Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR DETECTING MOLECULAR COMPLEXES
CROSS-REFERENCE TO RELATED APPLICATION
[001] This application claims benefit of provisional application Serial No.
60/813,838,
filed June 15, 2006, entitled A METHOD FOR MAPPING MOLECULAR
COMPLEXES, the entire contents of which are incorporated herein in their
entirety.
FIELD OF THE INVENTION
[002] This invention relates to the field of molecular biology and medicine
and
specifically to assays for detecting molecular interactions.
BACKGROUND OF THE INVENTION
[003] Proteins continuously interact with one another in a highly regulated
fashion to
determine cell fate, such as proliferation, differentiation, or death. Protein-
protein
interactions represent an enormous and diverse group of targets for
therapeutic
intervention. Complicated protein-protein interaction can also be regulated
through post-
translational modification (glycosylation, phosphorylation, etc). In addition
to post-
translational modification and alternative splicing, the formation of protein-
protein
complexes contributes to both the exceedingly large size of and the dizzying
complexity of
the proteome. Not only can proteins exist as single entities whose functions
are
determined by any number of post-translational modification mechanisms, but
proteins
can also exist in any number of complexes with other proteins. Indeed, the
study of
protein-protein interactions has become increasingly important and many
researchers are
devising new ways to study protein-protein interactions and their associated
functions
(Russel, RB et al. 2004. Curr Opin Struct Biol. 14:313-324.; Zhu, H. et al.
2002. Curr
Opin Cell Biol. 14:173-179).
[004] Protein-protein interactions have been implicated in cell signaling
(Gastel, M.
2006. Handb Exp Pharmacol. 172:93-109), and the scientific community has long
been
interested in studying the signaling pathways that control cellular functions.
To this end,
they have devised approaches to study both the genes which code for the
signaling
pathways and the proteins which comprise the signaling pathways. Because of
post
translational modifications, gene function is regulated not only by its
genomic code, but
also by the proteins interacting with the gene product. To further decipher
which proteins
interact to determine functionality of the proteins of interest, a variety of
approaches have
been devised to investigate protein-protein interactions. The most common
methodologies
of examining protein complexes are yeast two- or three- hybrid screens,
affinity tagging
coupled with mass spectrometry, and protein array methodologies (Zhu, H. et
al. 2002.
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Curr Opin Cell Biol. 14:173-179). Although these techniques are useful, there
is currently
no high throughput methodology to study protein-protein interactions.
[005] As protein-protein complexes are involved in all cell signaling
pathways, protein-
protein complexes are also implicated in all phenotypes. Unique protein-
protein
interactions have been discovered to be associated not only with certain organ
systems, but
also with particular diseases, including cancer (Fry, DC et al. 2005. J Mol
Med. 83:955-
963.), CNS diseases (Rabiner, CA et al. 2005. Neuroscientist 11:148-160),
diseases of the
platelet and associated cardiovascular diseases (Macaulay, IC et al. 2005. J
Clin Invest
115:3370-3377), and others (Houtman JD et al. 2005. FEBS 272:5426-5435).
[006] If a protein elicits an antibody-based immune response, antibodies
(immunoglobin
or "Ig") against those proteins can be generated. Such proteins are known as
"self'
proteins or "auto-antigens," and the antibodies generated against auto-
antigens are called
"auto-antibodies." For example, it is known that auto-antigens MUCl and p53
elicit an
auto-immune response, i.e., these proteins elicit the corresponding auto-
antibodies in a
subject (Finn OJ, Jerome KR, Henderson RA, Pecher G, Domenech N, Magarian-
Blander
J, Barratt-Boyes SM, Immunol Rev. 1995 Jun;145:61-89). Such auto-antibodies
may
occur because the auto-antigen is an altered form of a protein, so an
individual mounts a
limited immune response against that auto-antigen. Auto-antigens indicative of
a tumor
are known as tumor-associated antigens or "TAAs." In this instance, it is
known to detect
and measure the presence of the corresponding auto-antibody rather than the
auto-antigen.
This is a way to identify or detect tumor using the immune response, rather
than detecting
a protein secreted from a tumor. Detection of the immune response is believed
to be a
better method than detection of secreted proteins because B-cells will create
many auto-
antibodies and, thus, a high concentration of auto-antibodies circulate in the
bloodstream.
Additionally, because antibodies generally are more stable than secreted
proteins, there is
a consistent concentration of antibodies over a longer period of time. In
addition to tumor
detection, this method could be used to detect an immune response for any auto-
immune
disease, e.g., rheumatoid arthritis.
[007] One known method for detecting auto-antibodies includes creating arrays
of peptides
or proteins (produced using a variety of methods), incubating the arrays with
serum, and
using anti-immunoglobin to detect the presence of immunoglobulin at any of the
protein
spots. This method, however, is dependent on creating a universal form of the
peptide auto-
antigen. That is, recombinant proteins and peptides are spotted on an array
but do not
necessarily carry the same sequence variations or post-translational
modifications as the auto-
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antigen in a sample under examination. Auto-antigens from different
individuals may have
different immunogenic alterations, so the isolation and use of auto-antigen
material from one
individual does not necessarily provide auto-antigen material that is
immunoreactive with
auto-antibody for other individuals. For example, an auto-antigen (e.g.,
protein X) from one
subject may include a mutation that has triggered an auto-immune response. A
comparable
protein X' from another individual may be immunogenic due to a slightly
different mutation.
Thus, an auto-antibody that binds to protein X' will not necessarily bind to
protein X. Using
this prior method, an array including auto-antigens from one individual will
bind auto-
antibodies in only a relatively small subset of the population, e.g., 30% of
the population. As
such, this method can create a significant number of false negatives. Thus,
some researchers
have used arrays with bound auto-antigens that were isolated directly from
individual
samples, but such auto-antigen proteins are difficult to purify completely and
difficult to
confirm in follow-up studies.
[008] An additional complex of interest is the interaction between a protein
and a small
molecule (e.g., a drug). A known method for the detection of drug-protein
interactions
includes mass spectrometry to identify proteins isolated with a drug. Mass
spectrometry can
be effective, but is expensive, low-throughput, and not quantitative. Drug
interactions with
specific proteins can be directly tested with such methods if recombinant
proteins are
available. In many cases, however, proteins may need to be in their native
state or interacting
with certain other factors in order to bind a drug, making the use of
recombinant proteins
ineffective. Furthermore, it is otherwise generally difficult or expensive to
obtain a wide
range of purified proteins.
SUMMARY OF THE INVENTION
[009] The present invention includes various methods for detecting molecular
complexes. These methods are high through-put and are based on the use of
microarray
technology (in particular, using bound anti-protein capture antibodies) to
detect a
molecular complex in its native form.
[0010] More specifically, the present invention includes a method of analyzing
a protein
complexed with another molecule, comprising: providing a first microarray
slide, wherein
the first microarray slide includes a first array that is attached to the
microarray slide and a
second array that is attached to the first microarray slide and which second
array is
separated from the first microarray, wherein the first array includes a first
capture antibody
for specifically binding to a first protein and a second capture antibody for
specifically
binding to a second protein, wherein the second array includes a duplication
of the first
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array; providing a first biological sample that may contain the first and
second proteins in
their native form; incubating an aliquot of the first biological sample on the
first and
second arrays of the first microarray slide to permit any of the first protein
and second
protein in the first biological sample to bind to their respective capture
antibody; washing
off any unbound proteins from the first and second arrays of the first
microarray slide;
providing a first detection antibody that specifically binds to a first
molecule; providing a
second detection antibody that specifically binds to a second molecule;
incubating the first
detection antibody on the first array of the first microarray slide and
incubating the second
detection antibody on the second array of the first microarray slide to permit
the first
detection antibody and the second detection antibody to bind to their
respective molecules;
detecting the presence of any complex including the first molecule in the
first array of the
first microarray slide; detecting the presence of any complex including the
second
molecule in the second array of the first microarray slide; and determining
whether the
first protein is complexed with the second molecule in the first biological
sample and
whether the second protein is complexed with the first molecule in the first
biological
sample.
[0011] The method of the present invention also includes the first molecule or
second
molecule selected from the group consisting of a protein, carbohydrate, lipid,
nucleic acid,
or small molecule; or the first biological sample is serum (e.g., blood) or a
cell lysate from
either cell culture or tissue. Additionally, the first protein and the first
molecule may be
the same and the first capture antibody and the first detection antibody may
be specific to
different epitopes on the first protein. Similarly, the second protein and the
second
molecule may be the same and the second capture antibody and the second
detection
antibody may be specific to different epitopes on the second protein. The
method of the
present invention further comprises determining the quantity in the first
array of any
complex that includes the first molecule, or determining the quantity in the
second array of
any complex that includes the second molecule.
[0012] Various embodiments of the invention include the first detection
antibody tagged
with a first tag and the second detection antibody tagged with a second tag;
and the first
and second detection antibodies are biotinylated. Further, one embodiment of
the
invention includes incubating the first and second arrays with streptavidin-
phycoerythrin
and scanning the first detection antibody and the second detection antibody
for
fluorescence. Other detection methods of the present invention include
scanning the first
detection antibody and the second detection antibody for chemoluminescence or
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calorimetrically detecting the presence of the first detection antibody and
the second
detection antibody.
[0013] Another method of the present invention includes providing a second
microarray
slide that includes the same first array and the same second array as the
first microarray
slide; providing a second biological sample that may contain the first and
second proteins
in their native form; incubating the second biological sample on the first and
second arrays
of the second microarray slide to permit any of the first protein and second
protein in the
second biological sample to bind to their respective capture antibody; washing
off any
unbound proteins from the first and second arrays of the second microarray
slide;
incubating the first detection antibody on the first array of the second
microarray slide and
incubating the second detection antibody on the second array of the second
microarray
slide to permit the first detection antibody and the second detection antibody
to bind to
their respective molecules; detecting the presence of any complex including
the first
molecule in the first array of the second microarray slide; detecting the
presence of any
complex including the second molecule in the second array of the second
microarray slide;
and determining whether the first protein is complexed with the second
molecule in the
second biological sample and whether the second protein is complexed with the
first
molecule in the second biological sample.
[0014] In an additional embodiment the first biological sample originates from
a healthy
person and the second biological sample originates from a diseased person, the
first
biological sample is treated with a drug and the second biological sample is
not treated
with the drug, or the first biological sample is exposed to a hormone and the
second
biological sample is not exposed to the hormone. In each embodiment, any
complexing in
the first biological sample is compared with any complexing in the second
biological
sample.
[0015] Another method of the present invention includes detecting an immune
response in
a subject, comprising: providing a microarray slide, wherein the microarray
slide includes
an antibody array that is attached to the microarray slide, wherein the array
includes a first
capture antibody that specifically binds a first auto-antigen and a second
capture antibody
that specifically binds a second auto-antigen; providing a biological sample
that may
contain the first or second auto-antigens in their native form; incubating the
biological
sample on the array of the microarray slide to permit any of the first and
second auto-
antigens in the biological sample to be captured by their respective capture
antibody;
washing off the microarray slide; providing a detection antibody that
specifically binds to
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an auto-antibody; incubating the detection antibody on the array of the
microarray slide to
permit the detection antibody to bind to any auto-antibody that is complexed
with the first
or second auto-antigens; and detecting the presence of any auto-antibody in
the array of
the microarray slide. Additionally, the subject may be a mammal or, more
specifically, a
human; the first and second auto-antigens may be different isoforms of the
same antigen;
and the first and second auto-antigens may be tumor antigens. In other
embodiments of
the present invention, the method further provides that the presence of any
auto-antibody
that is complexed with the first or second auto-antigens is indicative of an
auto-immune
disease or cancer. In other embodiments of the invention, the first and second
capture
antibodies are specific to different epitopes of an auto-antigen or to
different isoforms of
an auto-antigen.
[0016] Another method of the present invention includes detecting a protein
interaction
with a small molecule, comprising: providing a microarray slide; wherein the
microarray
slide includes an antibody array that is attached to the microarray slide;
wherein the array
includes a first capture antibody that specifically binds a first protein and
a second capture
antibody that specifically binds a second protein; providing a biological
sample that has
been exposed to a small molecule and that may contain the first or second
proteins in their
native form; incubating the biological sample on the array of the microarray
slide to
permit any of the proteins in the biological sample to be captured by their
respective
capture antibody; washing off the microarray slide; providing a detection
antibody that
specifically binds to the small molecule; incubating the detection antibody on
the array of
the microarray slide to permit the detection antibody to bind to any small
molecule that is
complexed with the first or second proteins; and detecting the presence of any
small
molecule in the array of the microarray slide. Additionally, the small
molecule may be a
drug.
[0017] A further method of detecting a protein interaction with a small
molecule includes:
providing a microarray slide, wherein the microarray slide includes an
antibody array that
is attached to the microarray slide and wherein the array includes a first
capture antibody
that specifically binds a first protein and a second capture antibody that
specifically binds
a second protein; providing a biological sample that has been exposed to a
small molecule
that includes a radioactive label, and which biological sample may contain the
first or
second proteins in their native form; incubating the biological sample on the
array of the
microarray slide to permit any of the proteins in the biological sample to be
captured by
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their respective capture antibody; and detecting any radioactivity at each
location of the
antibody array of the microarray slide.
[0018] Another method of detecting a protein interaction with a small molecule
includes:
providing a microarray slide, wherein the microarray slide includes an
antibody array that
is attached to the microarray slide and wherein the array includes a first
capture antibody
that specifically binds a first protein and a second capture antibody that
specifically binds
a second protein; providing a biological sample that has been exposed to a
tagged small
molecule, and which biological sample may contain the first or second proteins
in their
native form; incubating the biological sample on the array of the microarray
slide to
permit any of the proteins in the biological sample to be captured by their
respective
capture antibody; and detecting the tag of the small molecule at each location
of the
antibody array of the microarray slide. In a further embodiment, the tag of
the small
molecule may be a biotin or a FLAG tag.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figures lA and B show antibody-array interaction mapping. Figure lA is
a
schematic representation of an experimental strategy. Figure 1B shows
molecular-level
detail on the detection of hypothetical interactions, and shows an interaction
between
Protein 1 and Protein 3 and detection of Protein 1-Protein 3 complexes.
[0020] Figure 2 shows representative antibody arrays from a pooled serum
sample,
detected on separate arrays using the indicated detection antibodies. Some of
the relevant
capture antibody spots are labeled. Each antibody was spotted in three
adjacent spots, and
a biotin-labeled BSA positive control appears in the lower right of each
array.
[0021] Figures 3A and B show clusters of interaction levels between antibody
targets.
Each cluster shows the results from a set of 47 arrays, comprising 47
different detection
antibodies, using one serum sample pool. For each array, the intensities of
the capture
antibodies were ranked from highest (48) to lowest (1). Each rank was
multiplied by the
rank of the interaction when the capture and detection antibodies were
switched. The
matrix of rank-products was log-transformed, median centered, and clustered.
The results
from two different serum pools are presented, showing by variation and
consistency in the
patterns of interactions.
[0022] Figure 4 shows RT-PCR analysis of the expression levels of 11 genes in
the
pancreatic cancer cell lines. PCR products were analyzed by 1% agarose gel
electrophoresis and visualized by ethidium bromide staining.
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[0023] Figure 5 shows CEACAM6 co-immunoprecipitates with IL-lbeta in the cell
culture system. CEACAM6 was released from BxPC3 cell surface by phospholipase
C
digestion. The soluble CEACAM6 was mixed with conditioned media collected from
different cell lines and immunoprecipitated with anti-CEACAM6 antibody. The
immuno-
complexes were separated by SDS-PAGE, transferred to PVDF membrane, and
blotted
with anti-CEACAM6 or anti-IL-lbeta antibodies.
[0024] Figures 6A-C show protein and glycan detection in cell culture media
using
antibody arrays. Figure 6A shows detection of cell culture media on antibody
arrays using
CA 19-9 (left) and IL-8 (right) antibodies. Some of the capture antibodies are
labeled.
Figure 6B shows detection of cell culture media from six different cell lines
using a
MUCl antibody. Figure 6C shows RT-PCR levels of the MUCl transcript in
pancreatic
cancer cell lines.
[0025] Figure 7 shows the preparation of antibody arrays. Antibody arrays are
printed
onto microscope slides: 48 arrays on each slide (only 9 arrays are shown),
with 48
antibodies in each array.
[0026] Figure 8 shows the application of a serum sample to the arrays.
Hypothetical
complexing for proteins 1, 2, and 3 is shown, which bind proteins to the three
antibodies.
[0027] Figure 9 shows probing of the arrays. The first three arrays are probed
with
antibodies targeting proteins 1, 2, and 3, respectively.
[0028] Figure 10 shows detecting bound antibody. The amount of antibody bound
at each
spot in each array is detected using the streptavidin-phycoerythrin reagent,
followed by
scanning for fluorescence.
[0029] Figure 11 shows binding profiles of three detection antibodies. The
fluorescence
intensities (y-axes) at each antibody spot (x-axes) are shown for the PSA, HC-
II, and
protein S detection antibodies. All three detection antibodies show signal at
the PSA, HC-
II, and protein S capture antibodies, indicating a possible complex between
these three
proteins.
[0030] Figure 12 shows an interaction cluster. For each detection antibody,
the level of
binding at each capture was ranked (48=highest binding, 1=lowest). Each of
those ranks
was multiplied by the rank of the reverse sandwich - when the capture and
detection
antibodies were reversed. Therefore, interactions that were high in both
directions had
high scores. The scores were logged, median centered, and clustered.
[0031] Figure 13 show a network of interactions found in the serum sample.
Arrows
indicate binding between proteins.
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[0032] Figure 14 shows validation of complexes using protein arrays. Purified
proteins are
spotted onto slides and incubated with other proteins. Protein-protein
interactions are
detected using antibodies targeting the protein that was incubated.
[0033] Figure 15 shows binding levels of incubated PSA (solid bars) or TBS
buffer (white
bars, negative control) at various proteins (x-axis). PSA bound to HC-II, but
not the other
proteins.
[0034] Figure 16 shows HC-II bound to PSA but not the other proteins.
[0035] Figure 17 shows protein S bound to HC-II but not the other proteins.
[0036] Figure 18 shows a PSA-heparin cofactor 11-protein S complex model.
Heparin
cofactor II is in the core of the complex. As to the PSA and heparin cofactor
II
interaction: PSA is a serine protease, while heparin cofactor II is a serine
protease inhibitor
(Serpin), which binds to the serine protease covalently. As to the heparin
cofactor II and
protein S interaction: heparin cofactor binds to sugar groups, while the
protein S is a
glycoprotein, the binding may occur through the heparin cofactor II sugar
binding domain.
[0037] Figure 19 shows an IP-westem blot validation of the complex. Serum was
immuno-precipitated with different antibodies, indicated in the different
lanes. The
precipitates were separated by gel electrophoresis, blotted onto
nitrocellulose, and protein
with anti-HC-II. HC-II was detected in its own IP and in the IPs from PSA and
protein S,
indicating it was pulled down with PSA and protein S.
[0038] Figure 20 is a graph showing changes in CRP-kininogen complexing and
CRP-
bradykinin complexing in induced myocardial ischemia over time.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] The preferred embodiments of the present invention may be understood
more
readily by reference to the following detailed description of preferred
embodiments and
Examples included hereafter.
[0040] Definitions
[0041] As used in the present application, "a" can mean one or more, depending
on the
context with which is it used.
[0042] "Antibody" refers to a polypeptide comprising a framework region from
an
immunoglobulin gene or fragments thereof that specifically binds and
recognizes an
antigen. The term also includes genetically engineered forms such as chimeric
antibodies
(e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g.,
bispecific
antibodies). A "chimeric antibody" is an antibody molecule in which (a) the
constant
region, or a portion thereof, is altered, replaced or exchanged so that the
antigen binding
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site (variable region) is linked to a constant region of a different or
altered class, effector
function and/or species, or an entirely different molecule which confers new
properties to
the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug,
etc.; or (b)
the variable region, or a portion thereof, is altered, replaced or exchanged
with a variable
region having a different or altered antigen specificity. The recognized
immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant
region
genes, as well as the myriad immunoglobulin variable region genes. Light
chains are
classified as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha,
delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM,
IgA, IgD
and IgE, respectively. Typically, the antigen-binding region of an antibody
will be most
critical in specificity and affinity of binding. An exemplary immunoglobulin
(antibody)
structural unit comprises a tetramer. Each tetramer is composed of two
identical pairs of
polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy"
chain
(about 50-70 kD). The N-terminus of each chain defines a variable region of
about 100 to
110 or more amino acids primarily responsible for antigen recognition. The
terms variable
light chain (VL) and variable heavy chain (VH) refer to these light
and heavy
chains respectively. The term "antibody" also includes antigen binding forms
of
antibodies, including fragments with antigen-binding capability (e.g., Fab',
F(ab')2, Fab, Fv
and rIgG) and recombinant single chain Fv fragments (scFv). Antibodies exist,
for
example, as intact immunoglobulins or as a number of well-characterized
fragments
produced by digestion with various peptidases. Thus, for example, pepsin
digests an
antibody below the disulfide linkages in the hinge region to produce
F(ab)'2, a dimer
of Fab which itself is a light chain joined to VH-CHl by a disulfide
bond. The
F(ab)'2 may be reduced under mild conditions to break the disulfide
linkage in the
hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
The Fab'
monomer is essentially Fab with part of the hinge region (see Fundamental
Immunology
(Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms
of the
digestion of an intact antibody, one of skill will appreciate that such
fragments may be
synthesized de novo either chemically or by using recombinant DNA methodology.
Thus,
the term antibody, as used herein, also includes antibody fragments either
produced by the
modification of whole antibodies, or those synthesized de novo using
recombinant DNA
methodologies (e.g., single chain Fv) or those identified using phage display
libraries (see,
e.g., McCafferty et al., Nature 348:552-554 (1990)).
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[0043] "Biological sample" means any fluid or other material derived from the
body of a
normal or diseased subject, such as blood, serum, plasma, lymph, urine,
saliva, tears,
cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus, sputum,
stool, urine, and
the like. Also included within the meaning of the term "biological sample" is
an organ or
tissue extract, cultured cells (e.g., primary cultures, explants, and
transformed cells), and
culture fluid in which any cells or tissue preparation from a subject has been
incubated. A
biological sample is typically obtained from a eukaryotic organism, most
preferably a
mammal such as a primate (e.g., chimpanzee or human), cow, dog, cat, rodent
(e.g.,
guinea pig, rat, mouse), rabbit, bird, reptile, or fish.
[0044] The term "cell" is used in its usual biological sense, and does not
refer to an entire
multicellular organism. The cell, for example, can be in vitro, e.g., in cell
culture, or
present in a multicellular organism, including, e.g., birds, plants and
mammals such as
humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be
prokaryotic
(e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
[0045] "Protein", "polypeptide", or "peptide" refer to a polymer in which the
monomers
are amino acids and are joined together through amide bonds, alternatively
referred to as a
polypeptide. When the amino acids are .alpha.-amino acids, either the L-
optical isomer or
the D-optical isomer can be used.
[0046] The term "specific" or "specifically" refers to a binding reaction
which is
determinative of the presence of the protein in the presence of a
heterogeneous population
of proteins and/or other biomolecules. For example, under designated
conditions, a
specified antibody preferentially binds to a particular protein and does not
bind in a
significant amount to other proteins present in a sample.
[0047] The term "tumor cell" refers to a cancerous, pre-cancerous or
transformed cell,
either in vivo, ex vivo, and in tissue culture, that has spontaneous or
induced phenotypic
changes that do not necessarily involve the uptake of new genetic material.
Although
transformation can arise from infection with a transforming virus and
incorporation of new
genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise
spontaneously
or following exposure to a carcinogen, thereby mutating an endogenous gene.
The term
"tumor" includes at least one tumor cell.
[0048] All references, patents, patent publications, articles, and databases,
referred to in
this application are incorporated-by-reference in their entirety, as if each
were specifically
and individually incorporated herein by reference.
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[0049] The present invention utilizes antibody microarray technology.
Microarrays are
orderly arrangements of spatially resolved samples or probes (in the present
invention, the
probes are antibodies of known specificity to a particular protein).
[0050] The underlying concept of antibody microarray depends on binding
between
proteins and antibodies specific to proteins. Microarray technology adds
automation to the
process of resolving proteins of particular identity present in an analyte
sample by
labeling, preferably with fluorescent labels and subsequent binding to a
specific antibody
immobilized to a solid support in microarray format. An experiment with a
single
antibody microarray chip is highly throughput, i.e., the chip can provide
simultaneous
information on protein levels of many genes. Antibody microarray experiments
employ
common solid supports such as glass slides, upon which antibodies are
deposited at
specific locations (addresses).
[0051] Antibody microarray analysis generally involves injecting a
fluorescently tagged
sample of proteins into a chamber on a microarray slide to bind with
antibodies having
specific affinity for those proteins; laser excitation at the interface of the
array surface and
the tagged sample; collection of fluorescence emissions by a lens; optical
filtration of the
fluorescence emissions; fluorescence detection; and quantification of
intensity.
[0052] Antibodies used in connection with the present invention are
commercially
available or may be synthesized by standard methods known in the art. For
preparation of
antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many
techniques
known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497
(1975);
Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss,-Inc. (1985); Coligan, Current
Protocols in
Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and
Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The
genes
encoding the heavy and light chains of an antibody of interest can be cloned
from a cell,
e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma
and used
to produce a recombinant monoclonal antibody. Gene libraries encoding heavy
and light
chains of monoclonal antibodies can also be made from hybridoma or plasma
cells.
Random combinations of the heavy and light chain gene products generate a
large pool of
antibodies with different antigenic specificity (see, e.g., Kuby, Immunology
(3rd ed.
1997)). Techniques for the production of single chain antibodies or
recombinant
antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted
to produce
antibodies to polypeptides. Also, transgenic mice, or other organisms such as
other
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WO 2007/147141 PCT/US2007/071382
mammals, may be used to express humanized or human antibodies (see, e.g., U.S.
Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks
et al.,
Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994);
Morrison,
Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51
(1996);
Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern.
Rev.
Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used
to
identify antibodies and heteromeric Fab fragments that specifically bind to
selected
antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et
al.,
Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific,
i.e., able to
recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al.,
EMBO J.
10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)).
Antibodies can also be heteroconjugates, e.g., two covalently joined
antibodies, or
immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO 92/200373;
and EP
03089).
[0053] Preferably, antibodies employed to practice the present invention bind
to a selected
target antigen with an affinity (association constant) of greater than or
equal to 107 M-i.
When an antibody is referred to as specific for a particular antigen, it means
that the
binding reaction is determinative of the presence of the antigen in a
heterogeneous
population of proteins and other biologics. Thus, under suitable conditions,
the specified
antibodies bind to a particular protein sequences at least two times the
background and
more typically more than 10 to 100 times the background.
[0054] One method of the present invention is shown in Figure 1. Multiple
antibody
arrays are prepared, each array containing multiple antibodies targeting
proteins that
potentially interact. Figure 1 shows 48 antibody microarrays printed on one
microscope
slide. The center-to-center spacing of the arrays is 4.5 mm (same as the wells
of a 384-
well microtiter plate), and each array contains 48 different antibodies
spotted in triplicate.
The antibodies are printed at a concentration of 0.5 mg/ml in a phosphate-
buffered saline
(PBS) buffer. Lower concentrations of antibodies, down to about 0.1 mg/ml can
also work
if the antibody is high affinity, and concentrations up to about 1 mg/ml would
also work.
A hydrophobic wax line is printed around the borders of each array, so that a
different
sample can be placed on each array without cross-contamination between arrays.
[0055] Because the present invention uses an antibody microarray, it is high
throughput,
allowing for the analyses of multiple protein complexes simultaneously. A
native, non-
denatured biological sample, such as a serum sample, is incubated on each
array (a portion
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of the same sample is used on each array), e.g., 3 microliters of sample per
array. Lysates
from cultured or fresh tissue could be used, or bodily fluids such as blood,
urine or
cerebral spinal fluid would also be applicable. The sample is prepared so that
the proteins
are not denatured and the protein-protein interactions are not greatly
disrupted. After
washing away unbound proteins, each array is probed with a different
biotinylated
detection antibody, andeach detection antibody matched to one of the capture
antibodies
on the arrays. That is, each array is incubated with one detection antibody.
For example,
a first detection antibody is incubated with a first copy of the antibody
microarray, a
second detection antibody is incubated with a second copy of the antibody
microarray, and
a third detection antibody is incubated with a third copy of the antibody
microarray.
Because a protein can complex with a molecule other than another protein, a
detection
antibody also may be used to target a molecule other than a protein, e.g., a
carbohydrate,
lipid, nucleic acid, or small molecule (e.g., a drug) can be analyzed
according to the
methods of the present invention.
[0056] Each detection antibody binds wherever its targeted protein was
retained, whether
at the corresponding capture antibody that targets the same protein, or at
another antibody
that captured a protein in complex with the targeted protein targeted. The
detection
antibody could target an epitope of the protein that is different than the
epitope targeted by
the capture antibody.
[0057] The arrays can be incubated with streptavidin-phycoerythrin and scanned
for
fluorescence to detect the detection antibody and thereby determine that the
target of the
detection antibody is complexed with the protein captured by the capture
antibody.
Moreover, the level of such complexing can be determined by measuring the
amount of
fluorescence of the detection antibody. One could also use another affinity
tag besides
biotin, such as digoxigenin, or a different fluorophore besides phycoerythrin,
such as Cy3
or Cy5. Alternately, other methods besides fluorescence could be used to
detect the tagged
and bound antibodies. For example, horseradish peroxidase or alkaline
phosphatase could
be linked to streptavidin, and the addition of the appropriate substrate for
those enzymes
would lead to the generation of a detectable signal, as is commonly used for
ELISA
assays. In this way, one can observe all potential protein-protein
interactions within a
group of proteins. Since it is a native sample, multi-protein complexes may
exist, and
some of the observed interactions actually may occur through other proteins.
[0058] In one embodiment, if a protein 1 is complexed with proteins 7, 10, and
12,
detection antibody 1 binds to the molecule captured by capture antibodies 1,
7, 10, and 12.
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Likewise, detection antibodies 7, 10, and 12 will bind to the proteins bound
to capture
antibody 1.
[0059] The method of the present invention also can reveal networks of
multiple protein
complexes, e.g., protein 1 might be complexed with proteins 3, 5, and 7, but
also might be
complexed with protein 12. In this instance, using the methods of the present
invention,
one can detect protein 12 binding with protein 1 at capture antibody 1,
protein 1 binding
with protein 12 at capture antibody 12, but protein 12 will not be detected as
binding to
proteins 3, 5, and 7 (protein 12 will only be detected as binding to protein 1
at capture
antibodies 1 and 12). Molecular complexes formed from many proteins can be
examined
with the present method, i.e., the detection method is not limited to
detecting binary
interactions.
[0060] Using the method of the present invention, changes in protein complexes
under
various conditions can be examined. These analyses may involve examining one
biological sample on one microarray slide according to the method described
hereinabove,
and examining a different biological sample on a second microarray slide
according to the
methods described hereinabove. The complexing revealed in the two samples then
can be
compared. Tested conditions might be in cell culture, by treatment with a
drug, hormone,
or growth factor. Further, using the present invention, changes in protein
complexes can
be examined as between healthy persons and persons having a disease, such as
cancer.
[0061] The invention also would be useful to study interactions within a
family of
proteins. For example, one could study, using the present method, coagulation
pathways
relating to blood disorders (such as hemophilia, bleeding disorders, and
coagulation
disorders), because these processes are regulated by complex networks of
protein-protein
interactions in the blood. The ability to examine families could yield new
interactions
related to disease.
[0062] In addition to using the methods of the present invention to detect the
presence of
protein complexes, the levels of the complexes also can be quantitated.
[0063] Another method of the present invention includes the detection of auto-
antigen
interactions with auto-antibodies (auto-immune complexes). In this method, a
biological
sample is incubated on an array of capture antibodies targeting potential auto-
antigens.
After the auto-antigens have bound the corresponding capture antibodies, the
array is
washed and probed with a labeled anti-immunoglobulin detection antibody. This
anti-Ig
detection antibody is used to detect whether any auto-antibodies are complexed
with any
of the auto-antigens that have been captured by the capture antibodies. This
method
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depends upon those auto-antigen/auto-antibody complexes that remain complexed
in a
biological sample. When the biological sample is from an individual, the
individual's own
auto-antigens are used as the basis to detect (and quantify) the level of auto-
antibodies
present in the biological sample. In one embodiment of the present method, for
each auto-
antigen of interest, capture antibodies are prepared for several isoforms of
each auto-
antigen. These isoforms could be splice variants, cleavage variants, or
proteins or peptides
resulting from various post-translational modifications of the auto-antigen.
By using many
different forms of capture antibody against one auto-antigen, one could
determine particular
forms of the auto-antigen that are more immunogenic. In another embodiment,
one could
utilize multiple captive antibodies against the same auto-antigen, each
capture antibody
targeting a different epitope of the auto-antigen, to determine whether
certain epitopes are
present at a higher level in certain conditions, such as cancer. An array
including the
capture antibodies for multiple epitopes of an auto-antigen is highly likely
to capture an
auto-antigen (and consequently any auto-antigen/auto-antibody complexes) in a
large
cross-section of the population. This method provides a significant advance
over known
methods which utilize a common or universal auto-antigen to attempt to detect
auto-
antibodies in biological samples.
[0064] Using the present method, one could use ten captive antibodies against
a certain
protein, and only one of them may bind to a region of the auto-antigen that
was mutated in
cancer (not previously known). By looking at the binding of all combinations
of capture
and detection antibodies for detecting the molecule in both healthy and cancer
samples,
one could observe that one particular combination using the antibody in
question showed
much different binding in the cancer samples than in the healthy samples. From
that it
could be concluded that this antibody must bind a cancer-associated epitope.
[0065] Another method of the present invention includes probing capture
antibody arrays
with affinity reagents to target other types of molecules. These methods can
detect
interactions between a protein (or set of proteins) and a small molecule,
e.g., a drug. More
specifically, this method includes providing an affinity reagent (e.g., an
antibody, single-
chain antibody, etc) against the drug, peptide, or small molecule of interest.
In this
method, for example, a biological sample is treated with a drug to be studied.
Proteins
from the sample are incubated on an array of antibodies that target various
proteins that
may interact with the drug. The array is then washed and probed with an
affinity reagent
that targets the drug. The array location of binding of that affinity reagent
reveals the
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proteins with which the drug interacts. This method could be useful for
optimizing doses,
or for biological studies.
[0066] In the absence of a suitable affinity reagent, a protein-drug complex
can be
detected by tagging the drug prior to treatment. For example, the drug could
be tagged
with biotin and then streptadavin attached to a flourophore could be used to
detect the
drug. Additionally, a peptide tag, such as a FLAG (Sigma) tag also could be
used, or the
drug could be synthesized to include a radioactive label.
[0067] The novel detection methods of the present invention are high
throughput and can be
used to quantitatively measure changes over conditions, or in many different
samples. They
could be useful in disease monitoring or diagnosis, or to determine whether a
drug is
efficacious. For example, if a drug was expected to prevent signaling through
a certain
pathway which was characterized by protein A and protein B forming a complex,
one could
examine biological samples over the course of treatment with the drug, or at
different doses,
and determine how the complexing changes at different times (see Example 5
below) or with
different doses.
[0068] Example 1: Analysis of Pancreatic Cancer Samples Using the Method of
the
Invention
[0069] The present inventive method was applied to the study of 12 different
samples.
Each sample was a pool of serum samples from 26-30 different patients. Four
pools were
from pancreatic cancer patients, four were from patients with benign
pancreatic disease,
and four were from healthy controls. The use of pools allowed a view of
averages in the
population without running dozens of samples, which was impractical at this
early stage of
development. Each of the 12 samples was applied to all of the 48 arrays on one
slide, and
the 48 arrays were each detected with a different detection antibody (as in
Figure 1).
Representative images from one of the slides are shown in Figure 2. Some of
the detection
antibodies bound only at the location of their corresponding capture antibody,
such as anti-
kininogen. Other detection antibodies bound at the location of not only their
corresponding capture antibody, but and also at the location of several other
capture
antibodies, indicating protein-protein interactions between the target of the
detection
antibody and the target of the other capture antibodies. Some of these
observed protein-
protein interactions were previously known, which supports the validity of the
present
method. For example, haptoglobin was shown to strongly interact with
hemoglobin, and
vice versa, which is a known interaction. Platelet factor 4 (PF4) is known to
interact with
IL-8 (56), which was seen on the arrays. The CA 19-9 antibody bound at
glycoproteins
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known to carry that epitope, such as MUCl, CEACAM6, and CA 125. Other
potential
interactions had not been seen before and thus can be further studied.
[0070] Example 2: Validation of Protein-Protein Interactions
[0071] Newly-observed interactions could be validated using another method,
since non-
specific cross-reactivity of an antibody could give rise to false-positive
results. In one
respect, the use of an array contains many internal negative controls. The
binding of a
detection antibody to its proper target without binding to many of the other
antibodies on
array gives some indication that the antibody is specific. Further, if an
interaction is
observed using either antibody in a pair as the capture or detection antibody,
or if more
than one capture or detection antibody give the same result, the interaction
is more likely
to be real. Western blots on serum samples are useful to show a general level
of cross
reactivity of an antibody. Clean, single bands in the western blot of
biological samples
indicate a detection antibody has low general cross-reactivity. Protein-
protein interactions
could be validated by binding assays between purified proteins, by
immunoprecipitation-
western blot, by immunoprecipitation-mass spectrometry, or by the use of
additional,
different antibodies targeting the same proteins. All these methods have been
used to
validate some of the newly-observed interactions described above.
[0072] Immunoprecipitation/mass spectrometry was used to validate a CRP-
kininogen
interaction. Biotinylated anti-CRP was incubated with a serum pool and
precipitated using
streptavidin-coated beads. The eluent from those beads was analyzed using mass
spectrometry, and one of the top identifications after CRP was kininogen.
Negative control
beads, using normal mouse IgG instead of anti-CRP, showed no kininogen in the
identifications. Both the CRP and kininogen antibodies showed single bands in
western
blots of serum samples (not shown).
[0073] Another newly-observed interaction was validated using protein arrays.
Strong
interactions were seen between PSA, heparin cofactor II, and protein S
(Figures 11, 15-
18). Recombinant versions of these proteins were obtained and printed in
microarrays,
along with some negative control proteins. The individual proteins were
incubated on the
arrays, and the binding level of the proteins to each spotted protein was
detected using a
biotinylated antibody followed by streptavidin-phycoerythrin. The PSA and the
heparin
cofactor II bound each other, but the protein S only bound heparin cofactor
II. This
validated the direct interaction between PSA and heparin cofactor II and also
indicated
that the observed interaction between protein S and PSA may be mediated
through heparin
cofactor II.
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[0074] Another interaction, between IL-lbeta and CEACAM6, was validated by
observing consistent results on the arrays with two different IL-lbeta
antibodies and by
immunoprecipitation-western blot (Figure 5). These validation studies show
that the
present invention is useful for discovering new protein-protein interactions.
[0075] Example 3: Interaction Clustering
[0076] Some of the observed interactions seemed to take place among clusters
of proteins,
in which all the proteins interacted with each other. The inventor used a
cluster method to
better visualize the grouping of interactions among the set of antibodies. An
interaction
between two proteins is measured twice in this method: once using one of the
antibodies
from the pair as the detection antibody, and again using the other antibody
from the pair as
the detection antibody. The strongest interactions will show good signal using
either
antibody as the detection antibody. Therefore, the level of interaction
between two
proteins was scored by multiplying the two types of measurements. All of the
interaction
scores were clustered (Figures 3A and 3B) to look at the relative strengths of
interactions
as well as the higher patterns of interactions. The inventor made a cluster of
all 12 serum
pools, of which two representatives are shown in Figures 3A and 3B. The values
on the
diagonal are the sandwich assays, measuring the level of a single protein, and
the clusters
are symmetric about the diagonal. A fair amount of diversity was seen in the
cluster
patterns between the 12 serum pools. Some showed tight clusters of
interactions (Figure
3A), and others had more diffuse interactions (Figure 3B). Interactions among
the group of
proteins identified by arrows in Figures 3A and 3B were regularly seen. The
cluster in
Figure 3A indicates that all members of that cluster interacted with all the
other members.
The group is less tight in Figure 3B, but some level of interaction is still
seen among the
members.
[0077] Example 4: Cell Culture Studies
[0078] Cell culture experiments were designed to more thoroughly study the
interactions
among IL-lbeta, CEACAM6, MUCl, tenascin C, CRP, 90k, and CA 19-9-containing
proteins. Having a cell culture system to study these interactions is useful
since it is a less
complex system than serum and because the expression of individual proteins
can be
manipulated. Many different pancreatic cancer cell lines were tested for their
expression of
these proteins. The cell lines tested are shown in Table 1.
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[0079] Table 1
Cell line Morphology Derived from metastatic Tumorogenic
site
Hs 766T Epithelial Lymph node Yes, in immunosuppressed
mice
HPAF-11 Epithelial - Yes, in athymic mice which
resemble the original tumor
PANC-1 Epithelial - The cells will grow in soft
agar
SW1990 Epithelial Spleen Yes, forms tumors in nude
mice
BxPC-3 Epithelial - Yes, tumor developed
within 21 days at 100%
frequency in nude mice
Capan-2 Polygonal - Yes, in nude mice; forms
well differentiated
adenocarcinoma
CFPAC-1 Epithelial - Yes, in nude mice
Capan-1 Epithelial Liver Yes, in nude mice; forms
adenocarcinoma
Su.86.86 Epithelial Liver Yes, in nude mice
MPanc-96 Epithelial - Yes, in SCID mice
HPAC Epithelial - Yes, the cells form tumors
in athymic nude mice
AsPC-2 Epithelial - ?
L3-3 Epithelial - Derived from COL0357,
highly metastatic
MIAPACA Epithelial - ?
[0080] Most of the cell lines tested are highly tumorigenic, and some were
derived from
metastases. Total RNA was collected from each, and first strand cDNAs were
synthesized.
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PCR primers were designed to probe the expression of the 11 different genes.
The primers
were designed to encompass an intron, so that amplification from contaminating
genomic
DNA would produce a product of the wrong size. Figure 4 shows the expression
levels of
the tested genes. Some were expressed in all cell lines, and others were more
variable.
This information should allow for identification of the cell culture media
which contains
certain groups of proteins, so that the interactions between those proteins
can be further
studied.
[0081] The protein CEACAM6 is normally membrane bound via a GPI anchor. In
order to
examine the interactions in the media, phospholipase C was used to cleave the
GPI anchor
at the juxta-membrane position and release it into the media. The cleavage
took place after
the normal media had been rinsed away. The CEACAM6 preparation was mixed with
the
media from several cells lines that either did or did not express IL-lbeta.
After the media
were mixed, CEACAM6 was immunoprecipitated. The eluent was probed by western
blot
for the presence of both CEACAM6 and IL-lbeta, which were predicted to
interact by the
present invention. CEACAM6 protein was found in all media, and the media that
was not
spiked with the CEACAM6 preparation showed very minimal levels, meaning that
some
CEACAM6 normally escapes into the media. The detection with anti-IL-lbeta (17
kD)
showed that is co-immunoprecipitated with CEACAM6 only when using the Hs766T
and
Su.86.86 cell lines. Those cell lines express high IL-lbeta (Figure 4), and
none of the cell
lines that showed low IL-lbeta showed co-IP in Figure 5. The BxPC3 cell line
expressed
high IL-lbeta showed no band in the co-IP, perhaps because something besides
the
presence of those two proteins regulated their interaction. This validates the
interaction
between IL-lbeta and CEACAM6 and also establishes cell culture models for
studying
interactions among the group of proteins observed in Figure 3.
[0082] Also, the detection of proteins expressed (in Figure 4) in the media of
these
cultures was tested. Several of the media were incubated on antibody arrays
and probed
with different detection antibodies. Figure 6A shows the detection of many of
the
glycoproteins in the media using the CA 19-9 detection antibody, and also
shows that
these glycoproteins bear the sialyl LewisA epitope in that cell culture. This
set of
experiments did not make use of the same pair of IL-lbeta antibodies used in
the
experiments of Figures 2 and 3. However, the ability to specifically detect
cytokines in
cell culture media was confirmed, as shown for IL-8. The abundance of MUC 1 in
the
media (Figure 6B) was closely correlated with the MUCl transcript levels
(Figure 6C).
For example, the cell line Su 86.86 showed no expression both in the media and
at the
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RNA level, and the cell line Capan-2 showed high levels in both. So, for MUCl,
media
levels could be predicted based on the expression levels. Another interesting
result from
this experiment was the high binding of the anti-MUCl antibody at the anti-
CA19-9
capture antibody in the media from the Capan-2 and BxPC3 cell lines but in
none others.
Therefore a proportionally higher level of the CA19-9 epitope is present on
MUCl in
those two cell lines. The identification cell lines with secreted MUC 1
bearing either high
or low CA 19-9 epitopes will be useful to study the functional consequences of
the
presence of that epitope.
[0083] Example 5: Testing the Time Dependence of C-Reactive Protein Levels
Over Time
[0084] Methods of the present invention have been used to test the time-
dependence of C-
reactive protein (CRP) over time. Specifically, a CRP-kininogen complex has
been
elucidated and a CRP-bradykinin complex also has been determined using the
present
invention. See the attached Figures.
[0085] CRP is known to be elevated in association with injury and infection
(acute-phase
reactin), it functions in innate immunity, and is a significant risk factor
for heart disease.
Kininogen circulates in complex with pre-kallikrein and factor XI; is
activated after injury,
coagulation; when cleaved by kallikrein, it releases the peptide bradykinin,
which is
involved in vascular muscular relaxation, edema, and pain; and is rapidly
cleaved by
circulating peptidases. Cleaved kininogen chains are involved in protease
inhibition and
angiogenesis inhibition. Recent reports also suggest the involvement of CRP in
bradykinin-related pathologies. Angioedema in patients treated with ACE-
inhibitor only
occurs when CRP is high. In CRP-transgenic mice, the effects of angiotensity
and
giotensin are greatly enhanced when CRP is high. CRP in kininogen can bind the
same
receptor on endothelial cells. CRP down-regulates eNOS, and bradykinin up-
regulates
eNOS.
[0086] The present invention was used to test the time-dependence of CRP-
interactions in
induced myocardial ischemia. Myocardial ischemia was induced in five patients
(three
samples each) by inflating a balloon in the left interior descending (LAD)
coronary artery,
thus occluding blood flow. Peripheral venous blood sampling was performed on
the
patients at baseline (before myocardial ischemia) and ten minutes and twenty-
four hours
after the myocardial ischemic event. Using the present method, it was
determined that
there is a negative correlation between CRP-bradykinin complexing and CRP-
kininogen
complexing over time (Figure 20).
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