Note: Descriptions are shown in the official language in which they were submitted.
WO 2016/003479 PCT/US2014/049038
SPECIFIC BIOMARKER SET FOR NON-INVASIVE DIAGNOSIS OF LIVER CANCER
Copyright Notice/Permission
A portion of the disclosure of this patent document contains material which is
subject to
copyright protection. The copyright owner has no objection to the facsimile
reproduction by
anyone of the patent document or the patent disclosure as it appears in the
Patent and Trademark
Office patent file or records, but otherwise reserves all copyright rights
whatsoever. The
following notice applies to the processes, experiments, and data as described
below and in the
drawings attached hereto: Copyright 0 2014, Vision Global Holdings Limited,
All Rights
Reserved.
Technical Field
[0002] The present disclosure describes a detection and quantification method
for a list of
specific and novel Hepatocellular Carcinoma (HCC) tumor biomarkers, by
measuring the
corresponding auto-antibodies in liver cancer patients' sera. The set of
biomarkers comprises
Bmil, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4,
Midkine, IL-17A and IL26. More specifically, this disclosure further describes
a design of a high
throughput and sensitive test kit readily available to take patients'
peripheral serum samples for
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detecting liver cancers early and in a non-invasive manner by measuring the
auto-antibodies
against at least one of the biomarkers selected from the biomarker set. The
present disclosure
further allows identification of signature biomarker patterns for staging, as
well as the detection
of recurrences during a monitoring period of post-chemotherapeutic treatment.
The present
disclosure would support automatic data analysis.
Background of Invention
[0003] Hepatocellular carcinoma (HCC) is the second most prevalent cancer in
China, which
covers 5.7 % of the total population [I]. Most HCC patients have rapid tumor
progressing
resulting in high mortality rate. In order to improve the overall survival,
early diagnosis of the
disease becomes essential. Currently, the most common way of detecting HCCs
are blood tests
that measure level of HCC tumor markers such as alpha fetoprotein (AFP). AFP
is a plasma
protein produced by yolk sac and liver during the development of fetus serving
as a form of
serum albumin. In normal condition, AFP level gradually decreases after birth
and remain in low
level in adults. Increased level of tumor markers indicates probability of
liver cancers. However,
the major problem of the AFP test is excessive false positive. It is because
HCC is not the only
cause for the AFP level elevation, but alcoholic hepatitis, chronic hepatitis
or cirrhosis also
associates with increase of AFP.
[0004] Despite AFP test is commonly suggested for diagnosis of liver cancers,
its result is not
conclusive. Suspected patients will need to go through ultrasound imaging, CT
scans or contrast
MRI scans for further confirmation. Liver biopsy will be taken to distinguish
whether the tumor
is benign or malignant. However, conventional detection of HCCs comes with
several limitations:
(a) About 20% of liver cancers does not produce elevated level of the commonly
used HCC
tumor markers [2]. (b) Viral cirrhosis produces false positive results on the
blood tests [3]. (c)
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Ultrasound is not able to detect small tumors [4]. (d) CT scans require high
radiation dose and
are insensitive to tumors less than 1 cm [5]. (e) MRI scans are expensive and
the procedure is
time consuming. Due to these limitations, there are needs to develop novel
biomarkers screen
with higher sensitivity and specificity for the purpose of early diagnosis of
HCC and/or
determining a prognosis of HCC to complement the conventional methods.
[0005] HCC tumor cells tend to produce a unique set of proteins when compared
to the normal
liver epithelial cells juxtaposed to the tumor. Evaluation of validated HCC
tumor biomarkers has
great potential to facilitate the diagnosis of HCC. However, not all
biomarkers themselves can be
found in serum or urine for convenient diagnosis. Alternatively, the auto-
antibodies which are
specifically against the biomarkers provide an opportunity to evaluate the
expression of the
biomarkers. It has been demonstrated in many cancers that the presence of
tumor biomarkers
couples the production of auto-antibodies against these tumor antigens [6-8].
Detection on auto-
antibodies in patients' sera would allow us to examine the presence of
biomarkers more
efficiently. Ideally, examination of auto-antibodies from peripheral blood
would be a testament
for detecting liver cancers early, and in a non-invasive manner. One common
hurdle hindering
clinical use of biomarkers is that they have not been validated after
discovery. But once validated,
such test would be cost effective and accurate. The design of the prototype
also supports high-
throughput screening. This may alleviate the cost required for conventional
liver cancer
diagnosis.
[0006] There follows a list of references that are occasionally cited in the
specification.
[1] Chen JG, Zhang SW. Liver cancer epidemic in China: past, present and
future. Semin Cancer
Biol. 2011; 21(1):59-69
[2] Okuda K, Peters RL. Human alpha-1 fetoprotein. Hepatocellular Carcinoma.
1976:353-67
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[3] Lok AS, Lai CL. Alpha-fetoprotein monitoring in Chinese patients with
chronic hepatitis E
virus infection: role in the early detection of hepatocellular carcinoma.
Hepatolog.y 1989;9:110-
115
[4] Colombo M, de Franchis R, Del Ninno E, Sangiovanni A, De Fazio C,
Tommasini M,
Donato MF, Piva A, Di Carlo V, Dioguardi N. Hepatocellular carcinoma in
Italian patients with
cirrhosis. N Engl J Med. 1991;325:675-80
[5] Sahani DV, Kalva SP. Imaging the Liver. The Oncologist. 2004; 9 (4): 385-
397
[6] Masutomi K, Kaneko S. Yasukawa M, Arai K, Murakami S, Kobayashi K.
Identification of
serum anti-human telomerase reverse transcriptase (hTERT) auto-antibodies
during progression
to hepatocellular carcinoma. Oncogene. 2002 Aug 29;21(38):5946-50.
[7] Karanikas V, Khalil S, Kerenidi T, Gourgoulianis KI, Germenis AE. Anti-
survivin antibody
responses in lung cancer. Cancer Lett. 2009 Sep 18;282(2):159-66.
[8] Wang YQ, Zhang HH, Liu CL, Xia Q, Wu H, Yu XH, Kong W. Correlation between
auto-
antibodies to survivin and MUC1 variable number tandem repeats in colorectal
cancer. Asian
Pac J Cancer Prey. 2012;13(11):5557-62.
Summary of Invention
[0007] In the present invention, a detection and quantification method
measuring the auto-
antibodies against a list of specific tumor biomarker aiming for diagnosing
and staging cancers is
provided. Comparing to the normal liver epithelial cells, HCC tumor cells tend
to produce a
unique set of proteins. The evaluation of the unique protein set, biomarkers,
will complement the
conventional diagnostic methods and facilitate early detection of cancers.
[0008] By using a Two-Dimensional/Mass Spectrometry based method, a set of
liver cancer
biomarkers from paired patients' biopsies (tumor biopsy versus juxtaposed
normal tissue) is
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identified in the present invention comprising Bmil, VCC1, SUMO-4, RhoA, TXN,
ET-1,
UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, Midkine, IL-17A and IL26.
[0009] Specificity and accuracy of this set of liver caner biomarkers are then
validated and taken
together for diagnosis of liver cancers. In the present invention, proteins of
the listed biomarkers
are expressed from cDNA clones, purified and coupled to fluorescent
microsphere beads with
different emission wavelengths. Auto-antibodies present in patients' sera
against the proteins
immunologically bind to the protein-bead conjugate. The auto-antibodies
subsequently interact
with PE-conjugated secondary antibodies. The specific fluorescence signal of
the microsphere
beads serves as an identifier for the conjugated biomarkers. By measuring the
fluorescent
intensity given by the PE-conjugated secondary antibodies at the complex, it
allows the detection
and quantification of the auto-antibodies. Since the auto-antibodies are
produced in the patients'
sera in proportion to the abundance of the biomarkers at HCC tumor cells, the
higher fluorescent
intensity resulted from higher concentration of auto-antibodies indicates the
higher expression of
the corresponding biomarkers. The lowest detection limit of each biomarker to
the total serum
auto-antibodies is about 0.15 ng/mL.
[0010] Comparing to sera from healthy subjects, the level of auto-antibodies
against the target
biomarkers is at a higher concentration in cancer patient. Moreover, comparing
different sera
from liver cancer patients at different stages, signature patterns for staging
may be generated.
Thus, the present invention allows the non-invasive evaluation of the targeted
liver cancer
biomarker. This enables the detection of HCC at early stages and the
identification of signature
biomarker patterns for staging, as well as the detection of recurrences during
a monitoring period
of post-chemotherapeutic treatment.
Brief Description of the Drawings
WO 2016/003479 PCT/US2014/049038
[0011] Embodiments of the present invention are described in more detail
hereinafter with
reference to the drawings, in which:
[0012] FIG. 1 shows the difference in protein expression pattern between tumor
biopsy and
juxtaposed normal tissue by two-dimensional/mass spectrometry leading to the
identification of
15 specific biomarkers up-regulated in liver cancer; arrows indicate location
of spots identified
on a 2-D gel of the mass spectrometry.
[0013] FIG. 2 shows the set of 15 validated liver cancer biomarkers and their
corresponding
molecular weight targeted and measured in the present invention.
[0014] FIG. 3 shows the workflow of expressing the biomarkers from cDNA
clones.
[0015] FIG. 4 shows the workflow of purification of the biomarkers expressed
from E. coli.
[0016] FIG. 5 shows the workflow of measuring the auto-antibodies by BioP1exTM
system.
[0017] FIG. 6 shows the conjugation of biomarker protein to BioPiexTm bead.
[0018] FIG. 7 shows illustration of the complex of biomarker-] BioP1exTM bead
conjugate
immunoreacting with primary antibody and PE-conjugated secondary antibody.
[0019] FIG. 8 shows the gel electrophoresis of the DNA insert released from
plasmid cut by
restriction enzymes HindIII and BamH1.
[0020] FIG. 9 shows the Coomassie Blue stained SDS-PAGE verifying the IPTG
induction of (a)
Bmil, (b) SOD] , (c) IL-17A, (d) TXN and (e) Midkine biomarkers.
[0021] FIG. 10 shows the elution profile of (a) Bmil, (b) SOD-1 and (e) IL-17A
in AKTA.
[0022] FIG. 11 shows the Coomassie Blue stained SDS-PAGE verifying the
purification of His-
tagged (a) Bmil, (b) SOD-1 and (d) IL-17A biomarkers; Fraction A is bacteria
without IPTG
induction; Fraction B is bacteria with IPTG induction; Fraction C is bacterial
lysate.
[0023] FIG. 12 shows the standard curve showing the fluorescence intensity
against the
concentration of anti-Bmil antibody.
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[0024] FIG. 13 is a schematic diagram showing the design of the test: Patient
serum containing
auto-antibodies are mixed to a well containing 15 types of beads corresponding
to the 15
biomarkers of the biomarker set, followed by the addition of PE-conjugated
secondary antibody.
Definitions
[0025] The term "biomarker" refers to the protein uniquely expressed or up-
regulated in the
tumor comparing to the normal epithelial cells.
[0026] The term "biomarker set" refers to the specific combination of the
biomarkers identified
from paired patients' biopsies (tumor biopsy versus juxtaposed normal tissue)
and is the target of
the measurement in the present invention.
[0027] The term "auto-antibodies" refers to the anti-bodies produced by the
patient body
coupling to the expression of the tumor biomarker and it is present in the
circulation and can be
collected in the peripheral serum.
[0028] Bmil (Polycomb Ring Finger) is a protein component of a Polycomb Group
(PcG)
multiprotein PRC 1-like complex. It is responsible for maintaining the
transcriptionally repressive
state of many genes, including Hox genes, throughout development. The
regulation is via
monoubiquitination of histone H2A 'Lys-119', which modifies histone and
remodels chromatin,
rendering the expression.
[0029] VCC I or CXCL17 (Chemokine (C-X-C Motif) Ligand 17) has an essential
role in
angiogenesis and possibly in the development of tumors. It is also suggested
that it is a
housekeeping chemokine regulating the recruitment of non-activated blood
monocytes and
immature dendritic cells into tissues. It may also play a role in the innate
defense against
infections. Malfunction of VCC1 is associated with duodenitis and cholera.
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[0030] SUMO-4 (Small Ubiquitin-Like Modifier 4) belongs to the family of small
ubiquitin-
related modifiers and located in the cytoplasm. It covalently attaches to the
target protein, IKBA,
in order to control its subcellular localization, stability, or activity. This
eventually leads to a
negative regulation of NF-kappa-B-dependent transcription of the IL12B gene.
[0031] RhoA (Ras Homolog Family Member A) regulates the signaling pathway
linking plasma
membrane receptors to the assembly of focal adhesions and actin stress fibers.
It also involves in
microtubule-dependent signaling essential during cell cycle cytokinesis, and
other signaling
pathways involved in stabilization of microtubules and cell migrations and
adhesion.
[0032] TXN (Thioredoxin) forms homodimer and is involved in redox reactions
through the
reversible oxidation of its active center dithiol to a disulfide and catalyzes
dithiol-disulfide
exchange reactions. It has been reported to be associated with breast mucinous
carcinoma.
[0033] ET-1 (Endothelin 1) is a potent vasoconstrictor produced by vascular
endothelial cells. It
binds to endothelin receptors widely expressed in all tissues, including non-
vascular structure
like epithelial cells, glia, and neurons. Apart from the main role in
maintenance of vascular tone,
it is also suggested to have co-mitogenic activity and potentiate the effects
of other growth
factors.
[0034] UBE2C (Ubiquitin-Conjugating Enzyme E2C) belongs to the family of E2
ubiquitin-
conjugating enzyme. This is one of the three enzymes involved in
ubiquitination, which is an
important cellular mechanism for targeting abnormal proteins for degradation.
More specifically,
UBE2C is required for the targeted degradation of mitotic cyclins and for cell
cycle progression.
Thus, it is believed that this protein may be also involved in cancer
progression.
[0035] HDGF2 is called hepatoma-derived growth factor 2. This protein which is
highly
expressed in a variety of tumors has been reported to play a pivotal role in
the development and
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progression of several tumors. Although the mechanism is yet to be identified,
it is suggested
that HDGF2 has mitogenic, angiogenic, neurotrophic and antiapoptotic activity.
[0036] FGF21 (Fibroblast Growth Factor 21) is a family member of the FGF
family which is
involved in vary biological processes including embryonic development, cell
growth,
morphogenesis, tissue repair, tumor growth and invasion. More specifically,
FGF21 stimulates
glucose update in differentiated adipocytes via the induction of glucose
transporter
SLC2A1/GLUT1 expression. It has been found that FGF21 is associated with fatty
liver disease.
[0037] LECT2 (Leukocyte Cell Derived Chemotaxin 1) is a secretory protein acts
as a
chemotactic factor to neutrophils and stimulates the growth of chondrocytes
and osteoblasts.
This protein is associated with acute liver failure.
[0038] SOD1 (Superoxide Dismutase 1) is a Cu/Zn-containing antioxidant enzyme
responsible
for destroying free superoxide radicals into molecular oxygen and hydrogen
peroxide in the
cytosol, the nucleus, and the intermembrane space of the mitochondria. It is
important for
maintaining low levels of superoxide in the cytosol, thus protecting the cell
from oxidative stress
and subsequent cell death.
[0039] STMN4 (Stathmin-Like 4) is a small regulatory protein which is believed
to have a role
in relaying integrating diverse intracellular signaling pathways, which in
turn, controls cell
proliferation, differentiation and functions. It is also shown that this
protein contributes to the
control of microtubule dynamics by inhibiting the polymerization of
microtubules and/or
favoring their depolymerization.
[0040] Midkine or NEGF2 (Neurite Growth-Promoting Factor 2) is a secretory
growth factor
that binds heparin and responsive to retinoic acid. Midkine promotes cell
growth, migration and
angiogenesis, in particular during tumorigenesis. It has already been
demonstrated to be
associated with breast adenocarcinoma and soft tissue sarcoma.
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[0041] IL-17A (Interleukin 17A) is a proinflammatory cytokine produced by the
activated T
cells. It regulates the activity of NF-kappaB and mitogen-activated protein
kinases, stimulates the
expression of 1L6 and cyclooxygenase-2, and enhances the production of nitric
oxide. Several
chronic inflammation and sclerosis are usually associated with IL-17A
elevation.
[0042] IL-26 (Interleukin 26) belongs to the IL-10 cytokine family and is
produced by the
activated T cells and targets epithelial cells for signal transduction. It
binds strongly to
glycosaminoglycans such as heparin, heparan sulphate, and dermatan sulfate on
cellular surfaces
which act similarly to coreceptors in order to enrich IL-26 on the surface of
producer and target
cells.
Detailed Description of Invention
[0043] In the following description, the biomarker,/biomarkers, the
corresponding embodiments
of the detection/validationlidentification/quantification methods are set
forth as preferred
examples. It will be apparent to those skilled in the art that modifications,
including additions
and/or substitutions, may be made without departing from the scope and spirit
of the invention.
Specific details may be omitted so as not to obscure the invention; however,
the disclosure is
written to enable one skilled in the art to practice the teachings herein
without undue
experimentation.
[0044] In the present invention, the set of liver tumor biomarkers for
detection and quantification
of liver cancer is first identified by two-dimensional/mass spectrometry
resolving the difference
in the pattern of proteins expression between the paired patients' biopsies
(tumor biopsy versus
juxtaposed normal tissue) (FIG. 1). The biomarkers are validated by
immunohistochemical
staining on paraffin-sectioned HCC blocks, and Western Blotting in HCC
patients' sera. This
WO 2016/003479 PCT/US2014/049038
results in a finalized list of 15 biomarkers to be evaluated in the present
invention for the liver
cancer diagnosis purpose (FIG. 2).
[0045] Based on the amino acid sequences of the targeted biomarkers,
commercially synthesized
cDNA clones are employed for the expression of the biomarker set (FIG. 3).
Proteins expressed
from the cDNA clones are then subjected to a series of steps of purifications
(FIG. 4). The
purified biomarkers are subsequently conjugated via stable amide bonds with
BioPlexim beads
(FIGS. 5, 6), a type of fluorescent microsphere beads and available in a panel
which give unique
fluorescent signals individually for identification at a multiplex set up. The
biomarkers on the
beads are recognized by the specific primary antibodies, which are
subsequently bound by an
anti-human secondary antibody conjugated with PE (FIG. 7). Thus the BioPIexTM
machine
simultaneously measures two signals from the complex. The fluorescence given
by the BioP1exTM
beads serves as an identifier, while the signal from the PE indicates the
presence of the
biomarker in the complex. This also helps differentiating the biomarker-bead
conjugates bound
by the anti-body cascade from those with no immuno-reactivity with antibodies.
[0046] To prove the significance of the biomarkers in the present invention,
the cDNA clones
are confirmed by restriction enzyme cut (FIG. 8). The transformed bacteria is
induced by IPTG
to express the biomarker proteins. The protein expression verified by SDS-PAGE
and Coomassie
Blue staining reveals the protein bands (FIG. 9 a-c). The His-tagged Bmil,
SOD1 and IL-17A
proteins are purified by AKTA (FIG. 10 a-c) and then verified by SDS-PAGE and
Coomassie
Blue staining (FIG. 11 a-c).
[0047] Sensitivity of the test is measured by spiking in a serial dilution of
the antibodies. The
lowest concentration of the antibody added that can give signal suggests the
sensitivity of that
particular biomarker. Meanwhile a standard curve is constructed showing the
fluorescence
intensity of the PE against the serial dilutions of the antibodies (FIG. 12).
The standard curve
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will be used for estimating the concentration of the biomarker specific auto-
antibodies in the
patient sera by comparing the PE intensity.
[0048] In the present invention, a multiplex of 15 different BioPlexTm beads
individually giving
unique fluorescence are conjugated with the biomarker set and preloaded in the
wells of a plate
(FIG. 13). To a well, patient serum containing auto-antibodies is loaded and
allowed to interact
with the biomarker conjugates. The PE-conjugated secondary antibodies are then
added and bind
to the auto-antibodies. In the machine, the excess secondary antibodies are
washed away, the
complex comprising the biomarker-bead conjugate and cascade of antibodies are
measured
individually. The unique fluorescence signal of the BioP1exTM bead identifies
the biomarkers, while
the PE signal from the same complex indicates the presence of the auto-
antibodies as the primary
antibody (FIG. 7). Taken together, the measurement will suggest the presence
of auto-antibodies
and the relative concentration in the presents' sera.
[0049] In a standard randomized trial design, the mean of the relative level
of auto-antibodies
between the healthy group and patients diagnosed with liver cancer is
compared. Student T test is
used to analyze the variation significance. The significant difference
indicates that the biomarker
is specific for liver cancer. After the verification trials, ranges of the
concentration of biomarker
specific auto-antibodies will be obtained for the liver cancer positive and
negative patients and
serve as reference point for the future diagnosis. Meanwhile, expression
pattern of the auto-
antibodies is also compared between liver cancer patients of different stages.
The signature
patterns of the biomarker expressions will indicate the HCC staging.
[0050] Taken together, the measurement of the relative auto-antibodies level
and the expression
pattern of the biomarkers, the present invention represents a different avenue
to complement
conventional liver cancer diagnosis. The present invention further enables non-
invasive detection
of auto-antibodies against the validated targets in patients' sera of the
present invention,
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identifying the extent and the characteristics of the disease. Apart from
early detection for stage I
liver cancers, the present invention also enables the generation of signature
patterns for staging,
and the detection of recurrences during a monitoring period of post-mastectomy
or post-
chemotherapeutic treatment.
Examples
[0051] The following examples are provided by way of describing specific
embodiments of this
invention without intending to limit the scope of this invention in any way.
[0052] Example la
[0053] Protein extraction from patients' biopsies
[0054] 500 mg of the paired patients' biopsies (tumor biopsy versus juxtaposed
normal tissue)
are collected and washed with PBS. The tissues are frozen by submerging into
liquid nitrogen
and immediately homogenized with pestle and mortar. To the homogenized
samples, lysis
solution (8M Urea, 4% CHAPS, 2% IPG Buffer, 0.2mg/m1 PMSF) is added, then
vortex for at
least 5 min until the tissues are completely dispersed. The lysates are then
clarified by
centrifugation at 14,000 rpm for 10 minutes at 4 C. The supernatants are
further cleaned up by
2D Clean Up kit (Amersham) to remove the salt and impurities. The pellets are
resuspended with
minimum volume of Rehydration Solution (No DTT & IPG Buffer added). The
protein
concentrations are then measured by Bio-Rad protein assay and aliquots of 200
g/per tube are
stored at -70 C.
[0055] Example lb
[0056] Resolving proteins by two-dimensional electrophoresis
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[0057] To 1 ml rehydration stock solution, 2.8 mg DTT, 5 1 pharmalyte or IPG
Buffer, and 2
p.1 bromophenol blue are added. 50 ¨ 100 lug of protein sample is added to the
13 cm Immobiline
DryStrip (IPG strip) containing 250 pA of rehydration solution. After removing
the protective
cover, the IPG strip is positioned in the strip holder with the gel side
facing down, and overlaid
with Cover Fluid to prevent dehydration during electrophoresis. The strip is
then placed on to
Ettan IPGphor (Amersham) for isoelectric focusing (first dimensional
electrophoresis).
[0058] After the first-dimensional electrophoresis, the IPG strip is
equilibrated with equilibrate
solution (6 M Urea 2 % SDS, 50 mM Tris HC1 pH
6.8, 30 % Glycerol,
0.002 % Bromophenol blue, 100 mg DTT per 10 ml buffer and 250 mg IAA per 10 ml
buffer),
and then washed with lx SDS running Buffer for 4 - 5 times. The IPG strip is
placed on top of
the second-dimension gel and overlaid with sealing solution (0.5 % Low Melting
agarose, 0.002
% Bromophenol Blue in 1 SDS running Buffer). The second-dimensional
electrophoresis is
then carried out at 30 mA for first 15 min followed by 60 mA for 3 - 4 h.
[0059] Upon the completion of the second dimensional electrophoresis, the gel
is removed from
the cassette, fixed and stained with silver nitrate. 15 spots representing 15
up-regulated proteins
are identified (FIG. 1). To identify the proteins (FIG. 2), the silver stained
gel slices are destained
and trypsinized to release the protein from the gel for MALDI-TOF analysis.
[0060] Example 2a (SEQ ID NO.1)
[0061] Amino acid sequence of Bmil
[0062] MHRTTRIKITELNPHLMCVLCGGYFIDATTIIECLHSFCKTCIVRYLETSKYCPICD
VQVHKTRPLLNIRSDKTLQDIVYKLVPGLFKNEMKRRRDFYAAHPSADAANGSNEDRG
EVADEDKRITTDDEIISLSIEFFDQNRLDRKVNKDKEKSKEEVNDKRYLRCPAAMTVMHL
RKFLRSKMDIPNTFQIDVMYEEEPLKDYYTLMDIAYIYTWRRNGPLPLKYRVRPTCKRM
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KISHQRDGLTNAGELESDSGSDKANSPAGGIPSTS SCLPSPSTPVQSPHPQFPHISSTMNGT
SNSPSGNHQSSFANRPRKSSVNGS SATS SG
[0063] Example 2b (SEQ ID NO.2)
[0064] Amino acid sequence of VCC1
[0065] MKVLIS SLLLLLPLMLMSMVS SSLNPGVARGHRDRGQASRRWLQEGGQECECK
D WFLRAPRRKF'MTV S GLPKKQCP CDHFKGN VKKTRHQRHHRKPNKHSRACQQFLKQC
QLRSFALPL
[0066] Example 2c (SEQ ID NO.3)
[0067] Amino acid sequence of SUMO-4
[0068] MANEKPTEEVKTENNNHINLKVAGQDGSVVQFKIKRQTPLSKLMKAYCEPRGLS
VKQIRFRFGGQPISGTDKPAQLEMEDEDTIDVFQQPTGGVY
[0069] Example 2d (SEQ ID NO.4)
[0070] Amino acid sequence of RhoA
[0071] MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVE
LALWDTAGQEDYDRLRPL SYPDTDVILM CF S ID SPD S L ENIPEKWTPEVKHF CPNVPIILV
GNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFE
MATRAALQARRGKKKSGCLVL
[0072] Example 2e (SEQ ID NO.5)
[0073] Amino acid sequence of TXN
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[0074] MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIF
L EVDVDD C QDVAS EC EVKC MP TFQFFKKGQKVGEF S GANKEKL EATINELV
[0075] Example 2f (SEQ ID NO.6)
[0076] Amino acid sequence of ET-1
[0077] MDYLLMIFSLLFVACQGAPETAVLGAELSAVGENGGEKPTPSPPWRLRRSKRCS
CSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRSKRALENLLPTKATDRENRCQCAS
QKDKKCWNFCQAGKELRAEDIMEKDWNNHKKGKDCSKLGKKCIYQQLVRGRKIRRSS
EEHLRQTRSETMRNSVKSSFHDPKLKGNPSRERYVTHNRAHW
[0078] Example 2g (SEQ ID NO.7)
[0079] Amino acid sequence of UBE2C
[0080] MAS QNRDPAAT SVAAARKGAEP S GGAARGPVGKRLQQEL MTLMM S GDKGI SA
FPESDNLFKWVGTIHGAAGTVYEDLRYKLSLEFPSGYPYNAPTVKFLTPCYHPNVDTQG
NICLDILKEKWSALYDVRTILLSIQSLLGEPNIDSPLNTHAAELWKNPTAFKKYLQETYSK
QVTSQEP
[0081] Example 2h (SEQ ID NO.8)
[0082] Amino acid sequence of HDGF2
[0083] MARPRPREYKAGDLVFAKMKGYPHWPARIDEL PEGAVKPPANKYPIFFFGTHET
AFLGPKDLFPYKEYKDKFGKSNKRKGFNEGLWEIENNPGVKFTGYQAIQQQSS SETEGE
GGNTADAS SEEEGDRVEEDGKGKRKNEKAGSKRKKSYTSKKSSKQSRKSPGDEDDKDC
KEEENKS SSEGGDAGNDTRNTTSDLQKTSEGT
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[0084] Example 21 (SEQ ID NO.9)
[0085] Amino acid sequence of FGF21
[0086] MDSDETGFEHSGLVVV SVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDA
Q QTEAHL EIRED GTVGGAAD Q SPE SLLQLKALKP GVI QILGVKT SRFLC QRPD GALYG SL
HFDPEAC SFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAL
PEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
[0087] Example 2j (SEQ ID NO.10)
[0088] Amino acid sequence of LECT2
[0089] MFSTKALLLAGLISTALAGPWANICAGKS SNEIRTCDRHGCGQYSAQRSQRPHQ
GVDVLC S A G STVYAPFTGMIVGQEKPYQNKNAINNGVRISGRGFCVKMFYIKPIKYKGP
IKKGEKLGTLLPLQKVYPGIQSHVHIENCDSSDPTAYL
[0090] Example 2k (SEQ ID NO.11)
[0091] Amino acid sequence of SOD1
[0092] MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDN
TAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDVISLSGDHCII
GRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ
[0093] Example 21 (SEQ ID NO.12)
[0094] Amino acid sequence of STMN4
[0095] MTLAAYKEKMKELPLVSLFC SCFLADPLNKSSYKYEADTVDLNWCVISDMEVIE
LNKCTSGQSFEVILKPPSFDGVPEFNASLPRRRDPSLEEIQKKLEAAEERRKYQEAELLKH
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LAEKREHEREVIQKAIEENNNFIKMAKEKLAQKMESNKENREAHLAAMLERLQEKDKH
AEEVRKNKELKEEASR
[0096] Example 2m (SEQ ID NO.13)
[0097] Amino acid sequence of Midkine
[0098] MQHRGFULTLLALLALTSAVAKKKDKVKKGGPGSECAEWAWGPCTPSSKDCG
VGFREGTCGAQTQRIRCRVPCNWKKEFGADCKYKFENWGACDGGTGTKVRQGTLKKA
RYNAQCQETIRVTKPCTPKTKAKAKAKKGKGKD
[0099] Example 2n (SEQ ID NO.14)
[00100] Amino acid sequence of IL-17A
[00101] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIH
NRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHM
NSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA
[00102] Example 2o (SEQ ID NO.15)
[00103] Amino acid sequence of IL-26
[00104] MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAA
WLKATIPEDRIKNIRLLKKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFVE
DFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIYKAISELDILLSWIKKLLESSQ
[00105] Example 3a
[00106] Expression of biomarker set
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[00107] His tagged plasmids containing cDNA inserts encoding the biomarker
set is
transformed into DH5 competent cells (301, FIG. 3). Single colony is picked
and allowed to
grow in bacterial culture (302). The number of plasmid is expanded and
extracted from the
bacteria by miniprep. The plasmid is further transformed into BL21DE3 or
BL21DE3pLysS
competent cells. Transformed bacteria are selected and grew in 2 X 100 ml LB
medium. When
the bacterial culture reaches the optical density of 0.06, 200 iM of IPTG is
added to 100 ml
bacterial culture (303). Another 100 ml of bacterial culture without IPTG is
used as negative
control. The bacterial cultures are incubated at 30 C with shaking. 500 jul
of the bacterial
cultures are saved and stored at -20 C 3 h after the incubation and in the
next morning after
incubating overnight.
[00108] Bacterial cultures with and without IPTG induction are mixed
together in a 500
ml centrifuge bottle. Bacterial cells are collected by centrifugation at 9000
rpm for 20 min at 4
C (304). 500 of supernatant is saved as another negative control and the
remaining
supernatant is discarded. The bacterial cultures and negative controls
collected in different points
are run on a SDS-PAGE to resolve the protein (305). The gel is then stained
with Coomassie
Blue overnight. After destaining the gel, the protein induction can be
confirmed by checking the
size and comparing with the negative controls.
[00109] Example 3b
[00110] Protein purification for biomarker set
[00111] The bacterial cell pellets are resuspended in 10 ml solubilization
buffer by vortex
at room temperature. Keeping the resuspended cells in 50 ml centrifuge tube on
ice, the cells are
completely lysed by sonication at amplitude 70 % 10 rounds of 30 s with
interval of 30 s (401,
FIG. 4). The lysed cells arc centrifuged at 10,000 rpm for 1 h at 4 C (402).
Supernatants are
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transferred into dialysis tubing and submerged in I L untittered starting
buffer for 4 - 6 h at 4 C
with constant stirring (403). Dialysis is continued with another 1 L starting
buffer overnight. The
supernatant is further filtered with 0.22 gm filter disc and syringe. To the
AKTA machine
equipped with 0.1M Nickel sulfate charged HiTrap chelating column (404),
filtered samples are
loaded (405). A program is set at the AKTA machine that the eluent is
collected in fractions
automatically (406). Proteins purified from different fractions are checked by
SDS-PAGE
analysis (407).
[00112] Example 4a
[00113] Protein coupling with BioPlexTM beads
[00114] The purified proteins of the biomarker set are coupled with
BioP1exTM beads (Bio-
Rad) (501) according to the manufacturer's manual. In brief, uncoupled bead is
vortexed for 30 s
and then sonicated for 15 s. 1,250,000 beads are collected in a reaction tube
by centrifugation of
100 gl bead at maximum speed for 4 min. After washing with 100 1 bead wash
buffer by
centrifugation, the beads are resuspended in 80 1 bead activation buffer. To
the beads 10 I 50
mg/ml freshly prepared S-NHS and 10 I 50 mg/m1 freshly prepared EDAC are
added, followed
by 20 min incubation in dark at room temperature (FIG. 6). The beads are then
washed with 150
I PBS twice.
[00115] To the washed beads, 10 g proteins are added and the total volume
is topped up
with PBS to 500 gl, and allowed to incubate for 2 h with shaking in dark.
Supernatant is
removed after centrifugation at maximum speed for 4 min. 250 I blocking
buffer is added to the
beads and shook in dark for 30 min, followed by centrifugation at maximum
speed for 4 mm and
removal of supernatant. The beads are briefly washed and then resuspended in
the storage buffer
for storage at 4 C. The numbers of the beads are counted with a
hemocytometer.
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[00116] Example 4b
[00117] Validation of protein-bead coupling
[00118] To a HTS 96 well plate, 50 1 of conjugated BioPlexTm beads (100
beads / 1) is
added to react with primary followed by secondary antibodies (502). A serial
dilution of the
commercially available primary anti-bodies against the biomarker set is
prepared as 8,000, 4,000,
1,000, 250, 62.5, 15.625, 3.906, 0.977, 0.244 and 0.061 ng/ml. 50 I of each
dilution is added to
each well. Two negative controls are performed by excluding the primary
antibodies, and both
primary and secondary antibodies in the wells. The plate is then sealed with a
foil and kept on a
shaker for 30 min at 350 rpm, avoiding exposure to light.
[00119] After incubation, the beads are washed three times with 150 I PBS.
50 pi of PE-
conjugated secondary antibody (8,000 ng/ml) is added into each well except
negative controls.
The plate is sealed again and incubated in dark for 30 min with shaking.
Excess antibodies are
then washed away by PBS. The BioP1exTM machine is calibrated with the
calibration kit and
validation kit. After the HTS plate is loaded to the machine, signals from
both the BioP1cxTM beads
and the PE conjugated at the secondary antibodies (503) are measured
(schematic diagram is
shown in FIG. 7). A calibration curve is generated by Logistic-5PL.
[00120] Example 4c:
[00121] Collection of serum samples and measurement of auto-antibodies by
BioPlexTm
system
[00122] Whole-blood samples are clotted by standing at 37 C for 1 h. Sera
containing the
auto-antibodies is collected at the supernatant after centrifugation at 1000g
room temperature for
min. The serum samples are diluted with PBS when necessary. To a HTS plate
preloaded with
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BioP1exTM beads conjugated with biomarker set, tne serum samples are loaded
and incubated for 30
mm with shaking (HG. 13). Similar to the steps described in Example 4b, to the
PBS washed
beads, 50 I of PE-conjugated secondary antibody (8000 ng/ml) is added,
followed by shaking
for another 30 min. After three rounds of washing, the plate is loaded to the
BioPlex-rm machine
and the fluorescence signal is measured (504). The concentration of the auto-
antibodies can then
be calculated from the standard curves.
[00123] The foregoing description of the present invention has been
provided for the
purposes of illustration and description. It is not intended to be exhaustive
or to limit the
invention to the precise forms disclosed. Many modifications and variations
will be apparent to
the practitioner skilled in the art.
[00124] The embodiments are chosen and described in order to best explain
the principles
of the invention and its practical application, thereby enabling others
skilled in the art to
understand the invention for various embodiments and with various
modifications that are suited
to the particular use contemplated.
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