U.S. patent application number 13/318804 was filed with the patent office on 2012-05-17 for means and methods for recognizing the development of cardiovascular disease in an individual.
This patent application is currently assigned to YEDA RESEARCH & DEVELOPMENT CO. LTD.. Invention is credited to Irun R. Cohen, Eytan Domany, Eli Sahar, Noam Shental.
Application Number | 20120122720 13/318804 |
Document ID | / |
Family ID | 43050575 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120122720 |
Kind Code |
A1 |
Cohen; Irun R. ; et
al. |
May 17, 2012 |
MEANS AND METHODS FOR RECOGNIZING THE DEVELOPMENT OF CARDIOVASCULAR
DISEASE IN AN INDIVIDUAL
Abstract
A method of recognizing the development of an Acute Myocardial
Infarction (AMI) process in an individual, wherein the method
comprises steps of: profiling specific antibody reactivities or
biomarkers associated with AMI susceptibility, the profiling
comprises steps of: attaching a set of defined antigens to a
substrate; obtaining a biological fluid derived specimen from an
individual, the specimen containing a specific antibody repertoire;
and binding said antibodies of the biological fluid specimen to the
attached antigens thereby forming bound antibody antigen complexes;
and analyzing results obtained, wherein the presence of the
complexes is indicative of AMI.
Inventors: |
Cohen; Irun R.; (Rehovot,
IL) ; Domany; Eytan; (Rehovot, IL) ; Sahar;
Eli; (Rehovot, IL) ; Shental; Noam; (Rehovot,
IL) |
Assignee: |
YEDA RESEARCH & DEVELOPMENT CO.
LTD.
Rehovot
IL
|
Family ID: |
43050575 |
Appl. No.: |
13/318804 |
Filed: |
May 5, 2010 |
PCT Filed: |
May 5, 2010 |
PCT NO: |
PCT/IL2010/000361 |
371 Date: |
January 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61175472 |
May 5, 2009 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/15;
506/17; 506/18; 506/19 |
Current CPC
Class: |
G01N 33/564 20130101;
G01N 2800/324 20130101; G01N 2333/47 20130101; G01N 2500/04
20130101; G01N 33/6893 20130101 |
Class at
Publication: |
506/9 ; 506/18;
506/17; 506/15; 506/19 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/12 20060101 C40B040/12; C40B 40/04 20060101
C40B040/04; C40B 40/10 20060101 C40B040/10; C40B 40/08 20060101
C40B040/08 |
Claims
1-86. (canceled)
87. A method of recognizing the development of an Acute Myocardial
Infarction (AMI) process in an individual, wherein said method
comprises steps of: a. profiling specific antibody reactivities or
biomarkers associated with AMI susceptibility, said profiling
comprises steps of: i. attaching a set of defined antigens to a
substrate; ii. obtaining a biological fluid derived specimen from
an individual, said specimen containing a specific antibody
repertoire; and, iii. binding said antibodies of said biological
fluid specimen to said attached antigens thereby forming bound
antibody antigen complexes; and, iv. analysing results obtained,
wherein the presence of said complexes is indicative of AMI.
88. The method according to claim 87, wherein said profiling
further comprises the steps of obtaining said defined antigens
characterized by association with a variety of conditions selected
from a group consisting of inflammation, angiogenesis, apoptosis or
any other condition involving immune regulation or any combination
thereof further wherein said profiling further comprises the steps
of selecting said biological fluid derived specimen from a group
consisting of plasma, serum, blood, cerebrospinal fluid, synovial
fluid, sputum, saliva, tears, lymph specimen, or any other
biological fluid known in the art.
89. The method according to claim 87, wherein said profiling
further comprises steps of selecting said substrate from a group
consisting of microarray chip, microarray wafers, microarray
strips, ELISA microwells, multiplex beads or any substrate known in
the relevant art, said method further comprising steps of selecting
said antigens from a group consisting of protein, polypeptide,
peptide, nucleic acids, lipids, glycosylated molecules,
polypeptides with modifications, polysaccharides or any other
biological molecule known in the art.
90. The method according to claim 87, further comprises the steps
of defining said antibody reactivities as IgG, IgM, IgA, IgE
antibody isotypes or more specific sub-isotypes of said
antibodies.
91. The method according to claim 87, wherein said analyzing
results obtained further comprises the steps of measuring said
bound antibody complexes further wherein said measuring
additionally comprises steps of labeling said bound antibody
complexes, said labeling further comprises the steps of selecting a
labeling signal from a group consisting of fluorescent,
chemiluminescent, absorptive, electronic, radioactive, paramagnetic
or any labeling signal known in the art, or combination thereof and
wherein said labeling step additionally comprises steps of reacting
said bound antibody complexes with labeled mixture of secondary
antibodies specific for human immunoglobulin types so as to obtain
labeled bound antibody antigen complexes.
92. The method according to claim 87, wherein said analyzing step
additionally comprises steps of: a. scanning the signal produced by
said bound antibody complexes so as to form a reactivity matrix; b.
measuring the intensity of said signal; c. generating and recording
results of data associated with said intensity of said signal; d.
producing said results in the form of a signature, profile, set,
pattern or matrix characteristic to said individual; e. comparing
said results with a databank containing data concerning said
antibody reactivities in populations susceptible to AMI thereby
identifying the development of an Acute Myocardial Infarction (AMI)
process in said individual further wherein said measuring and
scanning steps are selected from a group consisting of Surface
Plasmon Resonance (SPR), ellipsometry, laser scanning, light
detecting, photon detecting via a photon multiplier, photographing
with a digital camera based system or video system, radiation
counting, fluorescence detecting, electronic, magnetic detecting or
any other technique known in the art or combination thereof.
93. The method according to claim 87, wherein said analyzing step
further comprises the steps of defining said reactivity as a
relative intensity signal of a specific bound antibody as compared
to its intensity signal in healthy control individuals.
94. The method according to claim 87, wherein said analyzing step
further comprises the steps of defining said reactivity as a
relative intensity signal of a specific bound antibody as compared
to its intensity signal in individuals who have had a stroke
event.
95. The method according to claim 87, wherein said analyzing step
further comprises the steps of identifying a set of informative
antigens possessing higher or a lower intensity reactivity matrix
signals as compared to said intensity signals of said antibodies in
healthy control individuals or wherein said analyzing step further
comprises the steps of identifying a set of informative antigens
possessing higher or a lower intensity reactivity matrix signals as
compared to said intensity signals of said antibodies in
individuals who have had a stroke event.
96. The method according to claim 87 wherein said method is adapted
to recognize the development of a stroke process in an individual,
further wherein said method comprises steps of: a. profiling
specific antibody reactivities or biomarkers associated with stroke
susceptibility, said profiling comprises steps of : i. attaching a
set of defined antigens to a substrate; ii. obtaining a biological
fluid derived specimen from an individual, said specimen containing
a specific antibody repertoire; and, iii. binding said antibodies
of said biological fluid specimen to said attached antigens thereby
forming bound antibody antigen complexes; and, b. analysing results
obtained, wherein the presence of said complexes is indicative of
the development of a stroke process in an individual.
97. A set of informative antigens useful for recognizing the
development of an Acute Myocardial Infarction (AMI) process in an
individual said set of antigens selected from a group consisting of
Brain-1 oligo-dendrocyte transcription factor, Synaptotagmin 1,
Tryptophan hydroxylase peptide 1, Tryptophan hydroxylase peptide 2,
BSP (Brain specific protein), myelin associated glycoprotein,
Fibronectin, Caspase 3, Myelin-associated metalloproteinase, CD68,
Bone morphogenic protein 4 and D4GDI (GDP-dissociation inhibitor),
said informative antigens are identified by: a. profiling means for
profiling specific antibody reactivities or biomarkers associated
with AMI susceptibility, said profiling means further comprises: i.
attaching means for attaching a set of defined antigens to a
substrate; ii. a biological fluid derived specimen from an
individual, said specimen containing a specific antibody
repertoire; and, iii. binding means for binding said antibodies of
said biological fluid specimen to said attached antigens thereby
forming bound antibody antigen complexes; and, b. analyzing means
for analyzing results obtained. wherein said antigens are further
characterised by their specific antibody reactivity with human
antibodies associated with susceptibility to AMI.
98. The set of informative antigens according to claim 97, wherein
said defined antigens characterized by association with a variety
of conditions selected from a group consisting of inflammation,
angiogenesis, apoptosis or any other condition involving immune
regulation or any combination thereof.
99. The set of informative antigens according to claim 97, wherein
at least one of the following is true; said defined antigens are
attached to a substrate selected from a group consisting of
microarray chip, microarray wafers, microarray strips, ELISA
microwells, multiplex beads or any substrate known in the relevant
art; said defined antigens are selected form a group consisting of
protein, polypeptide, peptide, nucleic acids, lipids, glycosylated
molecules, polypeptides with modifications, polysaccharides or any
other biological molecule known in the art.
100. The set of informative antigens according to claim 97, wherein
said antigens are further characterized by their specific
capability to discriminate between the development of an AMI
process in an individual and a healthy control individual or
wherein said set of informative antigens are further characterized
by their specific capability to discriminate between the
development of an AMI process and a stroke process in an
individual.
101. The set of informative antigens according to claim 97, wherein
at least one of the following is true; said human antibodies are
derived from a biological fluid specimen selected from a group
consisting of plasma, serum, blood, cerebrospinal fluid, synovial
fluid, sputum, saliva, tears, lymph specimen, or any biological
fluid known in the art; said set of informative antigens have said
antibody reactivities defined as IgG, IgM, IgA, IgE antibody
isotypes or more specific sub-isotypes of said antibodies further
wherein said specific antibody reactivity is analyzable by using
measuring means; said set of informative antigens have said
specific antibody reactivity is analyzable by using labeling means
further wherein said labeling means comprises labeling signal
selected form a group consisting of fluorescent, chemiluminescent,
absorptive, electronic, radioactive, paramagnetic or any labeling
signal known in the art, or combination thereof.
102. The set of informative antigens according to claim 101,
wherein said labeling means is adapted to label said human
antibodies with a mixture of secondary antibodies specific for
human immunoglobulin types to form a reactivity matrix.
103. The set of informative antigens according to claim 97, wherein
said specific antibody reactivity is further analyzable by: a.
using a scanning means for scanning said reactivity matrix signal;
b. using a measuring means for measuring said intensity of said
signal; c. using generating and recording means for generating and
recording results associated with said reactivity matrix signal; d.
using producing means for producing said results in the form of a
signature, profile, set, pattern or matrix characteristic to said
individual; and, e. using means for comparing said results with a
databank containing data concerning said antibody reactivities in
populations susceptible to AMI, thereby identifying the development
of an Acute Myocardial Infarction (AMI) process in said individual
further wherein said measuring and scanning means are selected from
a group consisting of Surface Plasmon Resonance (SPR),
ellipsometry, laser scanning, light detecting, photon detecting via
a photon multiplier, photographing with a digital camera based
system or video system, radiation counting, fluorescence detecting,
electronic, magnetic detecting or any other technique known in the
art or combination thereof.
104. The set of informative antigens according to claim 97, wherein
said reactivity is defined as a relative intensity of a specific
antibody bound antigen as compared to its intensity signal in
healthy control individuals or wherein said reactivity is defined
as a relative intensity of a specific antibody bound antigen as
compared to its intensity signal individuals who have had a stroke
event.
105. The set of informative antigens according to claim 97, wherein
said reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to said intensity signals of said antigens in said healthy
control individuals or wherein said reactivity is further defined
as a set of informative antigens possessing a higher or a lower
intensity reactivity signals as compared to said intensity signals
of said antigens in individuals who have had a stroke event or
wherein said set of antigens are useful as therapeutic agents or
vaccines against the inflammatory process in AMI and stroke
conditions in an individual.
106. A kit useful for recognizing the development of an Acute
Myocardial Infarction (AMI) process in an individual, said kit
comprising: a. a set of antigens selected from a group consisting
of Brain-1 oligo-dendrocyte transcription factor, Synaptotagmin 1,
Tryptophan hydroxylase peptide 1, Tryptophan hydroxylase peptide 2,
BSP (Brain specific protein), myelin associated glycoprotein,
Fibronectin, Caspase 3, Myelin-associated metalloproteinase, CD68,
Bone morphogenic protein 4 and D4GDI (GDP-dissociation inhibitor)
attached to a substrate, wherein said antigens are further
characterised by their specific ability to form bound antibody
antigen reactivity complexes with human specimen containing
antibodies associated with susceptibility to AMI; b. a mixture of
secondary labeled antibodies specific for human immunoglobulin
types; c. components or solutions useful for reacting said antigens
with said antibodies of said specimen; d. components or solutions
useful for reacting said antigen antibody complexes with said
secondary labeled antibodies; and, e. instructions for reacting
said antigens with said components and said complexes with said
secondary antibodies in vitro so as to obtain results useful for
identifying an Acute Myocardial Infarction (AMI) process in an
individual.
Description
FIELD OF THE INVENTION
[0001] The present invention pertains to methods of identifying the
development of a cardiovascular disease in an individual. More
specifically, the present invention discloses means and methods of
recognizing the development of CVD, including an Acute Myocardial
Infarction (AMI) process in an individual.
BACKGROUND OF THE INVENTION
Cardiovascular Disease
[0002] Cardiovascular disease refers to the class of diseases that
involve the heart or blood vessels (arteries and veins). While the
term technically refers to any disease that affects the
cardiovascular system (as used in MeSH), it is usually used to
refer to those related to atherosclerosis (arterial disease). These
conditions have similar causes, mechanisms, and treatments. In
practice, cardiovascular disease is treated by cardiologists,
thoracic surgeons, vascular surgeons, neurologists, and
interventional radiologists, depending on the organ system that is
being treated. There is considerable overlap in the specialties,
and it is common for certain procedures to be performed by
different types of specialists in the same hospital.
[0003] Most Western countries face high and increasing rates of
cardiovascular disease. Each year, heart disease kills more
Americans than cancer [2]. Diseases of the heart alone caused 30%
of all deaths, with other diseases of the cardiovascular system
causing substantial further death and disability. Two out of three
cardiac deaths occur without any diagnosis of cardiovascular
disease [3]. Up until the year 2008, it was the major cause of
death and disability in the United States and most European
countries. A large histological study (PDAY) showed vascular injury
accumulates from adolescence, making primary prevention efforts
necessary from childhood [4] [5].
[0004] In contrast with gradual built-up of vessel narrowing, the
major cause of heart attack (Acute Myocardial Infarction--AMI) or
stroke is vulnerable (atherosclerotic) plaques. A vulnerable plaque
is an athermatous plaque, an unstable collection of white blood
cells (primarily macrophages) and lipids (including cholesterol) in
the wall of an artery which is particularly prone to rupture,
producing sudden vessel blockage and major problems, such as a
heart attack. Researchers have found that inflammation in the
arteries leads to the development of "soft" or vulnerable plaque,
which when released aggressively promotes blood clotting. When this
inflammation is combined with other stresses, such as high blood
pressure (increased mechanical stretching and contraction of the
arteries with each heart beat), it can cause the thin covering over
the plaque to split, spilling the contents of the vulnerable plaque
into the bloodstream. The sticky cytokines on the artery wall
capture blood cells (mainly platelets) that accumulate at the site
of injury. When these cells clump together, they form a clot,
sometimes large enough to block the artery. The most frequent cause
of a cardiac event following rupture of a vulnerable plaque is
blood clotting on top of the site of the ruptured plaque that
blocks the lumen of the artery, thereby stopping blood flow to the
tissues the artery supplies.
[0005] By the time that heart problems are detected, the underlying
cause (atherosclerosis) is usually quite advanced, having
progressed for decades. There is therefore increased emphasis on
preventing atherosclerosis by modifying risk factors, such as
healthy eating, exercise and avoidance of smoking. Established
cardiovascular risk factors, including dyslipidemia, smoking,
hypertension, and diabetes mellitus, have been incorporated into
algorithms for risk assessment in the general population [1], [2]
but these characteristics do not fully explain cardiovascular risk
[3], [4], [5] and other factors are sought.
Biomarkers
[0006] A biomarker is a substance or measurement that indicates
important facts about a living organism, usually a patient. It is
objectively measured and evaluated as an indicator of normal
biologic processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention. In other words, biomarkers
can provide the physician with useful information about: [0007] 1.
Biologic state of an individual; [0008] 2. Disease risk; [0009] 3.
Disease diagnosis; [0010] 4. Disease progression; [0011] 5.
Treatments of choice; [0012] 6. Monitoring responses to treatment;
[0013] 7. Endpoints for assessing treatment efficacy.
[0014] Biomarkers thus allow the physician a preventive or
therapeutic jump on the individual's disease process.
[0015] An effective biomarker, as a high concentration of "bad"
cholesterol, for example, can inform us about associated
complexities related to genes, heredity, metabolism, diet, blood
vessel walls, and the risks of vascular embolism and occlusion [3].
The biomarker, in short, reflects and summarizes all the agents and
processes that are needed to produce it--however many and complex
these agents and processes may be. A simple biomarker is
informative when it faithfully signifies for us the complex factors
from which the biomarker emerges.
[0016] There is thus substantial interest in the use of newer
biomarkers to identify persons who are at risk for the development
of cardiovascular disease and who could be targeted for preventive
measures. Some biomarkers are thought to offer a more detailed risk
of cardiovascular disease. However, the clinical value of these
biomarkers is questionable [6]. Many individual biomarkers have
been related to cardiovascular risk in ambulatory persons,
including levels of C-reactive protein [7], [8], B-type natriuretic
peptide [9], fibrinogen [10], D-dimer [11] and homocysteine [12].
Measurement of several biomarkers simultaneously (the "multimarker"
approach) could enhance risk stratification of ambulatory persons.
This approach was tried by Wang et al. (NEJM v.355.2631-2639), by
testing the usefulness of 10 previously reported biomarkers for
predicting death and major cardiovascular events in a large
population. However, the results were unpromising.
[0017] Currently, biomarkers which may reflect a higher risk of
cardiovascular disease include:
Higher fibrinogen and PAI-1 blood concentrations; Elevated
homocysteine, or even upper half of normal; Elevated blood levels
of asymmetric dimethylarginine; High inflammation as measured by
C-reactive protein; and, Elevated blood levels of Brain Natriuretic
Peptide (also known as B-type) (BNP) [7].
[0018] A fairly recent emphasis is on the link between low-grade
inflammation that hallmarks atherosclerosis and its possible
interventions. C-reactive protein (CRP) is an inflammatory marker
that may be present in increased levels in the blood in patients at
risk for cardiovascular disease. Its exact role in predicting
disease is the subject of debate.
[0019] In light of the above, it is clear that a long felt and
unmet need exists for a reliable method of identifying at a very
early stage, the beginning of a cardiovascular disease in an
individual. Such a method would make it possible to act quickly
without having to wait for real-time events to take place. For
example, anticipating, treating and monitoring the state of the
individual would improve the ability to prevent a future heart
attack or an impending stroke event.
SUMMARY OF THE INVENTION
[0020] It is hence one object of the present invention to provide a
method of recognizing the development of an Acute Myocardial
Infarction (AMI) process in an individual. The aforementioned
method comprises steps of: profiling specific antibody reactivities
or biomarkers associated with AMI susceptibility, and analysing
results obtained. The profiling step further comprises steps of (a)
attaching a set of defined antigens to a substrate; (b) obtaining a
biological fluid derived specimen from an individual, said specimen
containing a specific antibody repertoire; and, (c) binding said
antibodies of said biological fluid specimen to said attached
antigens thereby forming bound antibody antigen complexes.
[0021] It is a further object of the present invention to disclose
the method as defined above, wherein said profiling further
comprises the steps of obtaining said defined antigens
characterized by association with a variety of conditions selected
from a group consisting of inflammation, angiogenesis, apoptosis or
any other condition involving immune regulation or any combination
thereof.
[0022] It is a further object of the present invention to disclose
the method as defined above, wherein said profiling further
comprising the steps of selecting said biological fluid derived
specimen from a group consisting of plasma, serum, blood,
cerebrospinal fluid, synovial fluid, sputum, saliva, tears, lymph
specimen, or any other biological fluid known in the art.
[0023] It is a further object of the present invention to disclose
the method as defined above, wherein said profiling further
comprises steps of selecting said substrate from a group consisting
of microarray chip, microarray wafers, microarray strips, ELISA
microwells, multiplex beads or any substrate known in the relevant
art.
[0024] It is a further object of the present invention to disclose
the method as defined above, further comprising the steps of
selecting said antigens from a group consisting of protein,
polypeptide, peptide, nucleic acids, lipids, glycosylated
molecules, polypeptides with modifications, polysaccharides or any
other biological molecule known in the art.
[0025] It is a further object of the present invention to disclose
the method as defined above, further comprising the steps of
defining said antibody reactivities as IgG, IgM, IgA, IgE antibody
isotypes or more specific sub-isotypes of said antibodies.
[0026] It is a further object of the present invention to disclose
the method as defined above, wherein said analyzing results
obtained further comprising the steps of measuring said bound
antibody complexes.
[0027] It is a further object of the present invention to disclose
the method as defined above, wherein said measuring additionally
comprises steps of labeling said bound antibody complexes.
[0028] It is a further object of the present invention to disclose
the method as defined above, wherein said labeling further
comprises the steps of selecting a labeling signal from a group
consisting of fluorescent, chemiluminescent, absorptive,
electronic, radioactive, paramagnetic or any labeling signal known
in the art, or combination thereof.
[0029] It is a further object of the present invention to disclose
the method as defined above, wherein said labeling step
additionally comprises steps of reacting said bound antibody
complexes with labeled mixture of secondary antibodies specific for
human immunoglobulin types so as to obtain labeled bound antibody
antigen complexes.
[0030] It is a further object of the present invention to disclose
the method as defined above, wherein said analyzing step
additionally comprises steps of: (a) scanning the signal produced
by said bound antibody complexes so as to form a reactivity matrix;
(b) measuring the intensity of said signal; (c) generating and
recording results of data associated with said intensity of said
signal; (d) producing said results in the form of a signature,
profile, set, pattern or matrix characteristic to said individual;
(e) comparing the results with a databank containing data
concerning said antibody reactivities in populations susceptible to
AMI thereby identifying the development of an Acute Myocardial
Infarction (AMI) process in said individual.
[0031] It is a further object of the present invention to disclose
the method as defined above, wherein said measuring and scanning
steps are selected from a group consisting of Surface Plasmon
Resonance (SPR), ellipsometry, laser scanning, light detecting,
photon detecting via a photon multiplier, photographing with a
digital camera based system or video system, radiation counting,
fluorescence detecting, electronic, magnetic detecting or any other
technique known in the art or combination thereof.
[0032] It is a further object of the present invention to disclose
the method as defined above, wherein said analyzing step further
comprises the steps of defining said reactivity as a relative
intensity signal of a specific bound antibody as compared to its
intensity signal in healthy control individuals.
[0033] It is a further object of the present invention to disclose
the method as defined above, wherein said analyzing step further
comprises the steps of defining said reactivity as a relative
intensity signal of a specific bound antibody as compared to its
intensity signal in individuals who have had a stroke event.
[0034] It is a further object of the present invention to disclose
the method as defined above, wherein said analyzing step further
comprises the steps of identifying a set of informative antigens
possessing higher or a lower intensity reactivity matrix signals as
compared to said intensity signals of said antibodies in healthy
control individuals.
[0035] It is a further object of the present invention to disclose
the method as defined above, wherein said analyzing step further
comprises the steps of identifying a set of informative antigens
possessing higher or a lower intensity reactivity matrix signals as
compared to said intensity signals of said antibodies in
individuals who have had a stroke event.
[0036] It is a further object of the present invention to disclose
the method as defined above, wherein said method further comprises
steps of: (a) obtaining said antigens attached to said substrate;
(b) packaging said substrate attached antigens in a kit containing
instructions; and, (c) following the steps according to said
instructions.
[0037] It is another core object of the invention to provide a set
of informative antigens useful for recognizing the development of
an Acute Myocardial Infarction (AMI) process in an individual.
According to certain embodiments of the present invention, some of
the herein disclosed informative antigens are specific peptides.
The aforementioned set of antigens is selected from a group
consisting of Brain-1 oligo-dendrocyte transcription factor,
Synaptotagmin 1, Tryptophan hydroxylase peptide 1, Tryptophan
hydroxylase peptide 2, BSP (Brain specific protein), myelin
associated glycoprotein, Fibronectin, Caspase 3, Myelin-associated
metalloproteinase, CD68, Bone morphogenic protein 4 and D4GDI
(GDP-dissociation inhibitor). The aforementioned antibody
reactivities are profiled by: profiling means for profiling
specific antibody reactivities or biomarkers associated with AMI
susceptibility, and analyzing means for analyzing results obtained.
The aforementioned profiling means further comprising: (a)
attaching means for attaching a set of defined antigens to a
substrate; (b) a biological fluid derived specimen from an
individual, said specimen containing a specific antibody
repertoire; and, (c) binding means for binding said antibodies of
said biological fluid specimen to said attached antigens thereby
forming bound antibody antigen complexes.
[0038] It is a core aspect of the invention that the antigens are
further characterised by their specific antibody reactivity with
human antibodies associated with susceptibility to AMI.
[0039] Moreover, it is a core aspect of the present invention to
provide means and methods to differentiate diagnoses of stroke and
AMI. The finding that stroke and AMI differ in their antigen chip
signature is highly novel, even though both conditions have been
assumed to involve similar atherosclerotic processes. The
diagnostic methods for stroke provided herein are particularly
useful in differentiating from brain tumors for example, which may
present with similar initial symptoms.
[0040] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
defined antigens characterized by association with a variety of
conditions selected from a group consisting of inflammation,
angiogenesis, apoptosis or any other condition involving immune
regulation or any combination thereof.
[0041] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
defined antigens are attached to a substrate selected from a group
consisting of microarray chip, microarray wafers, microarray
strips, ELISA microwells, multiplex beads or any substrate known in
the relevant art.
[0042] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
defined antigens are selected form a group consisting of protein,
polypeptide, peptide, nucleic acids, lipids, glycosylated
molecules, polypeptides with modifications, polysaccharides or any
other biological molecule known in the art.
[0043] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
antigens are further characterized by their specific capability to
discriminate between an individual harboring the development of an
AMI process and a healthy control individual.
[0044] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
antigens are further characterized by their specific capability to
discriminate between the development of an AMI process and a stroke
process in an individual.
[0045] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
human antibodies are derived from a biological fluid specimen
selected from a group consisting of plasma, serum, blood,
cerebrospinal fluid, synovial fluid, sputum, saliva, tears, lymph
specimen, or any biological fluid known in the art.
[0046] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
antibody reactivities are defined as IgG, IgM, IgA, IgE antibody
isotypes or more specific sub-isotypes of said antibodies.
[0047] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
specific antibody reactivity is analyzable by using measuring
means.
[0048] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
specific antibody reactivity is analyzable by using labeling
means.
[0049] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
labeling means is adapted to label said human antibodies with a
mixture of secondary antibodies specific for human immunoglobulin
types to form a reactivity matrix.
[0050] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
labeling means comprises labeling signal selected form a group
consisting of fluorescent, chemiluminescent, absorptive,
electronic, radioactive, paramagnetic or any labeling signal known
in the art, or combination thereof.
[0051] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
specific antibody reactivity is further analyzable by: (a) using a
scanning means for scanning said reactivity matrix signal; (b)
using a measuring means for measuring the intensity of said signal;
(c) using generating and recording means for generating and
recording results associated with said reactivity matrix signal;
(d) using producing means for producing said results in the form of
a signature, profile, set, pattern or matrix characteristic to said
individual; and, (e) using means for comparing the results with a
databank containing data concerning said antibody reactivities in
populations susceptible to AMI, thereby identifying the development
of an Acute Myocardial Infarction (AMI) process in said
individual.
[0052] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
measuring and scanning means are selected from a group consisting
of Surface Plasmon Resonance (SPR), ellipsometry, laser scanning,
light detecting, photon detecting via a photon multiplier,
photographing with a digital camera based system or video system,
radiation counting, fluorescence detecting, electronic, magnetic
detecting or any other technique known in the art or combination
thereof
[0053] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
reactivity is defined as a relative intensity of a specific
antibody bound antigen as compared to its intensity signal in
healthy control individuals.
[0054] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
reactivity is defined as a relative intensity of a specific
antibody bound antigen as compared to its intensity signal
individuals who have had a stroke event.
[0055] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to said intensity signals of said antigens in said healthy
control individuals.
[0056] It is a further object of the present invention to disclose
the set of informative antigens as defined above, wherein said
reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to said intensity signals of said antigens in individuals
who have had a stroke event.
[0057] It is another core object of the invention to provide a
reactivity matrix useful for recognizing the development of an
Acute Myocardial Infarction (AMI) process in an individual,
comprising at least one substrate embedded with defined antigens.
According to certain embodiments of the present invention, some of
the herein disclosed defined antigens are specific peptides. The
aforementioned defined antigens are selected from a group
consisting of Brain-1 oligo-dendrocyte transcription factor,
Synaptotagmin 1, Tryptophan hydroxylase peptide 1, Tryptophan
hydroxylase peptide 2, BSP (Brain specific protein), myelin
associated glycoprotein, Fibronectin, Caspase 3, Myelin-associated
metalloproteinase, CD68, Bone morphogenic protein 4 and D4GDI
(GDP-dissociation inhibitor), wherein said antigens are further
characterised by their specific antibody reactivity with human
specimen containing antibodies associated with susceptibility to
AMI.
[0058] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said human
antibodies are derived from a biological fluid specimen selected
from a group consisting of plasma, serum, blood, cerebrospinal
fluid, synovial fluid, sputum, saliva, tears, lymph specimen, or
any biological fluid known in the art.
[0059] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said antigens are
attached to a substrate selected from a group consisting of
microarray chip, microarray wafers, microarray strips, ELISA
microwells, multiplex beads or any substrate known in the relevant
art.
[0060] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said antigens are
selected form a group consisting of protein, polypeptide, peptide,
nucleic acids, lipids, glycosylated molecules, polypeptides with
modifications, polysaccharides or any other biological molecule
known in the art.
[0061] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said antibody
reactivities are defined as IgG, IgM, IgA, IgE antibody isotypes or
as more specific sub-isotypes of said antibodies.
[0062] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said specific
antibody reactivity is analyzable by using measuring means.
[0063] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said specific
antibody reactivity is analyzable by using labeling means.
[0064] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said labeling means
is adapted to label said human antibodies with a mixture of
secondary antibodies specific for human immunoglobulin types to
form a reactivity matrix.
[0065] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said labeling means
comprises labeling signal selected form a group consisting of
fluorescent, chemiluminescent, absorptive, electronic, radioactive,
paramagnetic or any labeling signal known in the art, or
combination thereof.
[0066] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said specific
antibody reactivity is further analyzable by: (a) using a scanning
means for scanning said reactivity matrix signal; (b) using a
measuring means for measuring the intensity of said signal; (c)
using generating and recording means for generating and recording
results associated with said reactivity matrix signal; (d) using
producing means for producing said results in the form of a
signature, profile, set, pattern or matrix characteristic to said
individual; and, (e) using means for comparing the results with a
databank containing data concerning said antibody reactivities in
populations susceptible to AMI, thereby identifying the development
of an Acute Myocardial Infarction (AMI) process in said
individual.
[0067] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said measuring and
scanning means are selected from a group consisting of Surface
Plasmon Resonance (SPR), ellipsometry, laser scanning, light
detecting, photon detecting via a photon multiplier, photographing
with a digital camera based system or video system, radiation
counting, fluorescence detecting, electronic, magnetic detecting or
any other technique known in the art or combination thereof.
[0068] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said reactivity is
defined as a relative intensity of a specific antibody bound
antigen as compared to its intensity signal in healthy control
individuals.
[0069] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said reactivity is
defined as a relative intensity of a specific antibody bound
antigen as compared to its intensity signal in individuals who have
had a stroke event.
[0070] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said reactivity is
further defined as a set of informative antigens possessing a
higher or a lower intensity reactivity signals as compared to said
intensity signals of said antigens in said healthy control
individuals.
[0071] It is a further object of the present invention to disclose
the reactivity matrix as defined above, wherein said reactivity is
further defined as a set of informative antigens possessing a
higher or a lower intensity reactivity signals as compared to said
intensity signals of said antigens in individuals who have had a
stroke event.
[0072] It is another core object of the invention to disclose a
system for recognizing the development of an Acute Myocardial
Infarction (AMI) process in an individual. The aforementioned
system comprising: (a) a substrate embedded with antigens; (b)
biological fluid derived specimen containing a specific antibody
repertoire; (c) means for physically contacting said specimen with
the surface of said substrate; (d) means for binding said
antibodies of said specimen to said antigens embedded in said
substrate so as to form bound antibody antigen complexes; (e) a
measuring device for quantifying the intensity signal of said bound
antibodies; and, (f) a data processor for producing said intensity
signals in the form of a signature, profile, set, pattern or matrix
characteristic to said individual. According to certain embodiments
of the present invention, some of the herein disclosed antigens are
specific peptides. The aforementioned antigens, are selected from a
group consisting of Brain-1 oligo-dendrocyte transcription factor,
Synaptotagmin 1, Tryptophan hydroxylase peptide 1, Tryptophan
hydroxylase peptide 2, BSP (Brain specific protein), myelin
associated glycoprotein, Fibronectin, Caspase 3, Myelin-associated
metalloproteinase, CD68, Bone morphogenic protein 4 and D4GDI
(GDP-dissociation inhibitor), further wherein said antigens are
characterised by their specific reactivity with said specimen
containing said antibodies associated with susceptibility to AMI,
when bound under defined conditions.
[0073] It is a further object of the present invention to disclose
the system as defined above, wherein said human antibodies are
derived from a biological fluid specimen selected from a group
consisting of plasma, serum, blood, cerebrospinal fluid, synovial
fluid, sputum, saliva, tears, lymph specimen, or any biological
fluid known in the art.
[0074] It is a further object of the present invention to disclose
the system as defined above, wherein said antigens are attached to
a substrate selected from a group consisting of microarray chip,
microarray wafers, microarray strips, ELISA microwells, multiplex
beads or any substrate known in the relevant art.
[0075] It is a further object of the present invention to disclose
the system as defined above, wherein said antigens are selected
form a group consisting of protein, polypeptide, peptide, nucleic
acid, lipid, glycosylated molecules, polypeptides with
modifications, polysaccharides or any other biological molecule
known in the art.
[0076] It is a further object of the present invention to disclose
the system as defined above, wherein said antibody reactivities are
defined as IgG, IgM, IgA, IgE antibody isotypes or as more specific
sub-isotypes of said antibodies.
[0077] It is a further object of the present invention to disclose
the system as defined above, wherein said specific antibody
reactivity is analyzable by using measuring means.
[0078] It is a further object of the present invention to disclose
the system as defined above, wherein said specific antibody
reactivity is analyzable by using labeling means.
[0079] It is a further object of the present invention to disclose
the system as defined above, wherein said labeling means is adapted
to label said human antibodies with a mixture of secondary
antibodies specific for human immunoglobulin types to form a
reactivity matrix.
[0080] It is a further object of the present invention to disclose
the system as defined above, wherein said labeling means comprises
labeling signal selected form a group consisting of fluorescent,
chemiluminescent, absorptive, electronic, radioactive, paramagnetic
or any labeling signal known in the art, or combination
thereof.
[0081] It is a further object of the present invention to disclose
the system as defined above, wherein said specific antibody
reactivity is further analyzable by: (a) using a scanning means for
scanning said reactivity matrix signal; (b) using a measuring means
for measuring the intensity of said signal; (c) using generating
and recording means for generating and recording results associated
with said reactivity matrix signal; (d) using producing means for
producing said results in the form of a signature, profile, set,
pattern or matrix characteristic to said individual; and, (e) using
means for comparing the results with a databank containing data
concerning said antibody reactivities in populations susceptible to
AMI, thereby identifying the development of an Acute Myocardial
Infarction (AMI) process in said individual.
[0082] It is a further object of the present invention to disclose
the system as defined above, wherein said measuring and scanning
means are selected from a group consisting of Surface Plasmon
Resonance (SPR), ellipsometry, laser scanning, light detecting,
photon detecting via a photon multiplier, photographing with a
digital camera based system or video system, radiation counting,
fluorescence detecting, electronic, magnetic detecting or any other
technique known in the art or combination thereof.
[0083] It is a further object of the present invention to disclose
the system as defined above, wherein said reactivity is defined as
a relative intensity signal of a specific antibody bound antigen as
compared to its intensity signal in healthy control
individuals.
[0084] It is a further object of the present invention to disclose
the system as defined above, wherein said reactivity is defined as
a relative intensity signal of a specific antibody bound antigen as
compared to its intensity signal in individuals who have had a
stroke event.
[0085] It is a further object of the present invention to disclose
the system as defined above, wherein said reactivity is further
defined as a set of informative antigens possessing a higher or a
lower intensity reactivity signals as compared to said intensity
signals of said antigens in said healthy control individuals.
[0086] It is a further object of the present invention to disclose
the system as defined above, wherein said reactivity is further
defined as a set of informative antigens possessing a higher or a
lower intensity reactivity signals as compared to said intensity
signals of said antigens in individuals who have had a stroke
event.
[0087] It is another core object of the invention to disclose a kit
useful for recognizing the development of an Acute Myocardial
Infarction (AMI) process in an individual. The aforementioned kit
comprising: (a) a set of antigens selected from a group consisting
of Brain-1 oligo-dendrocyte transcription factor, Synaptotagmin 1,
Tryptophan hydroxylase peptide 1, Tryptophan hydroxylase peptide 2,
BSP (Brain specific protein), myelin associated glycoprotein,
Fibronectin, Caspase 3, Myelin-associated metalloproteinase, CD68,
Bone morphogenic protein 4 and D4GDI (GDP-dissociation inhibitor)
attached to a substrate, wherein said antigens are further
characterised by their specific ability to form bound antibody
antigen reactivity complexes with human specimen containing
antibodies associated with susceptibility to AMI; (b) a mixture of
secondary labeled antibodies specific for human immunoglobulin
types; (c) components or solutions useful for reacting said
antigens with said antibodies of said specimen; (d) components or
solutions useful for reacting said antigen antibody complexes with
said secondary labeled antibodies; and, (e) instructions for
reacting said antigens with said components and said complexes with
said secondary antibodies in vitro so as to obtain results useful
for identifying an Acute Myocardial Infarction (AMI) process in an
individual.
[0088] It is a further object of the present invention to disclose
the kit as defined above, wherein said antigens characterized by
association with a variety of conditions selected from a group
consisting of inflammation, angiogenesis, apoptosis or any other
condition involving immune regulation or combination thereof.
[0089] It is a further object of the present invention to disclose
the kit as defined above, wherein said human antibodies are derived
from a biological fluid specimen selected from a group consisting
of plasma, serum, blood, cerebrospinal fluid, synovial fluid,
sputum, saliva, tears, lymph specimen, or any biological fluid
known in the art.
[0090] It is a further object of the present invention to disclose
the kit as defined above, wherein said antigens are attached to a
substrate selected from a group consisting of microarray chip,
microarray wafers, microarray strips, ELISA microwells, multiplex
beads or any substrate known in the relevant art.
[0091] It is a further object of the present invention to disclose
the kit as defined above, wherein said antigens are selected form a
group consisting of protein, polypeptide, peptide, nucleic acid,
lipid, glycosylated molecules, polypeptides with modifications,
polysaccharides or any other biological molecule known in the
art.
[0092] It is a further object of the present invention to disclose
the kit as defined above, wherein said antibody reactivities are
defined as IgG, IgM, IgA, IgE antibody isotypes or as more specific
sub-isotypes of said antibodies.
[0093] It is a further object of the present invention to disclose
the kit as defined above, wherein said specific antibody reactivity
is analyzable by using measuring means.
[0094] It is a further object of the present invention to disclose
the kit as defined above, wherein said specific antibody reactivity
is analyzable by using labeling means.
[0095] It is a further object of the present invention to disclose
the kit as defined above, wherein said labeling means are adapted
to label said human antibodies with a mixture of secondary
antibodies specific for human immunoglobulin types to form a
reactivity matrix.
[0096] It is a further object of the present invention to disclose
the kit as defined above, wherein said labeling means comprises
labeling signal selected form a group consisting of fluorescent,
chemiluminescent, absorptive, electronic, radioactive, paramagnetic
or any labeling signal known in the art, or combination
thereof.
[0097] It is a further object of the present invention to disclose
the kit as defined above, wherein said specific antibody reactivity
is further analyzable by: (a) using a scanning means for scanning
said reactivity matrix signal; (b) using a measuring means for
measuring the intensity of said signal; (c) using generating and
recording means for generating and recording results associated
with said reactivity matrix signal; (d) using producing means for
producing said results in the form of a signature, profile, set,
pattern or matrix characteristic to said individual; and, (e) using
means for comparing the results with a databank containing data
concerning said antibody reactivities in populations susceptible to
AMI, thereby identifying the development of an Acute Myocardial
Infarction (AMI) process in said individual.
[0098] It is a further object of the present invention to disclose
the kit as defined above, wherein said measuring and scanning means
are selected from a group consisting of Surface Plasmon Resonance
(SPR), ellipsometry, laser scanning, light detecting, photon
detecting via a photon multiplier, photographing with a digital
camera based system or video system, radiation counting,
fluorescence detecting, electronic, magnetic detecting or any other
technique known in the art or combination thereof.
[0099] It is a further object of the present invention to disclose
the kit as defined above, wherein said reactivity is defined as a
relative intensity of a specific antibody bound antigen as compared
to its intensity signal in healthy control individuals.
[0100] It is a further object of the present invention to disclose
the kit as defined above, wherein said reactivity is defined as a
relative intensity of a specific antibody bound antigen as compared
to its intensity signal in individuals who have had a stroke
event.
[0101] It is a further object of the present invention to disclose
the kit as defined above, wherein said reactivity is further
defined as a set of informative antigens possessing a higher or a
lower intensity reactivity signals as compared to said intensity
signals of said antigens in said healthy control individuals.
[0102] It is a further object of the present invention to disclose
the kit as defined above, wherein said reactivity is further
defined as a set of informative antigens possessing a higher or a
lower intensity reactivity signals as compared to said intensity
signals of said antigens in individuals who have had a stroke
event.
[0103] It is another aspect of the invention to provide antigens
such as synaptotagmin 1, tryptophan hydroxylase peptide 1,
tryptophan hydroxylase peptide 2, BSP (Brain Specific Protein), as
therapeutic agents or vaccines against the inflammatory process in
AMI and stroke. Tryptophan hydroxylase is an exemplar of an
informative antigen which may be useful for treating
atherosclerosis. Mouse models are being employed for this
purpose.
[0104] It is another core aspect of the invention to disclose a
method for arresting the development of an AMI process in a
patient. The aforementioned method comprising steps of: (a)
profiling the specific antibody reactivities or biomarkers
associated with AMI susceptibility, (b) determining the specific
AMI associated antigens bound by said antibody repertoires of said
individuals specimens; (c) designing a drug comprising at least one
of said specific antigens or at least one epitope of said specific
antigens; and, (d) administering said drug to said patient thereby
arresting the development of an AMI process in a patient. The
aforementioned profiling step comprises steps of: (i) attaching a
set of defined antigens to a substrate; (ii) obtaining biological
fluid derived specimens from a plurality of individuals in a
population, each of said specimens containing their specific
antibody repertoire; (iii) binding said antibody repertoire of each
of said specimens to said attached antigens thereby forming bound
antibody antigen complexes; and, (iv) analysing results
obtained.
[0105] It is another core aspect of the invention to disclose a
method for screening for candidate drugs for arresting the
development of an AMI process, in a patient. The aforementioned
method comprising the steps of: (a) profiling the specific antibody
reactivities associated with AMI susceptibility of individuals, (b)
determining the specific AMI associated antigens bound by said
antibody repertoires of said individuals; and, (c) selecting said
candidate drugs as appropriate for administration as immunotherapy
for arresting the development of an AMI process, in an individual,
wherein said candidate drug contains at least one of said specific
AMI associated antigens or at least one epitope of said specific
antigens. The aforementioned profiling step further comprising the
steps of: (i) attaching a set of defined antigens to a substrate;
(ii) obtaining biological fluid derived specimens from a plurality
of individuals in a population, each of said specimens containing
their specific antibody repertoire; (iii) binding said antibody
repertoire of each of said specimens to said attached antigens
thereby forming antibody antigen complexes; and, (iv) analysing
results obtained.
BRIEF DESCRIPTION OF THE FIGURES
[0106] In order to understand the invention and to see how it may
be implemented in practice, a plurality of embodiments is now
described, by way of non-limiting example only, with reference to
the accompanying drawings, wherein:
[0107] FIG. 1 is a flow diagram presenting the different phases
leading to the elucidation of the AMI informative antigens of the
present invention.
[0108] FIG. 2 is a graphic illustration representing the antibody
reactivity levels of Tryptophan hydroxylase (peptide 1) informative
antigen in each of the three specimen groups;
[0109] FIG. 3A is a graphic illustration representing the antibody
reactivity levels of Tryptophan hydroxylase (peptide 1) antigen in
Acute Myocardial Infarction (AMI) versus the Healthy control
specimen groups;
[0110] FIG. 3B is a graphic illustration representing the antibody
reactivity levels of Tryptophan hydroxylase (peptide 1) antigen in
Acute Myocardial Infarction (AMI) versus the Healthy control
specimen groups;
[0111] FIG. 3C is a graphic illustration representing the
correlated antibody reactivity levels of Tryptophan hydroxylase
peptides 1 and peptide 2 in Acute Myocardial Infarction (AMI)
versus the Healthy control specimen groups;
[0112] FIG. 3D is a graphic illustration representing the antibody
reactivity levels of synaptotagmin1 antigen in Acute Myocardial
Infarction (AMI) versus the Healthy control specimen groups;
[0113] FIG. 4A is a graphic illustration representing the antibody
reactivity distribution of Tryptophan hydroxylase (peptide 2)
antigen in the AMI versus the Stroke specimen groups;
[0114] FIG. 4B is a graphic illustration representing the antibody
reactivity distribution of Tryptophan hydroxylase (peptide 1)
antigen in the AMI versus the Stroke specimen groups;
[0115] FIG. 4C is a graphic illustration representing the antibody
reactivity distribution of Synaptotagmin1 antigen in the AMI versus
the Stroke specimen groups;
[0116] FIG. 4D is a graphic illustration representing the antibody
reactivity distribution of Myelin-Associated Metalloproteinase
antigen in the AMI versus the Stroke specimen groups;
[0117] FIG. 4E is a graphic illustration representing the antibody
reactivity distribution of Bone Morphogenic Protein 4 (BMP4)
antigen in the AMI versus the Stroke specimen groups; and,
[0118] FIG. 4F is a graphic illustration representing the antibody
reactivity distribution of GDP-Dissociation Inhibitor (D4GDI)
antigen in the AMI versus the Stroke specimen groups.
DETAILED DESCRIPTION OF THE INVENTION
[0119] In the following description, various aspects of the
invention will be described. For the purposes of explanation,
specific configurations and details are set forth in order to
provide a thorough understanding of the invention. However, it will
be also apparent to one skilled in the art that the invention may
be practiced without specific details presented herein.
Furthermore, well-known features may be omitted or simplified in
order not to obscure the invention.
[0120] It is herein disclosed that the present invention provides
methods for identifying the development of an Acute Myocardial
Infarction (AMI) process in an individual. More specifically, the
current invention discloses a set of informative antibody
reactivities which surprisingly and unexpectedly, are capable of
identifying the development of an AMI process in an individual.
[0121] It is within the scope of the invention to disclose a set of
informative antigens which unpredictably can discriminate between
an AMI process and an impending stroke event in an individual.
[0122] As is known, Acute Myocardial Infarction (AMI), also known
as a heart attack, occurs when the blood supply to part of the
heart is interrupted. This is most commonly due to occlusion
(blockage) of a coronary artery following the rupture of a
vulnerable atherosclerotic plaque. The aforementioned plaque
consists of an unstable collection of lipids (like cholesterol) and
white blood cells (especially macrophages) in the wall of an
artery. The resulting ischemia (restriction in blood supply) and
oxygen shortage, if left untreated for a sufficient period, can
cause damage and/or death (infarction) of heart muscle tissue
(myocardium).
[0123] A stroke is the rapidly developing loss of brain functions
due to a disturbance in the blood vessels supplying blood to the
brain. This can be due to ischemia (lack of blood supply) caused by
thrombosis or embolism or due to a hemorrhage. As a result, the
affected area of the brain is unable to function, leading to
inability to move one or more limbs on one side of the body,
inability to understand or formulate speech or inability to see one
side of the visual field. Stroke is also referred to as
Cerebrovascular Event or CVE.
[0124] Thus, both AMI and stroke conditions are acute vascular
events which result from atherosclerotic processes but in different
locations in the vascular system.
[0125] Hence, the herein disclosed ability to discriminate between
Acute Myocardial Infarction (AMI) and stroke is significant and
unexpected. It is highly novel that the present invention can
discriminate between different variations of atherosclerosis
process.
[0126] As used herein the terms `autoimmunity`, or `autoreactivity`
or `autoantibodies` refer to the immune system which expresses both
the genetic endowment of the individual and the life experience of
the individual. The immune system in both its innate and adaptive
arms can be viewed as a type of biological health-maintenance
system. Health maintenance, performed by the immune system,
requires access of the system to information regarding the biologic
state of the body. Towards this aim, the immune system, like
health-maintenance systems generally, needs biomarkers.
[0127] The immune system must be sensitive to the state of the body
and intimately responsive to it, thus autoimmunity has to be built
into the system and a degree of autoimmunity must be
physiological.
[0128] As suggested by Prof. Cohen and Prof. Sahar's research
groups, many natural antibodies detected in healthy subjects are
autoantibodies; which bind self-molecules [3, 22]. The specific
self-molecules recognized by these autoantibodies appear to form
clinically defining signatures: some autoantibodies create a
pattern that heralds susceptibility to a future disease, e.g. an
autoimmune disease [3, 22], while a different autoantibody pattern
can mark resistance to the disease.
[0129] It has been proposed that the natural autoantibodies and
auto-reactive T cells in healthy individuals may be directed to a
specific and limited set of self-molecules; this selective
autoimmunity has been termed the immunological homunculus or
immunculus--the immune system's internal representation of the
body. An important function of the immunological homunculus is to
create and detect biomarkers.
[0130] The autoantibodies which are presented at birth in the human
cord blood--the congenital immunological homunculus--were studied
using an antigen microarray chip [2]. IgG, IgA and IgM antibody
types, binding to about 300 self-antigens, were surveyed. These
autoantibody reactivities that were found by the above-mentioned
research groups provides the immune system with the right kind of
biomarker information about body state, needed to manage a healthy
inflammatory program. Thus, it was proposed by the researches that
the immunological homunculus helps in maintaining health in the
following ways: [0131] Natural autoimmunity could help the body
remove troublesome waste molecules and cells [23]. [0132] Natural
autoimmune T cells and B cells and autoantibodies could provide an
early immune response to pathogens expressing molecules that are
cross-reactive with particular self-antigens. [0133] Natural
autoimmunity is also proposed to prevent pathogenic autoimmunity by
generating regulatory circuits or by blocking the access of
potentially pathogenic agents to key self-antigens [3].
[0134] It is within the scope of the present invention to disclose
the self-antigens recognized by homuncular agents identified as
biomarkers that inform the immune system of the state of the body,
both locally and globally. This enables to gain insight into health
and disease conditions of the body, by studying an individual's
patterns of autoimmunity.
[0135] It is herein disclosed that the immunological homunculus, as
a natural biomarker system for immune health-maintenance, might
serve as a medical health-maintenance system. Moreover, in
accordance with a further aspect of the present invention,
identifying the biomarker system of an individual might be useful
in adjusting correct medicine for a patient.
[0136] As used herein, the term `Cardiovascular disease` refers to
any disease that involves the heart or blood vessels (arteries and
veins), and more specifically it refers to atherosclerosis, which
is an arterial disease. Types of cardiovascular diseases include:
Aneurysm, Angina, Atherosclerosis, Cerebrovascular Accident
(Stroke), Cerebrovascular disease, Congestive Heart Failure,
Coronary Artery Disease and Myocardial infarction (Heart Attack).
These conditions have similar causes, mechanisms, and
treatments.
[0137] It is herein acknowledged that the present invention
discloses methods and means of recognizing the development of a
cardiovascular disease (CVD) in an individual. The aforementioned
CVD's arise from all types of cardiovascular diseases listed below
in a non limiting manner:
[0138] Aneurysm Angina Arrhythmia Atherosclerosis, Cardiomyopathy,
Cerebrovascular Accident (Stroke), Cerebrovascular disease,
Congenital heart disease, Congestive Heart Failure, Myocarditis,
Valve Disease, Coronary Artery Disease, Dilated cardiomyopathy,
Diastolic dysfunction, Endocarditis, High Blood Pressure
(Hypertension), Hypertrophic cardiomyopathy, Mitral valve
prolapsed, Myocardial infarction (Heart Attack), Venous
Thromboembolism Ischaemic.
[0139] Acute coronary CHD: CAD-Coronary thrombosis-Coronary
vasospasm-Coronary artery aneurysm Angina pectoris (Prinzmetal's
angina)-Myocardial infarction (heart attack) Myocardial
rupture-Dressler's syndrome Layers Pericardium Pericarditis (Acute,
Constrictive)-Pericardial effusion-Cardiac
tamponade-Hemopericardium Myocardium Myocarditis Cardiomyopathy:
Dilated (Alcoholic)-Hypertrophic-Restrictive (Loeffler
endocarditis, Cardiac amyloidosis) Arrhythmogenic right ventricular
dysplasia Endocardium/valves Endocarditis (Subacute bacterial
endocarditis, Libman-Sacks endocarditis, Nonbacterial thrombotic
endocarditis) mitral (regurgitation, prolapse, stenosis)-aortic
(stenosis, insufficiency)-tricuspid (stenosis,
insufficiency)-pulmonary (stenosis, insufficiency)
Conduction/arrhythmia. Heart block AV (1.degree., 2.degree.,
3.degree.)-Bundle branch (Left,
Right)-Bifascicular/Trifascicular-Sinoatrial-Sick sinus
syndrome-Adams-Stokes syndrome Pre-excitation syndrome
Wolff-Parkinson-White-Lown-Ganong-Levine.
[0140] Tachycardia Paroxysmal-Supraventricular (AV nodal reentrant,
Accelerated idioventricular rhythm, Sinus)-Ventricular (Torsades de
pointes, Catecholaminergic polymorphic) Premature contraction
Atrial-Ventricular Flutter/fibrillation Atrial flutter-Ventricular
flutter -Atrial fibrillation (Familial)-Ventricular fibrillation.
Pacemaker Wandering pacemaker-Ectopic pacemaker-Parasystole Long QT
syndrome Romano-Ward syndrome-Andersen-Tawil syndrome-Jervell and
Lange-Nielsen syndrome Cardiac arrest Sudden cardiac death-Asystole
Other Cardiomegaly-Ventricular hypertrophy (Left, Right/Cor
pulmonale) Heart failure (Cardiac asthma)-Rheumatic fever Arterial
occlusive disease/peripheral vascular disease Arteriosclerosis:
Atherosclerosis-Intermittent claudication-Atheroma-Monckeberg's
arteriosclerosis Stenosis (Renal artery stenosis, Carotid artery
stenosis)-Fibromuscular dysplasia-Degos disease-Aortoiliac
occlusive disease Raynaud's phenomenon/Raynaud's
disease-Erythromelalgia Aneurysm Aortic aneurysm/Abdominal aortic
aneurysm-Cerebral aneurysm-Coronary artery aneurysm-Intracranial
berry aneurysm-Dissection (Aortic, Carotid,
Vertebral)-Pseudoaneurysm Other Arteritis (Aortitis)-Buerger's
disease Arteriovenous fistula-Hereditary hemorrhagic telangiectasia
Nevus (Spider angioma, Halo nevus)
[0141] Venous thrombosis/Phlebitis/Thrombophlebitis primarily lower
limb (Deep vein thrombosis) abdomen (May-Thurner syndrome, Portal
vein thrombosis, Budd-Chiari syndrome, Renal vein thrombosis) upper
limb (Paget-Schroetter disease) head (Cerebral venous sinus
thrombosis) Post-thrombotic syndrome Varicose veins
Varicocele-Gastric varices-Portacaval anastomosis (Hemorrhoid,
Esophageal varices, Caput medusae) Other Superior vena cava
syndrome-Inferior vena cava syndrome-Venous ulcer Arteries or veins
Vasculitis-Thrombosis-Embolism (Pulmonary embolism, Cholesterol
embolism)-Angiopathy (Macroangiopathy, Microangiopathy) Lymphatic
disease Lymphadenitis-Lymphedema-Lymphangitis Blood pressure
Hypertension Hypertensive heart disease-Hypertensive
nephropathy-Secondary hypertension (Renovascular
hypertension)-Pulmonary hypertension Hypotension Orthostatic
hypotension Certain conditions originating in the perinatal period
Maternal factors and complications Umbilical cord prolapse-Nuchal
cord-Chorioamnionitis Cardiovascular Pneumopericardium-Persistent
fetal circulation Haemorrhagic and haematological/hematologic
disease Haemorrhagic disease of the newborn-Hemolytic disease of
the newborn-Rh disease-Hydrops fetalis-Hyperbilirubinemia
(Kernicterus, Neonatal jaundice) and other disorders
Periventricular leukomalacia-Gray baby syndrome-muscle tone
(Congenital hypertonia, Congenital hypotonia)-Perinatal infection
(Congenital rubella syndrome)-Velamentous cord
insertion-Omphalitis.
[0142] As used herein, the term `antibody reactivity` or
`autoantibody reactivity` refers to the specific antigen-antibody
binding which is elicited by the presence of a given antigen. The
antibody or collective of different antibodies can recognize and
bind to the antigen, specific epitopes on an antigen and/or
different conformations of the antigen. Individual sera can be
defined by multiplicities of different antibodies binding to
different antigens, each antigen being recognised by a different
antibody or different set of antibodies. The antibody reactivities
which are formed with a specific defined set of antigens,
associated with susceptibility to Acute Myocardial Infarction (AMI)
or Stroke, are predictive of AMI or Stroke in an individual.
[0143] The term `antibody repertoire` used herein refers to the
present repertoire of antibodies in an individual's sample. This
collection of antibodies encodes the history of the individual's
past immunological experience, as well as the present state of the
individual's body. It is within the scope of the invention to
disclose means and methods of predicting resistance or
susceptibility of an individual to the future development of an AMI
and/or stroke condition by profiling the present autoantibody
repertoire of an individual.
[0144] The term `substrate` used herein refers in a no limiting
manner to a microarray chip, microarray wafers, microarray strips,
ELISA microwells, multiplex beads or any other substrate material
known in the relevant art.
[0145] The term `biological fluids` used herein refers in a non
limiting manner to plasma, serum, blood, cerebrospinal fluid,
synovial fluid, sputum, saliva, tears, lymph specimen of an
individual or any other biological fluid known in the art.
[0146] The term `defined antigens` used herein refers to at least
740 body molecules associated with a variety of conditions selected
from a group consisting of inflammation, angiogenesis, apoptosis or
any other condition involving immune regulation or combination
thereof.
[0147] The term `informative antigens` used herein refers to
antigens whose participation in antibody binding can discriminate
between AMI, Stroke and Healthy control serum samples groups.
[0148] The term `intensity` used herein refers to the signal
strength generated by scanning, measuring or observing
antibody-antigen binding. For example, a collection of antibody
reactivities, expressed as signal intensities, makes up a profile
for any given individual. Elevated levels of antibody production
relative to the Healthy controls are picked up as elevated
intensities on a results matrix.
[0149] The term `susceptibility` used herein refers to the
likelihood or probability of a patient to suffer an AMI and/or
Stroke.
[0150] The term `epitope` used herein refers to antigenic
determinant, which is the part of a molecule that is recognized by
the immune system, specifically by antibodies, B cells, or T
cells.
[0151] The term `human immunoglobulin types` used herein refers to
different isotypes of the human antibodies.
[0152] The term `sensitivity` of a diagnostic test used herein
refers to the probability that an individual with a specific
medical condition will be defined as such by the diagnostic test;
i.e. the number of true positive test results divided by the number
of all individuals tested who have the condition. The term also
refers to the studies undertaken to establish useful informative
antigens and antibody reactivities referred to herein.
[0153] The term `specificity` of a diagnostic test used herein
refers to the probability that an individual not having a specific
medical condition will be correctly defined by the test; i.e. the
number of true negative test results divided by the number of
control specimens tested. The term also refers to the studies
undertaken to establish useful informative antigens and antibody
reactivities referred to herein.
[0154] The term `accuracy` of a diagnostic test used herein refers
to the fraction of correct results provided by the test. The term
also refers to the studies undertaken to establish useful
informative antigens and antibody reactivities referred to
herein.
[0155] The term `PPV` used herein refers to the probability that an
individual with a positive test result indeed has the medical
condition indicated by the test.
[0156] The term `NPV` used herein refers to the probability that an
individual with a negative test result is indeed free of the
medical condition indicated by the test.
[0157] Thus the present invention provides a method of recognizing
the development of an Acute Myocardial Infarction (AMI) process in
an individual. The aforementioned method comprises steps of
profiling specific antibody reactivities or biomarkers associated
with AMI susceptibility and analysing results obtained. The
aforementioned profiling step further comprises steps of: (a)
attaching a set of defined antigens to a substrate; (b) obtaining a
biological fluid derived specimen from an individual, said specimen
containing a specific antibody repertoire; and, (c) binding said
antibodies of said biological fluid specimen to said attached
antigens thereby forming bound antibody antigen complexes.
[0158] Moreover, the present invention provides means and methods
to differentiate diagnoses of stroke and AMI. The finding that
stroke and AMI differ in their antigen chip signature is highly
novel, even though both conditions have been assumed to involve
similar atherosclerotic processes. The diagnostic methods for
stroke provided herein are particularly useful in differentiating
from brain tumors for example, which may present with similar
initial symptoms.
[0159] It is within the scope of the invention that the defined
antigens which are attached to the substrate, as detailed above,
are characterized by their association with a variety of conditions
selected from a group consisting of inflammation, angiogenesis,
apoptosis or any other condition involving immune regulation or
combination thereof.
[0160] It is further within the scope of the invention to provide
antigens such as synaptotagmin 1, tryptophan hydroxylase peptide 1,
tryptophan hydroxylase peptide 2, BSP (Brain Specific Protein), for
their as therapeutic agents or vaccines against the inflammatory
process in AMI and stroke. Tryptophan hydroxylase is an exemplar of
an informative antigen which may be useful for treating
atherosclerosis. Mouse models are being employed for this purpose.
The references of Appendix I are included in their entirety.
EXAMPLES
[0161] In order to understand the invention and to see how it may
be implemented in practice, a plurality of preferred embodiments
will now be described by way of non-limiting example only, with
reference to the following examples.
Example 1
Identifying Autoantibody Reactivities Discriminating Between AMI
and Healthy Control Serum Samples
[0162] Reference is now made to FIG. 1 illustrating a flow diagram
describing the different phases of the study detailed below. The
study aimed to elucidate informative antigens which are associated
with the development of an AMI process in an individual. As
detailed in FIG. 1, the study includes the following phases:
1. Preliminary Phase
I. Aim
[0163] The rationale behind the study summarized herein is to
provide a classifier or an algorithm which can discriminate between
healthy individuals and individuals who are at high risk for the
development of an AMI process. Moreover, the study is aimed to
discover biomarkers which distinguish between individuals having an
AMI process and individuals with a stroke event. The aforementioned
study tests the capability of the antigen microarray chip to
discriminate in a blinded test between three groups of serum
samples: Acute Myocardial Infarction (AMI) and stroke samples, and
a group of healthy control samples. The results serve as a
disclosure of the present invention and as basis for further
variations and improvements of the invented method.
II. Materials and Methods
[0164] Specimens: Three groups of serum samples, each group
containing 15 samples. Patient specimens were collected between 1
and 4 months after an acute event. [0165] a. 15 patients with a
recent Acute Myocardial Infarction (AMI)--group 4. [0166] b. 15
patients with a recent Stroke--group 2. [0167] c. 15 Healthy
controls--group 0.
[0168] The aforementioned specimens were coded (labeled "0", "2",
"4"), so that the tester was blinded to the identity of each
specimen group during the testing and analysis of the results.
[0169] An Antigen microarray Chip (I-Chip) was used, bearing over
740 body molecules (herein antigens) related to a variety of
conditions--immune regulation, inflammation, angiogenesis,
apoptosis and more.
[0170] Reference is now made to Quintana et al., 2004 [1]
describing the preparation of antigen microarray chips and the
process of image and data analysis used for the identification of
predictive antibody repertoire associated with induced diabetes in
mice.
[0171] It is herein disclosed that the antigen microarray chips
were produced, and the specimens were tested, as described by
Quintana et al., 2004 [1].
Data Analysis:
[0172] The output of the experiment was a set of 45 (3 groups of 15
specimens in each group) scanned slide images. The images obtained
by laser scanning of each microarray slide, were automatically
preprocessed to quantify the antibody-binding signal for each
antigen spot on the array and produce a reactivity matrix. The
(i,j)'th entry in this matrix corresponds to the reactivity of the
i' th antibody in the j' th specimen.
[0173] This matrix was used to build a classifier that was able to
predict the class identity of unseen specimens. To validate the
performance of the classifier, we applied N-fold cross-validation,
i.e. a Leave-One-Out (LOO) procedure. In this procedure, a single
specimen is set aside, while the rest of the specimens are used to
build a classifier, which is then tested on the left-out specimen.
This process is repeated over all specimens, and the total number
of errors--the LOO error--estimates the performance of the
classifier.
[0174] A separate classifier was built in each LOO instance. More
specifically, feature selection was first performed over the
left-in specimens; this entailed choosing which of the antigens may
be suitable for discriminating between the classes, and then build
a classifier based on these antigens. In the present case, feature
selection was done by ranking the different antigens according to a
mutual information criterion. Classification was based on the
selected antigens, using a nearest-neighbor classifier, or a linear
support vector machine.
[0175] To estimate the statistical significance of the findings, a
permutation test was performed. The different specimens were
repeatedly assigned random labels, and the LOO procedure was then
performed. For each set of random labels, we estimated the LOO
error, and compared it to the LOO error based on the correct
labels. An estimated p-Value is then computed based on the number
of such random permutations whose LOO error is lower than or equal
to the case of correct labels.
III. Results
[0176] The individual samples in each of the groups were compared
in a pair-wise manner:
a. The AMI group ("4") versus the Healthy control group ("0"). b.
The Stroke group ("2") versus the Healthy control group ("0"). c.
The Stroke group ("2") versus the AMI Group ("4"). AMI group
f''4'') versus the Healthy control group ("0")--
[0177] Reference is now made to Table 1 presenting the analysis of
the antigen microarray chip.
TABLE-US-00001 TABLE 1 AMI ("4") versus Healthy control ("0")
CLINICAL DEFINITION 4 0 I-CHIP 4 14 2 16 0 1 13 14 15 15 30
SENSITIVITY 93.3 SPECIFICITY 86.7 PPV 87.5 NPV 92.9
[0178] The numbers provided in the table indicate the total number
of times the antigen microarray chip provided a "4" or a "0"
result. This is used in the calculation of the predictive values
(positive & negative).
[0179] Analysis of the results comparing between the stroke group
and the healthy control group as well as between the stroke group
and the AMI group is presented below in Example 2.
The Most Informative Antigen:
[0180] Interestingly, the results obtained revealed that a peptide
of Tryptophan hydroxylase (TPH) (peptide 2) was by itself able to
separate to a large extent between the three groups. FIG. 2 shows
the antibody reactivity levels of Tryptophan hydroxylase antigen in
the three specimen groups "0", "2" and "4".
[0181] As illustrated, the reactivity level against Tryptophan
hydroxylase (TPH) antigen is most significant in the AMI group
("4), intermediate in the Stroke group ("2") and almost non
detectable in the Healthy control group ("0"). Thus, according to
the described results, the antibody reactivity level for Tryptophan
hydroxylase antigen can effectively discriminate between AMI
Healthy control specimens, with only 3 errors in the LOO test; and
could separate the Stroke group from the Healthy control group with
only six errors.
IV. Conclusions:
[0182] An unsupervised analysis using the 740 microarray chip
antigens detects a highly correlated subgroup of informative
antigens formed by samples appearing in each of the 3 groups.
[0183] Both the AMI ("4") and the Stroke ("2") groups can be
discriminated from the Healthy control group ("0") with
sensitivities and specificities of about 90%. The most informative
antigen was a peptide of Tryptophan hydroxylase, to which the AMI
patients manifested an elevated level of antibody production
relative to the healthy controls.
2. Validation Phase
[0184] The aim of the study described herein was to validate the
results obtained in the preliminary phase, by testing additional
new 62 serum samples representing AMI and Healthy control groups,
using the same classifier as described in the preliminary
phase.
[0185] Analysis of the autoantibody reactivity patterns confirms
the novel finding that Tryptophan hydroxylase peptide 1 can
function as an informative antigen or biomarker for atherosclerotic
conditions and can be used to discriminate AMI patients from
healthy controls (FIG. 3A), as was found in the preliminary
phase.
[0186] Three additional antigens were also informative, all with
higher antibody reactivities in the AMI patients:
a second peptide of Tryptophan hydroxylase (peptide 2), as
presented in FIG. 3B; a peptide of synaptotagmin1, as presented in
(FIG. 3D); and, a peptide of Brain-1, an oligodendrocyte
transcription factor.
[0187] A classifier based on these four antigens provided the
following performance in a LOO test:
SENSITIVITY 93%
SPECIFICITY 87%
PPV 88%
NPV 91%
[0188] Thus, two different peptides of Tryptophan hydroxylase
(peptides 1 and 2) separated the AMI group from the healthy control
group with a high degree of accuracy as indicated in FIGS. 3A and
3B.
[0189] Reference is now made to FIG. 3C showing a comparison of the
reactivity levels of TPH peptides 1 and 2. As shown, most AMI
subjects respond strongly to one or both of the abovementioned
antigens and few AMI subjects have only low responses, in the range
of the healthy control subjects. These low-reactor AMI subjects
might have confounding factors that set them apart. For example,
the specimen marked with a red circle would be considered negative
according to peptide 1 (thus yielding a false negative result), but
reacts positively with peptide 2. Thus, some of the low responders
to one of the peptides have higher responses to the other peptide.
Therefore both peptides together have fewer false negatives.
[0190] Other antigens showing increased reactivity in the AMI group
as compared to the healthy control group are peptides of
synaptotagmin1 (see FIG. 3D), and a peptide of Brain-1, an
oligodendrocyte transcription factor. The association of both
antigens to cardiovascular diseases is novel.
[0191] Thus, the correlation between the presence of autoantibodies
against particular molecules and the nature of the expression of
these molecules and their function is an important outcome of these
experiments.
[0192] The finding of markedly increased reactivity against few
antigens such as Tryptophan hydroxylase suggests that it is
possible to construct a method of classification based on these
reactivities and not based on clustering. The importance of
Tryptophan hydroxylase in blood vessel pathophysiology and
cardiovascular disease is well known. The novelty of the present
invention is that the immune system uses Tryptophan hydroxylase as
an immune biomarker.
3. Training Phase
I. Introduction
[0193] The aim of this study was to refine the classification
algorithms based on autoantibody reactivity patterns of AMI and
healthy control groups by using ImmunArray's antigen chip
microarray. Towards this aim the 62 specimens analysed in the
validation phase were combined with the 30 specimens of the
preliminary phase to generate a new training set of AMI and Healthy
control specimens. All 92 specimens were re-analyzed as one,
expanded training group in order to identify additional informative
antibody reactivities and obtain a refined classification rule.
II. Results
[0194] The combined set of 92 specimens was analyzed (50 AMI and 42
Healthy controls) to search for additional antigens and to refine
the classification algorithm. Five antigens, in addition to the 4
defined above under "validation phase", were found to discriminate
between the AMI patients and the Healthy controls with statistical
significance (FDR>0.05). Some of the antigens elicited both IgG
and IgM production, providing in total 14 informative antibody
reactivities, which define 9 different informative antigens, as
listed in the following Table 2.
TABLE-US-00002 TABLE 2 Informative antibody reactivities of AMI
versus Healthy control groups. GI Immuno- Antibody Antigen
Accession# globulin level in Name type or reference type AMI
peptide Sequence Brain-1, oligo- peptide 2760445 IgG, IgM up
DPSSVKMVQSDFMQGAMAA dendrocyte transcription factor Synaptotagmin 1
peptide 1351176 IgG, IgM up NESFSFEVPFEQIQKVQVVV Tryptophan peptide
136122 IgG, IgM up PLLKSHTTVLSVDSPDQLP hydroxylase peptide 1
Tryptophan peptide 136122 IgG, IgM up KSITSAMNELRHDLDVVND
hydroxylase peptide 2 BSP (Brain peptide 206781 IgG, IgM up
TVSILCSTQSNPDPILTIF specific protein, myelin associated
glycoprotein) Fibronectin whole Sigma- IgG up NA molecule Aldrich
cat # F0895 Caspase 3 peptide Sigma- IgM down MACHKIPVEADFLYAYSTAP
Aldrich cat # C1224 Myelin- whole Sigma- IgM up NA associated
molecule Aldrich cat # metalloproteinase A9975 CD68 whole Prospec
cat # IgM up NA molecule PRO-293
[0195] The specimens were classified with the following
performance:
Sensitivity 87%
Specificity 82%
Accuracy 84%
IV. Summary & Discussion
[0196] Implementation of the above described preliminary phase, the
validation phase and the training phase enabled the discovery of
the novel 14 antibody reactivities, which recognize 9 informative
antigens. These reactivities characterize a recent Acute Myocardial
Infarction (AMI) in a patient and thus can recognize the
development of an AMI process in an individual. The following
conclusions may be drawn:
[0197] 1. The autoantibody reactivities that were found to
discriminate AMI patients from healthy control group were validated
in a blind test performed on an independent group of test set
specimens.
[0198] 2. The amplified training set made it possible to identify
additional antigen reactivities and resulted in an improved
classifier, with both sensitivity and specificity of over 80%.
[0199] 3. The informative antigen reactivities have not been
previously reported to be immunologically involved in the disease
pathogenesis, although some of the antigens are known to function
in vascular physiology. Thus the discovery of their ability to be
informative for vascular conditions is most significant. Moreover,
there appears to be biological reasons for the targeting of these
molecules by the immune system as immune biomarkers.
Example 2
Identifying Informative Autoantibody Reactivities Associated with
Stroke
[0200] The antibody reactivity profiles of the 15 Stroke specimens
of the above described preliminary phase of Example 1 were compared
to the Healthy control antibody reactivity profiles as well as to
the AMI specimen groups by analyzing the antigen microarray chip
results. The data analysis was performed as described in Example
1.
a. The Stroke Group ("2") Versus the Healthy Control Group
("0")
[0201] A set of antigens that separated the stroke group from the
healthy control group with 5 errors (83% accuracy) in a LOO test,
is listed in Table 3.
TABLE-US-00003 TABLE 3 Stroke group versus Healthy control group
Immuno- Antigen globulin up/down name Antigen full name Antigen
type sequence type regulated NOGO Nogo-66(1-40) peptide IgM down
antagonist peptide (Sigma cat # N7161) P53 p53 peptide
CTYSPPLNKLFCQLVKTCPV IgM down MAZ MYC-associated zinc peptide
RHEAIHTHLNSHVRQVHS IgG down finger protein (purine- binding
transcription factor) GAD Glutamic acid peptide
GTDSVILIKQKGFVPFLVSA IgM down decarboxylase C P450 Cytochrome C
P450 whole molecule NA IgG down P53 p53 mutant whole molecule NA
IgM down Tropomyosin Tropomyosin whole molecule NA IgG down ERK
Extracellular regulated peptide AAEPLQSVLWVKQQRCAVSL IgG up MAP
kinase
b. The Stroke Group ("2") Versus the AMI Group ("4"):
[0202] Table 4 lists 6 antigens that separate the AMI subjects from
the Stroke subjects.
TABLE-US-00004 TABLE 4 Stroke group ("2") versus AMI ("4") group
up/down regulated type Antigen name up IgG Tryptophan hydroxylase
(peptide 1) up IgG Tryptophan hydroxylase (peptide 2) up IgG
Synaptotagmin1 (peptide) up IgG Myelin-Associated Metalloproteinase
up IgM Bone morphogenic protein 4 up IgG D4GDI, GDP-dissociation
inhibitor
[0203] Reference is now made to FIG. 4 showing the corresponding
graphs of the antibody reactivity levels for each of the antigens.
As can be seen, the two peptides of Tryptophan hydroxylase
(peptides 1 and 2) are again prominent, as is synaptotagmin1.
[0204] Thus the above described results identified two antigens out
of the 740 microarray chip antigens that correctly classified 28 of
the 30 specimens of both groups (two errors in the LOO test). These
antigens were: 1. TPH 2. Synaptotagmin1.
[0205] Reference is now made to Table 5. The numbers provided in
the Table indicate the total number of times the antigen microarray
chip provided a "4" or a "2" result. This is used in the
calculation of the predictive values (positive & negative).
TABLE-US-00005 TABLE 5 Stroke group ("2") versus the AMI group
("4") CLINICAL DEFINITION 2 4 I-CHIP 2 14 1 15 4 1 14 15 15 15 30
SENSITIVITY 93.3 SPECIFICITY 93.3 PPV 93.3 NPV 93.3
[0206] A permutation test was performed 500 times. The average
error rate for the randomization was, as expected, 0.5 and the
lowest number of LOO errors obtained was 6, hence the corresponding
p-Value for the real result is p<0.002.
[0207] It is surprising and unexpected that AMI differs from Stroke
significantly in these reactivities. These results implicate that
these antigens are specific biomarkers for AMI and therefore, can
predict an attack of acute AMI, by comparing pre-AMI sera, and sera
taken at the time of attack. The presence of IgG antibodies on the
day of an attack implicate these antibodies have been formed by the
subject well in advance of the acute episode hence can be
considered as biomarkers for AMI subjects.
IV. Conclusions:
[0208] An unsupervised analysis using all 740 microarray chip
antigens detects a highly correlated subgroup of informative
antigens formed by samples appearing in each of the 3 groups.
[0209] Both the AMI ("4") and the Stroke ("2") groups can be
discriminated from the Healthy control group ("0") with
sensitivities and specificities of about 90%.
[0210] These results were obtained with ImmunArray's antigen
microarray chip.
[0211] The discrimination between AMI and stroke is significant
since both conditions result from the atherosclerotic process, but
in different locations in the vascular system. Thus it is
unexpected and highly novel that the antigen chip results can
discriminate between different variations of atherosclerosis.
[0212] Since the autoantibodies were IgG, it is likely that T-cell
reactivity to these antigens is also a factor.
[0213] It is herein acknowledged that additional information about
the subjects such as gender, subject age, range of ages; time
between the event and serum specimen collection; further increases
the efficacy of the disclosed method and is well within its
scope.
Example 3
[0214] It is herein acknowledged that some embodiments of the
invention include the use of Luminex xMAP Multiplexing Technology
as outlined below. The rapidly growing knowledge base in drug
discovery and protein research has placed increased pressure on
researchers to quickly gain a greater understanding of
protein-protein interactions, cell signaling pathways and markers
of metabolic processes. Increasingly, this information is difficult
or impractical to obtain using traditional "singleplex" protein
detection methods, such as Western blotting. Consequently, several
emerging technologies have been introduced that offer "multiplex"
detection of proteins from a single biological sample.
[0215] MILLIPLEX MAP is based on the Luminex xMAP technology--one
of the fastest growing and most respected multiplex technologies
offering applications throughout the life-sciences, including
clinical diagnostics.
[0216] Systems using xMAP technology perform a variety of bioassays
including immunoassays on the surface of fluorescent-coded beads
known as microspheres, which are then read in a compact analyzer.
Using two lasers and high-speed digital-signal processors, the
analyzer reads signals on each individual microsphere particle. The
capability of adding multiple conjugated beads to each sample
results in the ability to obtain multiple results from each sample.
Open-architecture xMAP technology enables multiplexing of many
types of bioassays reducing time, labor and costs over traditional
methods.
[0217] It is herein acknowledged that some embodiments of the
invention further include the use of Surface Plasmon Resonance
Imaging (SPRI) as outlined below.
[0218] The excitation of surface plasmons by light is denoted as a
surface plasmon resonance (SPR) for planar surfaces or localized
surface plasmon resonance (LSPR) for nanometer-sized metallic
structures.
[0219] This phenomenon is the basis of many standard tools for
measuring adsorption of material onto planar metal (typically gold
and silver) surfaces or onto the surface of metal nanoparticles. It
is behind many color based biosensor applications and different
lab-on-a-chip sensors. An aspect of the present invention is the
use of SPR1 technology in scanning the results of the
antigen-antibody reactions, although any scanning method known to
persons skilled in the art can be employed.
Example 4
Method for Profiling Autoantibody Reactivities Associated with a
Susceptibility to AMI with a Serum Sample of an Individual and
Analysing Results Obtained
[0220] It is herein disclosed a preferred protocol for profiling
autoantibody reactivities of AMI susceptibility biomarkers
associated with a susceptibility to AMI with a serum or plasma
sample of an individual: [0221] A set of selected antigens detailed
above are spotted onto the chip surface, each antigen in a discrete
location (address). The antigens may be adsorbed or covalently
bound to the chip surface. [0222] A blood serum or plasma specimen
is obtained. [0223] The specimen is brought into contact with the
surface carrying the antigens. A few microliters of the specimen is
dropped onto the surface and covered with a cover-slip to spread
the serum over the entire surface. [0224] Incubation under binding
conditions is carried out for an hour followed by several washes.
Drying is by centrifugation. [0225] Measuring the results of
autoantibody binding. The amount of antibody bound to each
antigen-spot is measured. This is done by a common sandwich
immunoassay technique, converting the amount of bound antibody into
a fluorescent signal using a fluorescently labeled second antibody,
specific for human immunoglobulin type (IgG, IgM etc.). This is
done as for the serum incubation step (use coverslip, incubate,
wash). [0226] The chip is scanned in a system that produces a
fluorescent image of the chip surface. A laser scanner is used in
some embodiments. In other embodiments camera based systems are
used. [0227] The image is then analyzed.
[0228] Reference is now made to the disclosed method of recognizing
the development of an Acute Myocardial Infarction (AMI) process in
an individual, wherein the method comprises steps of: (a) profiling
specific antibody reactivities or biomarkers associated with AMI
susceptibility, and, (b) analysing results obtained. The profiling
step comprises the steps of: (i) attaching a set of defined
antigens to a substrate; (ii) obtaining a biological fluid derived
specimen from an individual, the specimen containing a specific
antibody repertoire; and, (iii) binding the antibodies of the
biological fluid specimen to the attached antigens thereby forming
bound antibody antigen complexes.
[0229] Reference is now made to the aforementioned method, wherein
the profiling further comprises the steps of obtaining the defined
antigens characterized by association with a variety of conditions
selected from a group consisting of inflammation, angiogenesis,
apoptosis or any other condition involving immune regulation or any
combination thereof.
[0230] Reference is now made to the aforementioned method, wherein
the profiling further comprises the steps of selecting the
biological fluid derived specimen from a group consisting of
plasma, serum, blood, cerebrospinal fluid, synovial fluid, sputum,
saliva, tears, lymph specimen, or any other biological fluid known
in the art.
[0231] Reference is now made to the aforementioned method, wherein
the profiling further comprises steps of selecting the substrate
from a group consisting of microarray chip, microarray wafers,
microarray strips, ELISA microwells, multiplex beads or any
substrate known in the relevant art.
[0232] It is herein further disclosed that the aforementioned
method, further comprises the steps of selecting the antigens from
a group consisting of protein, polypeptide, peptide, nucleic acids,
lipids, glycosylated molecules, polypeptides with modifications,
polysaccharides or any other biological molecule known in the
art.
[0233] It is herein further disclosed that the aforementioned
method, further comprises the steps of defining the antibody
reactivities as IgG, IgM, IgA, IgE antibody isotypes or more
specific sub-isotypes of the antibodies.
[0234] Reference is now made to the aforementioned method, wherein
the analyzing results obtained further comprises the steps of
measuring the bound antibody complexes.
[0235] Reference is now made to the aforementioned method, wherein
the measuring step additionally comprises steps of labeling the
bound antibody complexes.
[0236] Reference is now made to the aforementioned method, wherein
the labeling further comprises the steps of selecting a labeling
signal from a group consisting of fluorescent, chemiluminescent,
absorptive, electronic, radioactive, paramagnetic or any labeling
signal known in the art, or combination thereof.
[0237] Reference is now made to the aforementioned method, wherein
the labeling step additionally comprises steps of contacting the
bound antibody complexes with labeled mixture of secondary
antibodies specific for human immunoglobulin types so as to obtain
labeled bound antibody antigen complexes.
[0238] Reference is now made to the aforementioned method, wherein
the analyzing step additionally comprises steps of: (a) scanning
the signal produced by the bound antibody complexes so as to form a
reactivity matrix; (b) measuring the intensity of the signal; (c)
generating and recording results of data associated with the
intensity of the signal; (d) producing the results in the form of a
signature, profile, set, pattern or matrix characteristic to the
individual; (e) comparing the results with a databank containing
data concerning the antibody reactivities in populations
susceptible to AMI thereby identifying the development of an Acute
Myocardial Infarction (AMI) process in the individual.
[0239] Reference is now made to the aforementioned method, wherein
the measuring and scanning steps are selected from a group
consisting of Surface Plasmon Resonance (SPR), ellipsometry, laser
scanning, light detecting, photon detecting via a photon
multiplier, photographing with a digital camera based system or
video system, radiation counting, fluorescence detecting,
electronic, magnetic detecting or any other technique known in the
art or combination thereof.
[0240] Reference is now made to the aforementioned method, wherein
the analyzing step further comprises the steps of defining the
reactivity as a relative intensity signal of a specific bound
antibody as compared to its intensity signal in healthy control
individuals.
[0241] Reference is now made to the aforementioned method, wherein
the analyzing step further comprises the steps of defining the
reactivity as a relative intensity signal of a specific bound
antibody as compared to its intensity signal in individuals who
have had a stroke event.
[0242] Reference is now made to the aforementioned method, wherein
the analyzing step further comprises the steps of identifying a set
of informative antigens possessing higher or a lower intensity
reactivity matrix signals as compared to the intensity signals of
the antibodies in healthy control individuals.
[0243] Reference is now made to the aforementioned method, wherein
the analyzing step further comprises the steps of identifying a set
of informative antigens possessing higher or a lower intensity
reactivity matrix signals as compared to the intensity signals of
the antibodies in individuals who have had a stroke event.
[0244] Reference is now made to the aforementioned method, wherein
the method further comprises steps of: (a) obtaining the antigens
attached to the substrate; (b) packaging the substrate attached
antigens in a kit containing instructions; and, (c) following the
steps according to the instructions.
[0245] The current invention further discloses a set of informative
antigens useful for recognizing the development of an Acute
Myocardial Infarction (AMI) process in an individual. According to
certain embodiments of the present invention, some of the herein
disclosed informative antigens are specific peptides. The
aforementioned set of antigens is selected from a group consisting
of Brain-1 oligo-dendrocyte transcription factor, Synaptotagmin 1,
Tryptophan hydroxylase peptide 1, Tryptophan hydroxylase peptide 2,
BSP (Brain specific protein), myelin associated glycoprotein,
Fibronectin, Caspase 3, Myelin-associated metalloproteinase, CD68,
Bone morphogenic protein 4 and D4GDI (GDP-dissociation inhibitor),
the antibody reactivities are profiled by: (a) profiling means for
profiling specific antibody reactivities or biomarkers associated
with AMI susceptibility, and, (b) analyzing means for analyzing
results obtained. The aforementioned profiling means further
comprises steps of: (i) attaching means for attaching a set of
defined antigens to a substrate; (ii) a biological fluid derived
specimen from an individual, the specimen containing a specific
antibody repertoire; and, (iii) binding means for binding the
antibodies of the biological fluid specimen to the attached
antigens thereby forming bound antibody antigen complexes.
[0246] It is the core of the invention that the antigens are
further characterised by their specific antibody reactivity with
human antibodies associated with susceptibility to AMI.
[0247] Reference is now made to the aforementioned set of
informative antigens, wherein the defined antigens characterized by
association with a variety of conditions selected from a group
consisting of inflammation, angiogenesis, apoptosis or any other
condition involving immune regulation or any combination
thereof.
[0248] Reference is now made to the aforementioned set of
informative antigens, wherein the defined antigens are attached to
a substrate selected from a group consisting of microarray chip,
microarray wafers, microarray strips, ELISA microwells, multiplex
beads or any substrate known in the relevant art.
[0249] Reference is now made to the aforementioned set of
informative antigens; wherein the defined antigens are selected
form a group consisting of protein, polypeptide, peptide, nucleic
acids, lipids, glycosylated molecules, polypeptides with
modifications, polysaccharides or any other biological molecule
known in the art.
[0250] Reference is now made to the aforementioned set of
informative antigens, wherein the antigens are further
characterized by their specific capability to discriminate between
the development of an AMI process in an individual and a healthy
control individual.
[0251] Reference is now made to the aforementioned set of
informative antigens, wherein the antigens are further
characterized by their specific capability to discriminate between
the development of an AMI process in an individual and an
individual who have had a stroke event.
[0252] Reference is now made to the aforementioned set of
informative antigens, wherein the human antibodies are derived from
a biological fluid specimen selected from a group consisting of
plasma, serum, blood, cerebrospinal fluid, synovial fluid, sputum,
saliva, tears, lymph specimen, or any biological fluid known in the
art.
[0253] Reference is now made to the aforementioned set of
informative antigens, wherein the antibody reactivities are defined
as IgG, IgM, IgA, IgE antibody isotypes or more specific
sub-isotypes of the antibodies.
[0254] Reference is now made to the aforementioned set of
informative antigens, wherein the specific antibody reactivity is
analyzable by using measuring means.
[0255] Reference is now made to the aforementioned set of
informative antigens, wherein the specific antibody reactivity is
analyzable by using labeling means.
[0256] Reference is now made to the aforementioned set of
informative antigens, wherein the labeling means is adapted to
label the human antibodies with a mixture of secondary antibodies
specific for human immunoglobulin types to form a reactivity
matrix.
[0257] Reference is now made to the aforementioned set of
informative antigens, wherein the labeling means comprises labeling
signal selected form a group consisting of fluorescent,
chemiluminescent, absorptive, electronic, radioactive, paramagnetic
or any labeling signal known in the art, or combination
thereof.
[0258] Reference is now made to the aforementioned set of
informative antigens, wherein the specific antibody reactivity is
further analyzable by: (a) using a scanning means for scanning the
reactivity matrix signal; (b) using a measuring means for measuring
the intensity of the signal; (c) using generating and recording
means for generating and recording results associated with the
reactivity matrix signal; (d) using producing means for producing
the results in the form of a signature, profile, set, pattern or
matrix characteristic to the individual; and, (e) using means for
comparing the results with a databank containing data concerning
the antibody reactivities in populations susceptible to AMI,
thereby identifying the development of an Acute Myocardial
Infarction (AMI) process in the individual.
[0259] Reference is now made to the aforementioned set of
informative antigens, wherein the measuring and scanning means are
selected from a group consisting of Surface Plasmon Resonance
(SPR), ellipsometry, laser scanning, light detecting, photon
detecting via a photon multiplier, photographing with a digital
camera based system or video system, radiation counting,
fluorescence detecting, electronic, magnetic detecting or any other
technique known in the art or combination thereof.
[0260] Reference is now made to the aforementioned set of
informative antigens, wherein the reactivity is defined as a
relative intensity of a specific antibody bound antigen as compared
to its intensity signal in healthy control individuals.
[0261] Reference is now made to the aforementioned set of
informative antigens, wherein the reactivity is defined as a
relative intensity of a specific antibody bound antigen as compared
to its intensity signal in individuals who have had a stroke
event.
[0262] Reference is now made to the aforementioned set of
informative antigens, wherein the reactivity is further defined as
a set of informative antigens possessing a higher or a lower
intensity reactivity signals as compared to the intensity signals
of the antigens in the healthy control individuals.
[0263] Reference is now made to the aforementioned set of
informative antigens, wherein the reactivity is further defined as
a set of informative antigens possessing a higher or a lower
intensity reactivity signals as compared to the intensity signals
of the antigens in individuals who have had a stroke event.
[0264] The current invention further discloses a reactivity matrix
useful for recognizing the development of an Acute Myocardial
Infarction (AMI) process in an individual, comprising at least one
substrate embedded with defined antigens. According to certain
embodiments of the present invention, some of the herein disclosed
defined antigens are specific peptides. The aforementioned antigens
are selected from a group consisting of Brain-1 oligo-dendrocyte
transcription factor, Synaptotagmin 1, Tryptophan hydroxylase
peptide 1, Tryptophan hydroxylase peptide 2, BSP (Brain specific
protein), myelin associated glycoprotein, Fibronectin, Caspase 3,
Myelin-associated metalloproteinase, CD68, Bone morphogenic protein
4 and D4GDI (GDP-dissociation inhibitor), wherein the antigens are
further characterised by their specific antibody reactivity with
human specimen containing antibodies associated with susceptibility
to AMI.
[0265] Reference is now made to the aforementioned reactivity
matrix, wherein the antigens characterized by association with a
variety of conditions selected from a group consisting of
inflammation, angiogenesis, apoptosis or any other condition
involving immune regulation or combination thereof.
[0266] Reference is now made to the aforementioned reactivity
matrix, wherein the human antibodies are derived from a biological
fluid specimen selected from a group consisting of plasma, serum,
blood, cerebrospinal fluid, synovial fluid, sputum, saliva, tears,
lymph specimen, or any biological fluid known in the art.
[0267] Reference is now made to the aforementioned reactivity
matrix, wherein the antigens are attached to a substrate selected
from a group consisting of microarray chip, microarray wafers,
microarray strips, ELISA microwells, multiplex beads or any
substrate known in the relevant art.
[0268] Reference is now made to the aforementioned reactivity
matrix, wherein the antigens are selected form a group consisting
of protein, polypeptide, peptide, nucleic acids, lipids,
glycosylated molecules, polypeptides with modifications,
polysaccharides or any other biological molecule known in the
art.
[0269] Reference is now made to the aforementioned reactivity
matrix, wherein the antibody reactivities are defined as IgG, IgM,
IgA, IgE antibody isotypes or as more specific sub-isotypes of the
antibodies.
[0270] Reference is now made to the aforementioned reactivity
matrix, wherein the specific antibody reactivity is analyzable by
using measuring means.
[0271] Reference is now made to the aforementioned reactivity
matrix, wherein the specific antibody reactivity is analyzable by
using labeling means.
[0272] Reference is now made to the aforementioned reactivity
matrix, wherein the labeling means is adapted to label the human
antibodies with a mixture of secondary antibodies specific for
human immunoglobulin types to form a reactivity matrix.
[0273] Reference is now made to the aforementioned reactivity
matrix, wherein the labeling means comprises labeling signal
selected form a group consisting of fluorescent, chemiluminescent,
absorptive, electronic, radioactive, paramagnetic or any labeling
signal known in the art, or combination thereof.
[0274] Reference is now made to the aforementioned reactivity
matrix, wherein the specific antibody reactivity is further
analyzable by: (a) using a scanning means for scanning the
reactivity matrix signal; (b) using a measuring means for measuring
the intensity of the signal; (c) using generating and recording
means for generating and recording results associated with the
reactivity matrix signal; (d) using producing means for producing
the results in the form of a signature, profile, set, pattern or
matrix characteristic to the individual; and, (e) using means for
comparing the results with a databank containing data concerning
the antibody reactivities in populations susceptible to AMI,
thereby identifying the development of an Acute Myocardial
Infarction (AMI) process in the individual.
[0275] Reference is now made to the aforementioned reactivity
matrix, wherein the measuring and scanning means are selected from
a group consisting of Surface Plasmon Resonance (SPR),
ellipsometry, laser scanning, light detecting, photon detecting via
a photon multiplier, photographing with a digital camera based
system or video system, radiation counting, fluorescence detecting,
electronic, magnetic detecting or any other technique known in the
art or combination thereof
[0276] Reference is now made to the aforementioned reactivity
matrix, wherein the reactivity is defined as a relative intensity
of a specific antibody bound antigen as compared to its intensity
signal in healthy control individuals.
[0277] Reference is now made to the aforementioned reactivity
matrix, wherein the reactivity is defined as a relative intensity
of a specific antibody bound antigen as compared to its intensity
signal in individuals who have had a stroke event.
[0278] Reference is now made to the aforementioned reactivity
matrix, wherein the reactivity is further defined as a set of
informative antigens possessing a higher or a lower intensity
reactivity signals as compared to the intensity signals of the
antigens in the healthy control individuals.
[0279] Reference is now made to the aforementioned reactivity
matrix, wherein the reactivity is further defined as a set of
informative antigens possessing a higher or a lower intensity
reactivity signals as compared to the intensity signals of the
antigens in individuals who have had a stroke event.
[0280] The current invention further discloses a system for
recognizing the development of an Acute Myocardial Infarction (AMI)
process in an individual. The aforementioned system comprising: (a)
a substrate embedded with antigens; (b) biological fluid derived
specimen containing a specific antibody repertoire; (c) means for
physically contacting the specimen with the surface of the
substrate; (d) means for binding the antibodies of the specimen to
the antigens embedded in the substrate so as to form bound antibody
antigen complexes; (e) a measuring device for quantifying the
intensity signal of the bound antibodies; and, (f) a data processor
for producing the intensity signals in the form of a signature,
profile, set, pattern or matrix characteristic to the individual.
It is within the core of the invention that some of the herein
disclosed antigens are specific peptides. The aforementioned
antigens are selected from a group consisting of Brain-1
oligo-dendrocyte transcription factor, Synaptotagmin 1, Tryptophan
hydroxylase peptide 1, Tryptophan hydroxylase peptide 2, BSP (Brain
specific protein), myelin associated glycoprotein, Fibronectin,
Caspase 3, Myelin-associated metalloproteinase, CD68, Bone
morphogenic protein 4 and D4GDI (GDP-dissociation inhibitor),
further wherein the antigens are characterised by their specific
reactivity with the specimen containing the antibodies associated
with susceptibility to AMI, when bound under defined
conditions.
[0281] Reference is now made to the aforementioned system, wherein
the antigens characterized by association with a variety of
conditions selected from a group consisting of inflammation,
angiogenesis, apoptosis or any other condition involving immune
regulation or combination thereof.
[0282] Reference is now made to the aforementioned system, wherein
the human antibodies are derived from a biological fluid specimen
selected from a group consisting of plasma, serum, blood,
cerebrospinal fluid, synovial fluid, sputum, saliva, tears, lymph
specimen, or any biological fluid known in the art.
[0283] Reference is now made to the aforementioned system, wherein
the antigens are attached to a substrate selected from a group
consisting of microarray chip, microarray wafers, microarray
strips, ELISA microwells, multiplex beads or any substrate known in
the relevant art.
[0284] Reference is now made to the aforementioned system, wherein
the antigens are selected form a group consisting of protein,
polypeptide, peptide, nucleic acid, lipid, glycosylated molecules,
polypeptides with modifications, polysaccharides or any other
biological molecule known in the art.
[0285] Reference is now made to the aforementioned system, wherein
the antibody reactivities are defined as IgG, IgM, IgA, IgE
antibody isotypes or as more specific sub-isotypes of the
antibodies.
[0286] Reference is now made to the aforementioned system, wherein
the specific antibody reactivity is analyzable by using measuring
means.
[0287] Reference is now made to the aforementioned system, wherein
the specific antibody reactivity is analyzable by using labeling
means.
[0288] Reference is now made to the aforementioned system, wherein
the labeling means is adapted to label the human antibodies with a
mixture of secondary antibodies specific for human immunoglobulin
types to form a reactivity matrix.
[0289] Reference is now made to the aforementioned system, wherein
the labeling means comprises labeling signal selected form a group
consisting of fluorescent, chemiluminescent, absorptive,
electronic, radioactive, paramagnetic or any labeling signal known
in the art, or combination thereof.
[0290] Reference is now made to the aforementioned system, wherein
the specific antibody reactivity is further analyzable by: (a)
using a scanning means for scanning the reactivity matrix signal;
(b) using a measuring means for measuring the intensity of the
signal; (c) using generating and recording means for generating and
recording results associated with the reactivity matrix signal; (d)
using producing means for producing the results in the form of a
signature, profile, set, pattern or matrix characteristic to the
individual; and, (e) using means for comparing the results with a
databank containing data concerning the antibody reactivities in
populations susceptible to AMI, thereby identifying the development
of an Acute Myocardial Infarction (AMI) process in the
individual.
[0291] Reference is now made to the aforementioned system, wherein
the measuring and scanning means are selected from a group
consisting of Surface Plasmon Resonance (SPR), ellipsometry, laser
scanning, light detecting, photon detecting via a photon
multiplier, photographing with a digital camera based system or
video system, radiation counting, fluorescence detecting,
electronic, magnetic detecting or any other technique known in the
art or combination thereof.
[0292] Reference is now made to the aforementioned system, wherein
the reactivity is defined as a relative intensity signal of a
specific antibody bound antigen as compared to its intensity signal
in healthy control individuals.
[0293] Reference is now made to the aforementioned system, wherein
the reactivity is defined as a relative intensity signal of a
specific antibody bound antigen as compared to its intensity signal
in individuals who have had a stroke event.
[0294] Reference is now made to the aforementioned system, wherein
the reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to the intensity signals of the antigens in the healthy
control individuals.
[0295] Reference is now made to the aforementioned system, wherein
the reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to the intensity signals of the antigens in individuals
who have had a stroke event.
[0296] The current invention further discloses a kit useful for
recognizing the development of an Acute Myocardial Infarction (AMI)
process in an individual. The aforementioned kit comprising: (a) a
set of antigens selected from a group consisting of Brain-1
oligo-dendrocyte transcription factor, Synaptotagmin 1, Tryptophan
hydroxylase peptide 1, Tryptophan hydroxylase peptide 2, BSP (Brain
specific protein), myelin associated glycoprotein, Fibronectin,
Caspase 3, Myelin-associated metalloproteinase, CD68, Bone
morphogenic protein 4 and D4GDI (GDP-dissociation inhibitor)
attached to a substrate, wherein the antigens are further
characterised by their specific ability to form bound antibody
antigen reactivity complexes with human specimen containing
antibodies associated with susceptibility to AMI; (b) a mixture of
secondary labeled antibodies specific for human immunoglobulin
types; (c) components or solutions useful for reacting the antigens
with the antibodies of the specimen; (d) components or solutions
useful for reacting the antigen antibody complexes with the
secondary labeled antibodies; and, (e) instructions for reacting
the antigens with the components and the complexes with the
secondary antibodies in vitro so as to obtain results useful for
identifying an Acute Myocardial Infarction (AMI) process in an
individual.
[0297] Reference is now made to the aforementioned kit, wherein the
antigens characterized by association with a variety of conditions
selected from a group consisting of inflammation, angiogenesis,
apoptosis or any other condition involving immune regulation or
combination thereof.
[0298] Reference is now made to the aforementioned kit, wherein the
human antibodies are derived from a biological fluid specimen
selected from a group consisting of plasma, serum, blood,
cerebrospinal fluid, synovial fluid, sputum, saliva, tears, lymph
specimen, or any biological fluid known in the art.
[0299] Reference is now made to the aforementioned kit, wherein the
antigens are attached to a substrate selected from a group
consisting of microarray chip, microarray wafers, microarray
strips, ELISA microwells, multiplex beads or any substrate known in
the relevant art.
[0300] Reference is now made to the aforementioned kit, wherein the
antigens are selected form a group consisting of protein,
polypeptide, peptide, nucleic acid, lipid, glycosylated molecules,
polypeptides with modifications, polysaccharides or any other
biological molecule known in the art.
[0301] Reference is now made to the aforementioned kit, wherein the
antibody reactivities are defined as IgG, IgM, IgA, IgE antibody
isotypes or as more specific sub-isotypes of the antibodies.
[0302] Reference is now made to the aforementioned kit, wherein the
specific antibody reactivity is analyzable by using measuring
means.
[0303] Reference is now made to the aforementioned kit, wherein the
specific antibody reactivity is analyzable by using labeling
means.
[0304] Reference is now made to the aforementioned kit, wherein the
labeling means are adapted to label the human antibodies with a
mixture of secondary antibodies specific for human immunoglobulin
types to form a reactivity matrix.
[0305] Reference is now made to the aforementioned kit, wherein the
labeling means comprises labeling signal selected form a group
consisting of fluorescent, chemiluminescent, absorptive,
electronic, radioactive, paramagnetic or any labeling signal known
in the art, or combination thereof.
[0306] Reference is now made to the aforementioned kit, wherein the
specific antibody reactivity is further analyzable by: (a) using a
scanning means for scanning the reactivity matrix signal; (b) using
a measuring means for measuring the intensity of the signal; (c)
using generating and recording means for generating and recording
results associated with the reactivity matrix signal; (d) using
producing means for producing the results in the form of a
signature, profile, set, pattern or matrix characteristic to the
individual; and, (e) using means for comparing the results with a
databank containing data concerning the antibody reactivities in
populations susceptible to AMI, thereby identifying the development
of an Acute Myocardial Infarction (AMI) process in the
individual.
[0307] Reference is now made to the aforementioned kit, wherein the
measuring and scanning means are selected from a group consisting
of Surface Plasmon Resonance (SPR), ellipsometry, laser scanning,
light detecting, photon detecting via a photon multiplier,
photographing with a digital camera based system or video system,
radiation counting, fluorescence detecting, electronic, magnetic
detecting or any other technique known in the art or combination
thereof
[0308] Reference is now made to the aforementioned kit, wherein the
reactivity is defined as a relative intensity of a specific
antibody bound antigen as compared to its intensity signal in
healthy control individuals.
[0309] Reference is now made to the aforementioned kit, wherein the
reactivity is defined as a relative intensity of a specific
antibody bound antigen as compared to its intensity signal in
individuals who have had a stroke event.
[0310] Reference is now made to the aforementioned kit, wherein the
reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to the intensity signals of the antigens in the healthy
control individuals.
[0311] Reference is now made to the aforementioned kit, wherein the
reactivity is further defined as a set of informative antigens
possessing a higher or a lower intensity reactivity signals as
compared to the intensity signals of the antigens in individuals
who have had a stroke event.
[0312] The current invention further discloses a method for
arresting the development of an AMI process in a patient, wherein
the method comprising steps of: (a) profiling the specific antibody
reactivities or biomarkers associated with AMI susceptibility, (b)
determining the specific AMI associated antigens bound by the
antibody repertoires of the individuals specimens; (c) designing a
drug comprising at least one of the specific antigens or at least
one epitope of the specific antigens; and, (d) administering the
drug to the patient thereby arresting the development of an AMI
process in a patient.
[0313] It is within the scope of the invention that the profiling
step comprises steps of: (i) attaching a set of defined antigens to
a substrate; (ii) obtaining biological fluid derived specimens from
a plurality of individuals in a population, each of the specimens
containing their specific antibody repertoire; (iii) binding the
antibody repertoire of each of the specimens to the attached
antigens thereby forming bound antibody antigen complexes; and,
(iv) analysing results obtained.
[0314] The current invention further discloses a method for
screening for candidate drugs for arresting the development of an
AMI process, in a patient. The aforementioned method comprising the
steps of (a) profiling the specific antibody reactivities
associated with AMI susceptibility of individuals, (b) determining
the specific AMI associated antigens bound by the antibody
repertoires of the individuals; and (c) selecting the candidate
drugs as appropriate for administration as immunotherapy for
arresting the development of an AMI process, in an individual,
wherein the candidate drug contains at least one of the specific
AMI associated antigens or at least one epitope of the specific
antigens. The profiling step further comprising steps of: (i)
attaching a set of defined antigens to a substrate; (ii) obtaining
biological fluid derived specimens from a plurality of individuals
in a population, each of the specimens containing their specific
antibody repertoire; (iii) binding the antibody repertoire of each
of the specimens to the attached antigens thereby forming antibody
antigen complexes; and, (iv) analysing results obtained.
REFERENCES
[0315] 1. Quintana F J, Hagedorn P H, Elizur G, Merbl Y, Domany E
and Cohen IR. Functional immunomics: Microarray analysis of IgG
autoantibody repertoires predicts the future response of mice to
induced diabetes. Proc Natl Acad Sci USA 2004; 101 Suppl
2:14615-21. [0316] 2. Merbl Y, Zucker-Toledano M, Quintana F J,
Cohen I R. Newborn humans manifest autoantibodies to defined self
molecules detected by antigen microarray informatics. J Clin
Invest. 2007 March; 117(3):712-8. [0317] 3. Cohen I R. (2007).
Biomarkers, self-antigens and the immunological homunculus. J
Autoimmun., 2007 December; 29(4):246-9. [0318] 4. Cohen I R. Real
and artificial immune systems: computing the state of the body. Nat
Rev Immunol. 2007 July; 7 (7):569-74. [0319] 5. Maton, Anthea
(1993). Human Biology and Health. Englewood Cliffs, N.J.: Prentice
Hall. ISBN 0-13-98 1176-1. [0320] 6. United States (1999). "Chronic
Disease Overview". United States Government. Retrieved on 2007 Feb.
7. [0321] 7. Informational page on cardiovascular disease at Itamar
Medical. [0322] 8. Rainwater D L, McMahan C A, Malcom G T, Scheer W
D, Roheim P S, McGill H C Jr, Strong J P. Lipid and apolipoprotein
predictors of atherosclerosis in youth: apolipoprotein
concentrations do not materially improve prediction of arterial
lesions in PDAY subjects. The PDAY Research Group. Arterioscler
Thromb Vasc Biol. 1999; 19: 753-61. [0323] 9. Mcgill, H C, Jr.,
Mcmahan, C A, Zieske, A W et al. Associations of coronary heart
disease risk factors with the intermediate lesion of
atherosclerosis in youth. The Pathobiological Determinants of
Atherosclerosis in Youth (PDAY) Research Group. Arterioscler.
Thromb. Vasc. Biol. 2000; 20: 1998-2004. [0324] 10. Wang T J, Gona
P, Larson M G, Tofler G H, Levy D, Newton-Cheh C, Jacques P F,
Rifai N, Selhub J, Robins S J, Benjamin E J, D'Agostino R B, Vasan
R S (2006). "Multiple biomarkers for the prediction of first major
cardiovascular events and death". N. Engl. J. Med. 355 (25):
2631-9. doi:10.1056/NEJMoa055373. PMID 17182988. [0325] 11.
Ramachandran Vasan, et al. " ". N. Engl. J. Lack Energy? Maybe It's
Your Magnesium Level [0326] 12. Comparison of Mechanism and
Functional Effects of Magnesium and Statin Pharmaceuticals Andrea
Rosanoff, PhD, Mildred S. Seelig, M D. Journal of the American
College of Nutrition, Vol. 23, No. 5, 501S-505S (2004). [0327] 13.
Franco, O, Bonneux, L, de Laet. C, Steyerberg, E, Mackenbach, J
(2004). "Franco, O, Bonneux, L, de Laet. C, Steyerberg, E,
Mackenbach, J". BMJ. 329: 1447-1450. doi:10.1136/bmj.329.7480.1447.
PMID 15604180. [0328] 14. Studer M, Briel M, Liemenstoll B, Blass T
R, Bucher H C. "Effect of different antilipidemic agents and diets
on mortality: a systematic review." Arch. Intern. Med. 2005;
165(7): 725-730. [0329] 15. Cook N R, Cutler J A, Obarzanek E, et
al. (2007). "Long term effects of dietary sodium reduction on
cardiovascular disease outcomes: observational follow-up of the
trials of hypertension prevention (TOHP)". Br Med J 334: 885.
doi:10.1136/bmj.39147.604896.55. PMID 17449506. [0330] 16. Elliott
P, Stamler J, Nichols R, et al. (1996). "Intersalt revisited:
further analyses of 24 hour sodium excretion and blood pressure
within and across populations. Intersalt Cooperative Research
Group". Br Med J 312 (7041): 1249-53. PMID 8634612. [0331] 17.
Godlee F (2007). "Editor's Choice: Time to talk salt". Br Med J 334
(7599): 0. doi:10.1136/bmj.39196.679537.47. [0332] 18. Oral Hygiene
and Cardiovascular Disease American Heart Association [0333] 19.
Real connection between oral health and heart disease University of
Michigan Jan. 22, 1999. [0334] 20. Vanhecke T E, Miller W M,
Franklin B A, Weber J E, McCullough P A. Awareness, knowledge, and
perception of heart disease among adolescents. European Journal of
Cardiovascular Prevention and Rehabilitation. October, 2006; 13(5):
718-723. ISSN 1741-8267. [0335] 21. Andraws R, Berger J S, Brown D
L. Effects of antibiotic therapy on outcomes of patients with
coronary artery disease. JAMA 2005; 293:2641-7. PMID 15928286.
[0336] 22. Quintana F J, Merbl Y, Sahar E, Domany E, Cohen I R.
Antigen-chip technology for accessing global information about the
state of the body. Lupus. 2006; 15(7):428-30. [0337] 23. Grabar P.
"Self" and "not-self" in immunology. Lancet. 1974 Jun. 29;
1(7870):1320-2.
APPENDIX I
[0337] [0338] 1. Elias D, Markovits D, Reshef T, van der Zee R,
Cohen I R. Induction and therapy of autoimmune diabetes in the
non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein.
Proc Natl Acad Sci USA 1990; 87(4):1576-80. [0339] 2. Elias D,
Reshef T, Birk O S, van der Zee R, Walker M D, Cohen I R.
Vaccination against autoimmune mouse diabetes with a T-cell epitope
of the human 65-kDa heat shock protein. Proc Natl Acad Sci USA
1991; 88(8):3088-91. [0340] 3. Cohen I R. Peptide therapy for Type
1 diabetes: the immunological homunculus and the rationale for
vaccination. Diabetologia. 2002 October; 45(10):1468-74. [0341] 4.
Raz I, Elias D, Avron A, Tamir M, Metzger M, Cohen I R. Beta-cell
function in new-onset type 1 diabetes and immunomodulation with a
heat-shock protein peptide (DiaPep277): a randomised, double-blind,
phase II trial. Lancet 2001; 358(9295):1749-53. [0342] 5. Huurman V
A, van der Meide P E, Duinkerken G, Willemen S, Cohen I R, Elias D,
Roep B O. Immunological efficacy of heat shock protein 60 peptide
DiaPep277 therapy in clinical type I diabetes. Clin Exp Immunol.
2008 June; 152(3):488-97. [0343] 6. Andromeda Biotech Announces
Successful Phase III Interim Results Of Its Lead Product, DiaPep277
For Type 1 Diabetes 2 Jan. 2009. [0344] 7. Andromeda Biotech, a
subsidiary of ClaI Biotechnology Industries (CBI), focused on the
development of innovative treatment for autoimmune diabetes
announces positive results following the Phase III interim analysis
for the company's lead product, DiaPep277 for the treatment of Type
1 diabetes.
[0345] An Independent Data Monitoring Committee (IDMC) carefully
evaluated the safety and efficacy of the drug for long-term
treatment and re-estimated the sample size needed to demonstrate
statistically significant efficacy at the completion of the
study.
[0346] The Committee reviewed results of 233 patients who completed
at least 12 months of therapy and recommended proceeding with the
study without any change to the design and continuing the enrolment
of patients as planned.
[0347] The results show that no significant drug related Adverse
Events or Serious Adverse Events were reported, hence there are no
safety concerns to continue the study. In addition, a clear
treatment effect was observed in different sub-group populations.
It was recommended that sub-group analyses (age, insulin intake,
HLA and C-peptide baseline level) be included in the final report
to confirm the observed effect. The Committee concluded that the
current number of patients planned to be recruited into the study
is expected to be sufficient to achieve a statistically significant
effect at the end of the trial as observed in the Interim
Analysis.
[0348] "The Interim Analysis represents a critical milestone in the
development of a novel and effective treatment to prevent the
development and progression of Type 1 diabetes using DiaPep 277,"
said Prof. Itamar Raz, Head of the Diabetes Unit at Hadassah
University Hospital who chairs the study Steering Committee. "This
is the largest international study being conducted for intervention
in newly diagnosed Type 1 diabetes using a product that addresses
an unmet clinical need."
[0349] Dr. Shlomo Dagan, CEO of Andromeda commented that "The
report is an achievement for us in the clinical development process
and we look forward to completing patient recruitment during the
first half of 2009
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