoresis is a method for separating large molecules
(such as DNA fragments or proteins) from a mixture of similar
molecules. An electric current is passed through a medium
containing the mixture, and each kind of molecule travels through
the medium at a different rate, depending on electrical charge and
size. Separation is based on these differences. Agarose and
acrylamide gels are the media commonly used for electrophoresis of
proteins and nucleic acids. Specific electrophoretic methods
include Northern blot, Southern blot, and Western blot. Agarose gel
electrophoresis is a type of electrophoresis that uses a matrix of
highly purified agar to separate large DNA and RNA molecules
(generally around 20,000 nucleotides in size). Capillary
electrophoresis is a technique for separating compounds; a sample
of a compound to be separated is placed in a capillary tube, which
is then subjected to a high voltage current that separates chemical
components. Disc electrophoresis (short for "discontinuous
electrophoresis") is a type of polyacrylamide gel electrophoresis.
This electrophoresis method uses gels of two different
concentrations of polyacrylamide (a synthetic polymer), the one of
lower concentration stacked on top of the one with higher
concentration, in order to better resolve bands of whatever is
being separated (DNA, RNA, or protein) that would otherwise be very
close together.
[0060] Isoelectric focusing is a technique used in electrophoresis
that separates molecules on the basis of their different
isoelectric points.
[0061] In one embodiment, method of fractionation is a preparative
2-D gel electrophoresis system, such as that described by Merrick
et al. (U.S. Pat. No. 5,534,121, issued Jul. 9, 1996). This method
is a single procedure for separation and isolation of preparative
amounts of proteins from complex biological preparations. The
system includes sized-up isoelectric focusing tube gels and slab
gel molds that allow for sample loads of between about 0.5 and 2 mg
or greater. Increased protein loads, resolution and electrotransfer
allow for subsequent sequencing of separated proteins by
conventional methods.
[0062] In another embodiment, the method of fractionation is a 2-D
PAGE, such as that described by Rabilloud et al. (Electrophoresis
15: 1552-1558, 1994). This method includes in-gel sample
rehydration of immobilized pH gradient strips to allow larger
sample volume, followed by isoelectric focusing in the first
dimension and SDS-PAGE in the second dimension. As used herein, the
term "High Capacity Two-Dimensional Polyacrylamide Gel
Electrophoresis" ("HiCap 2-D PAGE") refers to the use of this
method of fractionation on a sample that has been treated to remove
high abundant serum proteins. With the removal of abundant
proteins, a higher amount of low abundant proteins can be
fractionated. For example, HiCap 2-D PAGE of serum permits higher
amounts of low abundant proteins to be loaded following the removal
of albumin and immunoglobulin. HiCap 2-D PAGE also permits larger
sample load due to in-gel sample rehydration (volumes up to 400
.mu.L) . The combination of abundant protein removal by the
collapsible affinity matrix and HiCap 2-D PAGE produces highly
reproducible maps of low abundance serum proteins in liquid sample.
Following isoelectric focusing in the first dimension, SDS-PAGE in
the second dimension and silver stain visualization, computer image
analysis allows detection of very small amounts of protein.
[0063] HiCap 2-D PAGE can be used in the analysis of disease state
serum samples when compared to normal serum. This allows the
identification and characterization of a variety of novel markers
that may have diagnostic or therapeutic utility. The discovery of
novel biochemical serum markers for diagnosis or therapy aids
immensely in the management of many diseases. The advantage of
HiCap 2-D PAGE mapping is that a large number of distinct unknown
serum proteins (.about.4000), from a single patient sample, can be
identified (with respect to isoelectric point and molecular weight)
and quantitated at the same time. With the appropriate patient
sample set, concentrations of unknown proteins in serum can be
correlated to other known clinical measures, such as bone mineral
density, and serum and urinary biochemical markers. In this way,
clinical utility of a large number of unknown proteins can be
evaluated simultaneously. 2-D mapping of low abundance serum
proteins requires specific removal of highly abundant proteins,
such as human serum albumin and immunoglobulins.
[0064] For example, HiCap 2-D PAGE can be used for the discovery of
biochemical serum markers for Paget's disease. This method can be
applied to the analysis of thirty samples from healthy
postmenopausal women at baseline and after three months treatment
with an estrogen or selective estrogen receptor modulator. Results
showing considerable differences in the 2-D mapping of several
polypeptides between the treated and untreated patient samples are
described in EXAMPLE XII.
[0065] Kit
[0066] The materials for use in the assay of the invention are
ideally suited for the preparation of a kit. Such a kit may include
two or more containers, such as vials, tubes, and the like. Each of
the containers contains one of the separate elements to be used in
the method. The first container contains a monoclonal antibody
capable of immunoprecipitating albumin from serum, to which is
bound a first member of a high affinity binding pair. Such a
polypeptide affinity reagent may be biotinylated HSA2126NX.012. The
second container contains a second member of a high affinity
binding pair, for example, avidin, streptavidin, or
NeutrAvidin.TM..
[0067] The invention will be further described in the following
EXAMPLES, which do not limit the scope of the invention described
in the claims.
EXAMPLE I
PRODUCTION OF A MONOCLONAL ANTIBODY THAT CAN IMMUNOPRECIPITATE
HUMAN SERUM ALBUMIN
[0068] This EXAMPLE provides a description of how the monoclonal
antibody HSA2126NX.012 was produced. This antibody is unique in
that the antibody can immunoprecipitate albumin from serum. This
means that the antibody recognizes an epitope on the HSA molecule
that is not blocked by the myriad of serum proteins that are known
to bind HSA in serum.
[0069] Immunizations. Three BALB/C mice were immunized with Human
Serum Albumin (fatty acid free, globulin free, Sigma Chemical
Company, St. Louis, Mo.) according to the following protocol. 100
.mu.g antigen emulsified in Complete Freund's Adjuvant was
administered subcutaneously at multiple sites. After 3 weeks, each
mouse was immunized subcutaneously with 100 .mu.g of antigen in
Incomplete Freund's Adjuvant and this schedule continued for three
more intervals. The animals were tested for titer. A final
intravenous boost of immunogen in phosphate buffered saline was
administered to the chosen mouse and three days later the spleen
was harvested.
[0070] Fusion and screening. Mouse splenic cells were fused with
SP2/0 myeloma cells in accordance with standard procedures using
polyethylene glycol (PEG). Hybridoma supernatants were initially
screened by ELISA. Supernatant antibodies showing reactivity
towards the biotinylated HSA antigen (2 .mu.g/mL) on 96-well
streptavidin plates (Labsystems) were detected by anti-mouse
IgG-peroxidase conjugate (Zymed). Anti-HSA secreting hybridomas
were subcloned by limiting dilution. Subcloned hybridomas were
adapted to serum-free conditions (HB-Pro; Irvine Scientific) for
antibody production. HSA specific monoclonal antibodies were
screened a second time for their ability to immunoprecipitate HSA
from human serum. The clones capable of immunoprecipitation of
human serum albumin were subcloned and isotyped (IsoStrip,
Boehringer-Mannheim). The clone, HSA2126NX (IgG2b), was chosen for
continuing studies based on the ability to produce large quantities
of antibody that efficiently immunoprecipitated albumin from human
serum. Further subcloning lead to the choice of HSA2126NX.012.
Primary and secondary seedlots were frozen for production scale
work.
[0071] Screening clones for ability to immunoprecipitate HSA.
Various hybridoma culture supernatants were passed over an HSA
column to affinity purify anti-HSA monoclonal antibodies. Elution
was with 0.1M glycine, pH 3.0. The eluent was concentrated to
100-200 .mu.L for evaluation of HSA immunoprecipitation
capability.
[0072] Human serum, stripped of endogenous immunoglobulin by
previous incubation with protein A and gamma-bind, was incubated
overnight at 4.degree. C. with the various anti-HSA antibodies.
Gamma-bind was added and incubation took place at 4.degree. C. for
3 hours with rotation. The gamma-bind beads, washed two times with
PBS, were then mixed with reducing buffer and boiled. Boiling
removed the bound anti-HSA antibody as well as any human serum
albumin bound by the specific antibody. This material, run on a
sizing gel and stained with Coomaisse blue, displayed either
antibody bands only or antibody plus HSA.
[0073] Antibody Production. Monoclonal antibody HSA2126NX.012 was
produced from hybridoma cultures grown in HB Pro medium (Irvine
Scientific, Irvine, Calif.), that is serum free, contains no
albumin and is low in total protein content (1 .mu.g/mL). Flasks
were incubated at 37.degree. C. with humidity and 5% CO.sub.2. IgG
quantitation of supernatants from actively growing flask cultures
was in the 35-45 .mu.g/mL range. For larger scale production, a
CellMax.RTM. artificial capillary system (Spectrum, Germantown,
Md.) was used. Well-established bioreactor culture yields 1-3 mg
antibody per mL of supernatant. Eighty mL supernatant can be
obtained per week from three harvestings.
[0074] Purification of anti-HSA monoclonal antibody. HSA2126NX.012
(ATCC accession No. HB112464) cell culture supernatant was 0.2
.mu.m filtered and run over a Protein A Sepharose (Pharmacia)
column. The column was washed with 10 column volumes of phosphate
buffered saline (10 mM sodium phosphate, 150 mM sodium chloride, pH
7.0) Bound antibody was eluted with 0.1M glycine, pH 3.0 and
neutralized by the addition of (10% by volume) 1.2M Tris, pH 8.5.
The purified antibody was dialyzed into 50 mM sodium bicarbonate,
pH 8.5. A normal yield was 1 mg of purified antibody per mL of
bioreactor supernatant.
EXAMPLE II
BIOTINYLATIONS
[0075] Purified anti-HSA monoclonal antibody and recombinant
protein A (Scripps Laboratories) were biotinylated in the same
manner. Biotinylation was performed with a 20 fold excess of
sulfosuccinimidyl-6-(biotinamido) hexanoate (Immunopure.RTM.
NHS-LC-Biotin, Pierce Chemical Co., Rockford, Ill.) in 50 mM sodium
bicarbonate buffer, pH 8.5 at a protein concentration of 3-5 mg/mL.
The reaction was carried out for 2 hr at room temperature with
rotation. The labeled protein was dialyzed in 5 mM phosphate, 50 mM
sodium chloride, pH 7.0 overnight at 4.degree. C. and with a total
of two 5 L buffer changes. The biotinylated anti-HSA was
concentrated in a Centricon-30 apparatus (Amicon, Inc., Beverly,
Mass.) to a final concentration of 6-10 mg/mL. The biotinylated
protein A solution was not concentrated.
EXAMPLE III
FORMATION OF "COLLAPSIBLE AFFINITY MATRICES" SPECIFIC FOR HUMAN
SERUM ALBUMIN (HSA) AND SERUM IMMUNOGLOBULIN (Ig)
[0076] This EXAMPLE provides information on the formation of
collapsible affinity matrices specific for human serum albumin and
immunoglobulin.
[0077] In one test, a biotinylated protein A and avidin collapsible
affinity matrix for the removal of serum immunoglobulins was
prepared by combining 0.4-0.6 mg biotinylated protein A and 1.2 mg
avidin (200 mg/mL avidin in deionized water) per 100 .mu.L of human
serum to be treated. This material was vortexed, incubated for 10
minutes (min) and centrifuged at 5000 rpm for one min. The
supernatant was discarded and the biotinylated protein A and avidin
collapsible affinity matrix pellet was recovered.
[0078] In another test, a biotinylated anti-HSA and avidin
collapsible affinity matrix for the removal of human serum albumin
(HSA) was prepared by combining 10 mg biotinylated anti-HSA
monoclonal antibody and 15 mg avidin (200 mg/mL avidin in deionized
water) per 100 .mu.L of human serum to be treated. This material
was vortexed, incubated for 10 min and centrifuged at 5000 rpm for
one min. The supernatant was discarded and the biotinylated
anti-HSA and avidin collapsible affinity matrix pellet was
recovered. The pellet was washed once with 200 mM NaCl, 5 mM Tris,
pH 7.5 to remove any excess avidin.
[0079] Removal of Ig and HSA from a human serum sample. The volume
of human serum to be treated was added to the biotinylated protein
A and avidin pellet, vortexed and incubated 15 min. At this step,
immunoglobulins in the serum sample are bound by the biotinylated
protein A and avidin collapsible affinity matrix. The treated serum
was added to the biotinylated anti-HSA and avidin collapsible
affinity matrix pellet and vortexed. The transfer tube was rinsed
with 200 mM NaCl, 5 mM Tris, pH 7.5 and this wash was added as
well. Incubation was for 1 hr at room temperature with rotation. At
this step, HSA in the serum sample was bound by the biotinylated
anti-HSA and avidin collapsible affinity matrix. Centrifugation was
at 12,000 rpm to allow the collapsible affinity matrices to pellet,
thus depleting the serum sample of both HSA and Ig. The supernatant
was exchanged against deionized water to remove excess salts and
concentrated to less than 100 .mu.L in a Centricon-3 apparatus
(Amicon, Inc.). The treated sample was ready to be fractionated
without interference by the abundant proteins HSA and Ig.
EXAMPLE IV
HiCap 2-D PROCEDURE
[0080] The method of HiCap 2-D PAGE combines the use of a
collapsible affinity matrix to remove high abundant proteins from a
liquid sample with a modified 2-D PAGE procedure as described by
Rabilloud et al., supra. First, the HSA and Ig depleted serum
samples were adjusted to a final volume of 400 .mu.L with
rehydration buffer (8M urea, 4% CHAPS, 0.1% Pharmalytes 3-10, 0.2%
Triton X-100, 0.1% taurodeoxycholate and 10 mM DTT). The entire 400
.mu.L sample was used to rehydrate a 3 mm.times.18 cm immobilized
pH gradient (IPG) strip (3.3% total acrylamide/2.7% piperazine
diacrylyl as crosslinker; Immobiline concentrations as per
published recipes). Rehydration was overnight at room temperature
in a rehydration chamber. For the first dimension, the rehydrated
IPG strips were focused at 15.degree. C. and an upper voltage limit
of 6 kV for greater than 100 kV-hr. The focused IPG strips were
then reduced with DTT and alkylated with iodoacetamide while also
being equilibrated with SDS (equilibration buffer base: 30%
glycerol, 6M urea, 2.5% SDS, 0.15M BisTris, 0.1M HCl and
bromophenol blue). For the second dimension, the equilibrated IPG
strip was sealed to a 3% stacking/14% resolving gel (Prosieve 50;
FMC BioProducts, Rockland, Me.) with dimensions of
20.times.20.times.1.5 cm. Electrophoresis was at 4.degree. C. in
SDS/Tricine buffer until the tracking dye reached the bottom of the
gel. Upon completion of electrophoresis, the PAGE gels were fixed
and silver stained for polypeptide visualization. Dried gels were
scanned, digitized and analyzed using the GELLAB II.sup.+ software
package (Scanalytics; Billerica, Mass.).
EXAMPLE V
STATISTICS ON THE REMOVAL OF Ig AND HSA USING THIS CLARIFICATION
TECHNIQUE
[0081] Twelve serum samples were evaluated for total protein
concentration pre-removal and post-removal of HSA and Ig. The
protein concentrations were assayed using micro BCA (Pierce). The
results from the twelve samples were very consistent and showed a
mean total protein post-treatment=18.5.+-.2.6 mg/mL, compared with
a mean total protein pretreatment=65.3.+-.4.0 mg/mL.
[0082] Therefore, 46.8 mg of immunoglobulin and human serum albumin
was removed with the collapsible affinity matrix clarification
technique. The remaining protein (28% of initial) contains low
abundant serum proteins, that can then be analyzed by further
fractionation methods, for example, 2-D PAGE.
EXAMPLE VI
SAMPLE TREATMENT FOR REMOVAL OF HSA AND Ig
[0083] This EXAMPLE demonstrates a preparation of a serum sample
that is substantial depleted in serum albumin and immunoglobulin.
Human serum samples were treated with a monoclonal antibody
specific for HSA (biotinylated anti-HSA, HSA2126NX.012) and avidin
or Cibacron-blue dye. Both treated samples were subsequently
incubated with gamma-bind protein A to remove immunoglobulin. The
removal of serum albumin and immunoglobulin was done to enable
larger sample loads and higher quality 2-D PAGE gels of human
serum. FIGS. 1 and 2 demonstrate the effectiveness of the specific
removal by the monoclonal antibody and compare this method of
treatment to an alternative method, using immobilized Cibacron-blue
dye. FIG. 1 shows the 2-D maps (performed using standard
ampholine-based IEF) for volume normalized (5 .mu.L) human serum
samples that are: untreated (FIG. 1A); monoclonal treated (FIG. 1B)
and treated with immobilized Cibacron-blue (FIG. 1C).
[0084] The purpose of showing the volume normalized maps is to
demonstrate the increased quality of the gel image by removal of
HSA and Ig. Also, comparing maps 1B and 1C shows the increased
specificity of the monoclonal treatment versus Cibacron-blue. Gel
1B has 1200 polypeptide spots compared to 1000 spots on Gel 1C.
Treating human serum with the monoclonal HSA2126NX.012 resulted in
20% more spots when compared to treatment with Cibacron-blue,
indicating a significant increase in specificity. Another
indication of increased specificity comes from the observation that
Cibacron-blue treatment of serum quantitatively removes the serum
glycoprotein, HC gp-39, (as determined by immunoassay) while
treatment with the HSA2126NX.012 monoclonal results in>75%
recovery. FIG. 2 shows the 2-D maps that are normalized for the
total amount of protein load (250 .mu.g) after: no treatment (FIG.
2A); monoclonal treatment (FIG. 2B) and Cibacron-blue treatment
(FIG. 2C). The total protein normalized 2-D maps reveal the large
increase of information obtained by removing HSA and Ig when the
analytical technique is sensitive to the total protein load. When
HSA and Ig were removed from the sample, the number of polypeptide
spots detected increased approximately two-fold (FIG. 2A versus
FIG. 2B and FIG. 2C).
[0085] From the above results, it becomes clear that the specific
removal of serum albumin and immunoglobulin can greatly enhance the
information obtained from analytical 2-D PAGE gels.
EXAMPLE VII
A COMPARISON OF 2-D ELECTROPHORESIS USING UNTREATED SERUM,
MONOCLONAL ANTI-HSA ANTIBODY TREATMENT, AND CIBACRON-BLUE
TREATMENT, USING VOLUME NORMALIZED SAMPLES
[0086] The purpose of this EXAMPLE is to show the improved
performance of the method of the invention over existing methods.
Human serum samples were treated with a monoclonal antibody
specific for HSA (biotinylated anti-HSA, HSA2126NX.012) and avidin
or Cibacron-blue dye. Both treated samples were subsequently
incubated with gamma-bind protein A to remove the immunoglobulin.
The removal of albumin and immunoglobulin was done to enable larger
loads of the less abundant serum proteins and higher quality 2-D
PAGE gels of human serum.
[0087] FIG. 1 demonstrates the effectiveness of the specific HSA
removal by the monoclonal antibody treatment and compares this
method of treatment to an alternate method, using immobilized
Cibacron-blue dye. FIG. 1 shows the 2-D maps for volume normalized
(5 .mu.L) human serum samples that are: untreated (FIG. 1A);
treated with an anti-HSA monoclonal (FIG. 1B) and treated with
immoblilized Cibacron-blue (FIG. 1C).
[0088] The purpose of showing the volume normalized maps is to
demonstrate the increased quality of the gel image by removal of
HSA and Ig. Also, comparing the maps in gels 1B and 1C shows the
increased specificity of the monoclonal treatment versus
Cibacron-blue. Gel 1B has 1200 polypeptide spots compared to 1000
spots on Gel 1C. Treating human serum with the monoclonal
HSA2126NX.012 resulted in 20% more protein spots when compared to
treatment with Cibacron-blue, indicating a significant increase in
specificity.
[0089] Therefore, this method of the invention allowed the
detection of proteins that would otherwise not have been resolved
using established procedures that nonspecifically bind protein, as
does Cibacron-blue.
EXAMPLE VIII
A COMPARISON OF 2-D ELECTROPHORESIS USING UNTREATED SERUM,
MONOCLONAL ANTI-HSA ANTIBODY TREATMENT, AND CIBACRON-BLUE
TREATMENT, USING SAMPLES NORMALIZED FOR TOTAL PROTEIN LOAD
[0090] Human serum samples were treated with a biotinylated
monoclonal antibody specific for HSA (HSA2126NX.012) and avidin, or
Cibacron-blue dye. Both treated samples were subsequently incubated
with gamma-bind protein A to remove the immunoglobulin. The removal
of albumin and immunoglobulin was done to enable larger loads of
the less abundant serum proteins and higher quality 2-D PAGE gels
of human serum. FIG. 2 demonstrates the effectiveness of the
specific HSA removal by the monoclonal antibody treatment and
compares this method of treatment to an alternate method, using
immobilized Cibacron-blue dye. FIG. 2 shows the 2-D maps that are
normalized for the total amount of protein load (250 .mu.g) after:
no treatment (FIG. 2A), treatment with an anti-HSA monoclonal (FIG.
2B) and Cibacron-blue treatment (FIG. 2C). The total protein
normalized 2-D maps reveal the large increase of information
obtained by removing HSA and Ig when the analytical technique is
sensitive to the total protein load. If the albumin and Ig are
removed from the sample, the number of polypeptide spots detected
increases approximately two-fold (FIG. 2A versus FIG. 2B and FIG.
2C).
[0091] Thus, the specific removal of serum albumin and
immunoglobulin can greatly enhance the information obtained from
analytical 2-D PAGE gels.
EXAMPLE IX
NONSPECIFIC REMOVAL OF GLYCOPROTEIN, HC gp-39 FROM HUMAN SERUM BY
CIBACRON-BLUE, BUT NOT MONOCLONAL ANTIBODY
[0092] Another indication of increased specificity with the
monoclonal antibody method comes from the observation that
Cibacron-blue treatment of serum quantitatively removed the serum
glycoprotein, HC gp-39 (as determined by immunoassay), while
treatment with the anti-HSA specific antibody, HSA2126NX.012,
resulted in>75% recovery. This experiment demonstrates the
advantage of a specific HSA removal method over a non-specific
method.
EXAMPLE X
SPECIFIC HSA REMOVAL BY MONOCLONAL ANTIBODY ALLOWS ANALYSIS OF
HSA-BOUND PROTEINS
[0093] The purpose of this EXAMPLE is to show the usefulness of
specifically immunoprecipitating albumin from serum using the
monoclonal antibody HSA2126NX.012. HSA-associated proteins is
precipitated from serum by the addition of biotinylated
HSA2126NX.012 with streptavidin or avidin.
[0094] The pelleted precipitate, containing HSA, is then boiled and
analyzed by 1-D or 2-D gel electrophoresis, providing valuable
information about HSA-bound proteins. Analysis of the HSA-bound
proteins from individuals with various disease states assists in
the characterization of the diseases.
[0095] The benefits of using the anti-HSA specific monoclonal
antibody, rather than other methods, lie in the anti-HSA specific
monoclonal antibody specificity. This EXAMPLE shows that the
proteins being analyzed are associated with HSA versus being
nonspecifically pulled down or trapped in the void space of a
slurry matrix.
EXAMPLE XI
MONKEY SERUM ALBUMIN REMOVAL USING THE ANTI-HSA MONOCLONAL
ANTIBODY
[0096] The anti-HSA antibody, HSA2126NX.012, was evaluated for the
ability to efficiently immunoprecipitate albumin from monkey serum.
Due to the homology between human and monkey serum albumins, the
monoclonal was able to successfully remove the monkey serum
albumin. This MSA (monkey serum albumin) removal is a beneficial
step in the gel electrophoresis analysis of monkey serum proteins
in various disease models or drug treatment analysis.
[0097] This depletion of albumin can be performed with any number
of mammalian species after the production of a specific monoclonal
antibody that recognizes that particular albumin in the respective
serum.
EXAMPLE XII
CLINICAL RESULTS
[0098] One of the strategies for applying HiCap 2-D PAGE to the
discovery of disease related serum proteins is to perform
exhaustive analysis on patient samples in which large changes in
disease related proteins are expected. For instance, one would
expect bone resorption markers to be greatly amplified in Paget's
disease patients and people suffering from hyperparathyroid. TABLE
1 shows several examples of some polypeptide species that are
up-regulated in a Pagetic sample when compared to an age-matched
normal sample.
1TABLE I Serum Proteins Increased in Concentration in Pagetic
Patient Versus Normal Approximate concentration in Spot ID Pagetic
Sample Fold change from normal 118 200 ng/mL 5 224 100 ng/mL 6 192
300 ng/mL 4 1133 100 ng/mL >5 136 750 ng/mL 10
[0099] A key element in identifying potential disease related
proteins is their correlation with already existing diagnostics.
For example, BMD and NTx measurements could be used (with matching
serum samples) to identify proteins associated with bone
metabolism. This concept is demonstrated with polypeptide Spot ID
118, that follows the disease progression of a Pagetic patient, as
determined by NTx values (FIG. 6A). FIG. 6B shows how the spot ID
118 concentration and NTx concentration in serum correlate with one
another.
EXAMPLE XIII
A COMPARISON OF THE COLLAPSIBLE AFFINITY MATRIX WITH IMMOBILIZED
SEPHAROSE MATRIX FOR SPECIFIC REMOVAL OF HSA AND Ig FROM SERUM
[0100] Two aliquots of a serum sample were treated with our
biotinylated anti-HSA monoclonal antibody HSA2126NX.012 and
biotinylated protein A. The difference between the two samples was
in the removal step. To one aliquot was added a rigid
streptavidin-Sepharose matrix (Ultralink Immobilized Streptavidin
on 3M Emphage Biosupport Medium; Pierce) and to the other sample
was added soluble avidin (Scripps Labs; La Jolla, Calif.) to form a
collapsible matrix. After 1 hr incubation with mixing, the matrices
were separated from the solution by centrifugation. The resulting
solution contained serum proteins, but was highly depleted of HSA
and Ig. This material was analyzed by a modified 2-D PAGE procedure
described by Rabilloud et al., supra.
[0101] Visualization of the gels clearly showed more polypeptides
in the collapsible affinity matrix sample (FIG. 7A) versus the
immobilized matrix sample (FIG. 7B). Analysis of the most clearly
resolved area of the gels using GELLAB II.sup.+ software calculated
164 polypeptide spots for the collapsible affinity matrix versus
108 polypeptide spots for the immobilized matrix. Due to the
microporous nature of the rigid Sepharose beads, that possess an
inherent dead volume, low abundant proteins of interest are
trapped. By contrast, the collapsible, low void-volume, affinity
matrix does not trap low abundant proteins of interest.
[0102] The advantage is that the dead volume will "collapse" upon
centrifugation and hence yield a superior recovery of serum
proteins.
EXAMPLE XIV
HiCap 2-D PAGE ASSAY
[0103] The following HiCap 2-D procedure is a modification of the
method described by Rabilloud et al. (Electrophoresis 15:
1552-1558, 1994).
[0104] 1. Serum samples were treated with the collapsible affinity
matrix (as per EXAMPLE III) for removal of HSA and
immunoglobulin.
[0105] 2. The HSA and Ig depleted serum samples were adjusted to a
final volume of 400 .mu.L with rehydration buffer (8M urea, 4%
CHAPS, 0.1% Pharmalytes 3-10, 0.2% Triton X-100, 0.1%
taurodeoxycholate and 10 mM DTT). The entire 400 .mu.L sample was
used to rehydrate a 3 mm.times.18 cm immobilized pH gradient (IPG)
strip (3.3% total acrylamide/2.7% piperazine diacrylyl as
crosslinker; Immobiline concentrations as per published recipes).
Rehydration was overnight at room temperature in a replica of a
rehydration chamber described by Rabilloud et al.
[0106] 3. For the first dimension, the rehydrated IPG strips were
focused at 15.degree. C. and an upper voltage limit of 6 kV for
greater than 100 kV-hr.
[0107] 4. The focused IPG strips were then reduced with DTT and
alkylated with Iodoacetamide while also being equilibrated with SDS
(Equilibration buffer base: 30% glycerol, 6M urea, 2.5% SDS, 0.15M
BisTris, 0.1M HCl and bromophenol blue).
[0108] 5. For the second dimension, the equilibrated IPG strip was
sealed to a 3% stacking/14% resolving gel (Prosieve 50; FMC
BioProducts, Rockland, Me.) with dimensions of
20.times.20.times.1.5 cm. Electrophoresis was at 4.degree. C. in
SDS/Tricine buffer until the tracking dye reached the bottom of the
gel.
[0109] 6. Upon completion of electrophoresis, the PAGE gels were
fixed and silver stained by the method of Rabilloud
(Electrophoresis 13: 429-439, 1992) for polypeptide
visualization.
[0110] 7. Dried gels were scanned, digitized and analyzed using the
GELLAB II.sup.+ software package (Scanalytics; Billerica,
Mass.).
[0111] 8. Molecular weights and pIs for individual protein spots
were determined by calibration curves generated by using known
serum proteins as internal standards.
[0112] 9. Protein concentration for individual spots was
approximated as follows: the total protein concentration loaded
onto the first dimension (determined by BCA) was divided by the
integrated optical density (OD) of all spots on the gel to give an
average protein concentration per unit OD or Average Staining Unit
(ASU); using the ASU, several spots of different intensities were
selected and used to construct a calibration curve for estimating
the concentration of all protein spots in the gel.
[0113] The subject cultures (for HSA2126NX.012) are deposited under
conditions that assure that access to the cultures will be
available during the pendency of the patent application disclosing
them to one determined by the Commissioner of Patents and
Trademarks to be entitled thereto under 37 C.F.R. .sctn.1.14 and 35
U.S.C. .sctn.122. The deposits are available as required by foreign
patent laws in countries wherein counterparts of the subject
application, or its progeny, are filed. However, it should be
understood that the availability of a deposit does not constitute a
license to practice the subject invention in derogation of patent
rights granted by governmental action.
[0114] Further, the subject culture deposits will be stored and
made available to the public in accord with the provisions of the
Budapest Treaty for the Deposit of Microorganisms, i.e., they will
be stored with all the care necessary to keep them viable and
uncontaminated for a period of at least five years after the most
recent request for the furnishing of a sample of the deposits, and
in any case, for a period of at least 30 (thirty) years after the
date of deposit or for the enforceable life of any patent which may
issue disclosing the cultures plus five years after the last
request for a sample from the deposit. The depositor acknowledges
the duty to replace the deposits should the depository be unable to
furnish a sample when requested, due to the condition of the
deposits. All restrictions on the availability to the public of the
subject culture deposits will be irrevocably removed upon the
granting of a patent disclosing them.
[0115] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention. Other aspects, advantages, and
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *