U.S. patent application number 15/547252 was filed with the patent office on 2018-01-25 for methods and compositions for diagnosing brain injury or neurodegeneration.
This patent application is currently assigned to ImmunArray USA, Inc.. The applicant listed for this patent is ImmunArray USA, Inc.. Invention is credited to DONNA EDMONDS, KEREN JAKOBI, RACHEL SOREK.
Application Number | 20180024145 15/547252 |
Document ID | / |
Family ID | 56563546 |
Filed Date | 2018-01-25 |
United States Patent
Application |
20180024145 |
Kind Code |
A1 |
SOREK; RACHEL ; et
al. |
January 25, 2018 |
METHODS AND COMPOSITIONS FOR DIAGNOSING BRAIN INJURY OR
NEURODEGENERATION
Abstract
Methods and compositions for diagnosing brain injury,
neurodegeneration; or a predisposition thereto, in a subject are
provided. Particularly, the present invention relates to specific
antigen antibody reactivities useful in diagnosing brain injury,
neurodegeneration or a predisposition thereto, in a subject.
Inventors: |
SOREK; RACHEL; (ZAFARIA,
IL) ; JAKOBI; KEREN; (TEL AVIV, IL) ; EDMONDS;
DONNA; (RICHMOND, VA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ImmunArray USA, Inc. |
RICHMOND |
VA |
US |
|
|
Assignee: |
ImmunArray USA, Inc.
RICHMOND
VA
|
Family ID: |
56563546 |
Appl. No.: |
15/547252 |
Filed: |
February 1, 2016 |
PCT Filed: |
February 1, 2016 |
PCT NO: |
PCT/IL2016/050108 |
371 Date: |
July 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62112189 |
Feb 5, 2015 |
|
|
|
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
G01N 33/96 20130101;
G01N 2800/52 20130101; C07K 14/47 20130101; G01N 2800/56 20130101;
G01N 33/6896 20130101; G01N 2800/28 20130101; G01N 33/54366
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; G01N 33/543 20060101 G01N033/543; G01N 33/96 20060101
G01N033/96 |
Claims
1. A method for detecting brain injury in a subject, the method
comprising the steps of: (i) obtaining a sample from a subject;
(ii) contacting the sample with at least one antigen having an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or any combinations thereof to
determine the reactivity of said at least one antigen with
antibodies in the subject's sample; (iii) measuring the reactivity
of antibodies in the sample relative to the reactivity of
antibodies in a healthy control; and (iv) detecting brain injury in
the subject by measuring a difference in the reactivity of the
antibodies in the sample compared to the reactivity of the
antibodies in the healthy control.
2. (canceled)
3. The method of claim 1, wherein the difference in the
reactivities of the antibodies in the sample and the antibodies in
the healthy control comprises differential IgG and IgM
reactivities.
4. The method of claim 1, wherein said brain injury is selected
from the group consisting of: concussions, chronic traumatic
encephalopathy, mild traumatic brain injuries, moderate traumatic
brain injuries, severe traumatic brain injuries, head trauma,
concussive blasts and brain neurodegenerative condition.
5. (canceled)
6. The method of claim 4, wherein said neurodegenerative condition
is selected from Alzheimer's Disease, Huntington's Disease,
Parkinson's Disease, demyelinating disease, HTLV-1-associated
myelopathy (HAM), multiple sclerosis (MS), amyotrophic lateral
sclerosis, pathological neurological symptoms after injury or
trauma, loss of memory, loss of motor function, encephalopathy, or
viral encephalopathy.
7. The method of claim 1, wherein the sample is selected from the
group consisting of blood, serum, plasma, cerebrospinal fluid
(CSF), urine and saliva.
8. (canceled)
9. The method of claim 1, wherein the reactivity of a healthy
control is selected from the group consisting of a reactivity of at
least one healthy individual, a baseline sample from the same
individual, a panel of control samples from a set of healthy
individuals, and a stored set of data from healthy control
individuals.
10. The method of claim 1, comprising determining the reactivity of
antibodies in the sample to a plurality of antigens provided in the
form of an antigen probe set, an antigen array, or an antigen
chip.
11. (canceled)
12. The method of claim 1, further comprising measuring the levels
of one or more biomarkers in the sample; and comparing the levels
of the one or more biomarkers with predefined levels of the same
biomarkers in a subject having brain injury and predefined levels
of the same biomarkers in a healthy control, wherein a correlation
of the levels in the subject's sample to one of the predefined
levels provides the determination of brain injury in the
subject.
13. The method of claim 12, wherein the one or more biomarkers is
selected from the group consisting of glial fibrillary acidic
protein (GFAP), Synuclein beta (SNCB), Metallothionein-3 (MT3),
Neurogranin (NRGN), intercellular adhesion molecule-5 (ICAM5) and
Brain derived neurotrophic factor (BDNF), or citrullinated forms
thereof.
14. An article of manufacture comprising an antigen probe set
comprising a plurality of antigen probes selected from the group
consisting of SEQ ID NOs: 1-115, isoforms thereof,
post-translationally modified forms thereof, fragments thereof, or
any combinations thereof.
15. (canceled)
16. The article of manufacture of claim 14, further comprising one
or more biomarkers selected from the group consisting of glial
fibrillary acidic protein (GFAP), Synuclein beta (SNCB),
Metallothionein-3 (MT3), Neurogranin (NRGN), intercellular adhesion
molecule-5 (ICAM5) and Brain derived neurotrophic factor (BDNF), or
citrullinated forms thereof.
17. The article of manufacture of claim 14, which is in a format
selected from an antigen probe array, an antigen chip, a dipstick,
or a lateral flow test.
18. (canceled)
19. A method for qualifying brain injury status in a subject in
need thereof, the method comprising the steps of: (i) obtaining a
sample from a subject; (ii) determining the reactivity of
antibodies in the sample to at least one antigen having an amino
acid sequence selected from the group consisting of SEQ ID NOS:
1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or any combinations thereof by
contacting the sample with the at least one antigen; (iii)
measuring the reactivity of antibodies in the sample to a
predefined reactivity that identifies one or more brain injury
statuses selected from the group consisting of having brain injury,
not having brain injury, predisposition to brain injury, sub-acute
brain injury, acute brain injury, post-acute brain injury,
progressing brain injury, regressing brain injury, subclinical
brain injury, mild brain injury, moderate brain injury, severe
brain injury and chronic brain injury; and (iv) qualifying brain
injury status in the subject as indicated by the measured
reactivity in the subject's sample correlating to one of the
predefined reactivities.
20. The method of claim 19, further comprising measuring the levels
of one or more biomarkers in the sample; and comparing the levels
of the one or more biomarkers with predefined levels of the same
biomarkers that correlate to one or more brain injury statuses,
wherein a correlation to one of the predefined levels determines
the brain injury status of the subject.
21. The method of claim 20, wherein the one or more biomarkers is
selected from the group consisting of glial fibrillary acidic
protein (GFAP), Synuclein beta (SNCB), Metallothionein-3 (MT3),
Neurogranin (NRGN), intercellular adhesion molecule-5 (ICAM5) and
Brain derived neurotrophic factor (BDNF), or citrullinated forms
thereof.
22. A method of detecting recovery from brain injury in a subject,
the method comprising the steps of: (i) obtaining a sample from the
subject; (ii) determining the reactivity of antibodies in the
sample to at least one antigen having an amino acid sequence
selected from the group consisting of SEQ ID NOS: 10, 61, 104,
isoforms thereof, post-translationally modified forms thereof,
fragments thereof, or any combinations thereof by contacting the
sample with the at least one antigen; (iii) assaying the reactivity
of antibodies in the sample relative to a predefined reactivity
threshold; and (iv) detecting a difference in the reactivity of the
antibodies in the sample compared to the predefined reactivity
threshold, thereby indicating recovery from brain injury in said
subject.
23. (canceled)
24. The method of claim 22, wherein the difference in the
reactivities of the antibodies in the sample and the antibodies in
the healthy control comprises differential IgG and IgM
reactivities.
25. The method of claim 22, further comprising: measuring the
levels of one or more biomarkers selected from the group consisting
of glial fibrillary acidic protein (GFAP), Synuclein beta (SNCB),
Metallothionein-3 (MT3), Neurogranin (NRGN), intercellular adhesion
molecule-5 (ICAM5) and Brain derived neurotrophic factor (BDNF), or
citrullinated forms thereof in the sample; and comparing the levels
of the one or more biomarkers with predefined levels of the same
biomarkers that correlates with recovery from brain injury, wherein
a correlation to one of the predefined levels is indicative of
recovery from brain injury of the subject.
26. (canceled)
27. The method of claim 25, wherein said comparing is conducted by
using at least one classifier algorithm.
28. The method of claim 27, wherein said at least one classifier
algorithm is selected from the group consisting of a decision tree
classifier, logistic regression classifier (LR), nearest neighbor
classifier, neural network classifier, Gaussian mixture model
(GMM), Support Vector Machine (SVM) classifier, nearest centroid
classifier, linear regression classifier, linear discriminant
analysis (LDA) classifier, quadratic discriminant analysis (QDA)
classifier and random forest classifier.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods and compositions
for diagnosing brain injury, neurodegeneration; or a predisposition
thereto, and more specifically to clinical methods for determining
the presence and type of brain injury or neurodegeneration.
BACKGROUND OF THE INVENTION
[0002] Brain injuries are complex and can have multiple severe
clinical outcomes. Injury of the brain and spinal cord can result
from head trauma, stroke, traumatic birth, heart surgery, cardiac
arrest and patients requiring cardiovascular support with
ventricular assist devices or extracorporeal membrane oxygenation
(ECMO).
[0003] About 1.7 Million Americans sustain a traumatic brain injury
(TBI) each year, ranging from mild to severe, and this is in
addition to about 360,000 soldiers involved in combat operations
and public safety workers surviving terrorist attacks who develop
mild TBI secondary to explosive (concussive) blasts. It contributes
about 30% of all injury related deaths and costs about $60B per
year. At least 230,000 people are hospitalized due to TBI and
survive; more than a million are treated in an emergency department
(ED) for TBI and 80,000 to 90,000 Americans experience long-term
disability from TBIs.
[0004] Recent study was conducted to determine the dimensions of
TBI evaluation in US emergency department. TBI was evaluated during
4.8 million visits per year; and head CT scan was performed in 82%
of TBI evaluations (3.9 million visits per year). TBI was diagnosed
in 52% of evaluations (2.5 million visits per year). Among those
who received head CT scans, 9% had CT evidence of traumatic
abnormalities. Among patients evaluated for TBI who had a Glasgow
Coma Scale score recorded, 94.5% were classified as having mild
TBI, 2.1% as moderate TBI, and 3.5% as severe TBI. Among patients
with International Classification of Diseases, Ninth Revision,
Clinical Modification, codes permitting the calculation of head
Abbreviated Injury Scale scores 9.0%, 85.0%, 2.5%, 3.2%, 0.3%, and
0% had head Abbreviated Injury Scale scores of 1, 2, 3, 4, 5, and
6, respectively. Of patients evaluated for TBI, 31% had other
head/face/neck injuries, 10% had spine and back injuries, 7% had
torso injuries, and 14% had extremity injuries (Korley et al., Sep.
10, 2015, J Head Trauma Rehabil)
[0005] TBI is the result of a blunt blow, jolt or blast
overpressure to the head that disrupts brain function. The subset
of mild TBI (mTBI) has represented a harder segment of TBI to
diagnose. The severity of head injuries range from a brief change
in mental status or consciousness to extended unconsciousness and
amnesia. In severe or multiple concussion cases, personality
changes can occur with devastating results. Cognitive decline is
recognized as part of the post injury syndrome.
[0006] Proper treatment of TBI injury requires an accurate
diagnosis of the structures affected. The mechanisms of injury in
TBI cause a variety of abnormalities in the peripheral vestibular
mechanisms, central vestibular structures, ocular-motor tracts,
cerebellum, as well as all portions of the brain communicating with
these structures. The onset of vestibular deficits generally occurs
within seven to ten days post injury. While reported symptoms of
dizziness resolve after three months, 15% have persistent symptoms
one year later.
[0007] At present, one of the rather subjective and not totally
effective diagnostic procedures when traumatic brain injury is
suspected involves a number of examining techniques. The patient
receives a neurological examination which may consist of the
following: 1) mental status, 2) motor function, 3) sensory
examination, 4) deep tendon reflexes, 5) station, gait, and
equilibrium, and 6) cranial nerve function. The mental status
examination may include: a) level of consciousness, b) short and
long term memory, c) knowledge of patient and place and d)
questions about symptoms: headache, dizziness, blurry vision, etc.
In addition, the patient may also have radiological studies which
could include CT scan of the head, MRI, PET scan. It has been
reported that in the early stages of (especially mild) traumatic
brain injury, the imaging techniques may not be sufficiently
sensitive to detect an abnormality. Furthermore, the patient's
cognitive skills may not be impaired initially, and there may be
few, if any, symptoms. Patients are often observed over 24-48 hours
and are awakened at regular intervals (e.g., every 3-4 hours) to
assure that they are able to be aroused. Narcotics for headache or
other pain are not given, so that their effects do not cloud the
issue of the patient's arousal state. A computerized test which
determines level of cognition and reaction time is often employed
with repetitive examinations.
[0008] One of the problems with this approach in diagnosing
potential traumatic brain injuries is that it is not one which
always provides precise, timely, objective information. It is also
subject to individual variations from person-to-person. Further, if
the person is asymptomatic at the time, the conclusion might be
that there is no problem, and the individual might be encouraged to
go back to normal activities. Such guidance could potentially be
injurious to the person's health and could even lead to fatal
consequences.
[0009] Once a patient has been diagnosed with a brain injury, it
becomes important to treat the patient in a timely, effective
manner in order to minimize the risk of permanent injury or
death.
[0010] In spite of the foregoing known procedures, there remains a
very real and substantial need for a method of early and effective
determination as to whether an individual has suffered a brain
injury, how severe it might be, and upon finding the presence of
such an injury, effectively treating the patient.
SUMMARY OF THE INVENTION
[0011] The present invention provides methods and kits for
diagnosing brain injury or neurodegeneration. The present invention
further provides antigen probe arrays for practicing such a
diagnosis, and antigen probe sets for generating such arrays.
[0012] The antigen probe sets of the present invention can be used
to profile the antibody response to said antigens in patients
suffering from brain injury or neurodegenerative disease, due to
disruption of any or all components of the anatomic structure and
the ability to detect elements which cross the blood brain barrier.
This antibody response profile can be used for the diagnosis,
monitoring and management of brain injury. According to some
embodiments, the antibody profile reflects the patient status at
time of injury. The profile of the antibody response can be
measured on any platform including but not limited to a micro array
or any array chip.
[0013] The present invention is based, in part, on the unexpected
results obtained when testing the antibody reactivity of patients
suffering from brain injury compared to healthy controls.
Surprisingly, differential immunoglobulin G (IgG) and IgM
reactivities to specific antigens were found in the tested brain
injury patients, compared to healthy controls. The present
invention is also based on the discovery that analysis of the
pattern of an individual's antibody response to specific brain
related molecules in combination with markers of immune response
provides a novel and reliable method of ascertaining the nature and
extent of brain injury and of other neurodegenerative conditions.
Thus, the present invention provides unique antigens, indicative to
brain injury. The present invention further provides
antigen-autoantibody reactivity patterns relevant to brain injury.
In particular embodiments, the present invention provides highly
specific, reliable, accurate and discriminatory assays for
diagnosing and monitoring brain injury, based on the indicative
antigens, or on reactivity patterns thereof.
[0014] According to some embodiments, the `pre-existing state` of
the patient status at time of injury is monitored.
[0015] Thus, according to some embodiments of the invention, there
are provided novel methods for diagnosing and monitoring the
progression of brain injury. According to some embodiments of the
invention, the methods comprise determining the reactivity of
antibodies in a sample obtained or derived from a subject to at
least one antigen as described herein. The methods of the invention
further comprise a step of comparing the reactivity of antibodies
in the sample to the at least one antigen to a control reactivity
to said at least one antigen. According to certain embodiments, a
significantly differential reactivity of the antibodies in the
sample compared to the reactivity of the healthy control, or to the
reactivity of baseline samples from the same patient, is an
indication that the subject is afflicted with brain injury.
[0016] According to certain embodiments, the baseline samples from
the same patient may be used [for measurements over time] to
predict progression, resolution of event or remission of disease
course.
[0017] According to certain embodiments, the methods of the present
invention can discriminate which patients with brain injury require
a head CT scan to rule out intracranial hemorrhage versus
concussion alone. If implemented as an initial response (e.g., in
the emergency department (ED) setting) or later (e.g., neurology
department), the methods of present invention would decrease head
CT scan utilization, decreasing health care costs and radiation
exposure.
[0018] Thus, according to a first aspect, the present invention
provides a method of diagnosing brain injury in a subject, the
method comprising the steps of obtaining a sample from the subject,
determining the reactivity of antibodies in the sample to at least
one antigen selected from the groups consisting of SEQ ID
NOs:1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing; and comparing the reactivity of antibodies in the sample
to a reactivity of a healthy control; wherein a significantly
different reactivity of the antibody or antibodies in the sample
compared to the reactivity of the healthy control is an indication
that the subject is afflicted with brain injury.
[0019] In certain embodiments, the at least one antigen is selected
from the groups consisting of SEQ ID NOs: 2, 14, 28, 42, 85, and
86, or any combination thereof.
[0020] In certain embodiments, said method further comprising
measuring the levels of one or more biomarkers in the sample; and
comparing the levels of the one or more biomarkers with predefined
levels of the same biomarkers that correlate to a subject having
brain injury and predefined levels of the same biomarkers that
correlate to a healthy control, wherein a correlation to one of the
predefined levels provides the diagnosis.
[0021] In certain embodiments, said brain injury is selected from
the group consisting of: concussions, chronic traumatic
encephalopathy, mild traumatic brain injuries, moderate traumatic
brain injuries, severe traumatic brain injuries, head trauma,
concussive blasts and brain neurodegenerative condition.
[0022] In certain embodiments, said brain injury causes disruption
of the blood-brain barrier.
[0023] In certain embodiments, the brain neurodegenerative
condition further comprises loss of memory or motor function and
cognitive decline.
[0024] In certain embodiments, the neurodegenerative condition is
selected from the group consisting of: Alzheimer's disease,
Huntington's disease, Parkinson's disease, demyelinating disease,
HTLV-1-associated myelopathy (HAM), multiple sclerosis (MS),
amyotrophic lateral sclerosis (ALS), pathological neurological
symptoms after injury or trauma, encephalopathy and viral
encephalopathy.
[0025] In certain embodiments, a significantly higher reactivity of
the antibodies in the sample compared to the reactivity of the
healthy control is an indication that the subject is of increased
likelihood to be afflicted with brain injury. In other certain
embodiments, where the reactivity of the antibodies in the sample
compared to the reactivity of the healthy control is not
significantly higher, where the reactivity of the antibodies in the
sample compared to the reactivity of the healthy control is the
same, where the reactivity of the antibodies in the sample compared
to the reactivity of the healthy control is lower or where the
reactivity of the antibodies in the sample compared to the
reactivity of the healthy control is significantly lower, it is an
indication that the subject is of decreased likelihood to be
afflicted with brain injury. Each possibility represents a separate
embodiment of the present invention.
[0026] In certain embodiments of the methods of the present
invention, the methods are preceded by a step comprising obtaining
or deriving a sample from the subject. In certain embodiments, the
sample is obtained or derived from the subject by non-invasive
means or methods.
[0027] In certain embodiments, said obtaining is carried out within
two hours of the head trauma. In certain embodiments, said
obtaining is carried out within four hours of the head trauma. In
certain embodiments, said obtaining is carried out within 24 hours
of the head trauma. In certain embodiments, said obtaining is
carried out within 72 hours of the head trauma. In certain
embodiments, said obtaining is carried out during the post-acute
care.
[0028] In certain embodiments, the subject is conscious at the time
of said obtaining.
[0029] In certain embodiments, determining the reactivity of
antibodies in the sample to a plurality of antigens produces a
reactivity pattern, used for the diagnosis of brain injury in the
subject. Thus, according to exemplary embodiments of the invention,
the reactivity pattern of antibodies in the sample to the plurality
of antigens is compared to the reactivity pattern of antibodies in
a sample corresponding to healthy control subjects to said
plurality of antigens, wherein a significant difference between the
reactivity pattern of the sample and the reactivity pattern of
healthy controls indicates that the subject is afflicted with, or
in other embodiments has increased likelihood for having brain
injury. Conveniently, the reactivity patterns are calculated and
compared using e.g. learning and pattern recognition algorithms as
described herein.
[0030] According to another embodiment, the reactivity of
antibodies comprises IgG and IgM reactivities. According to another
embodiment, the significantly higher reactivity of the antibodies
in the sample comprises differential IgG and/or IgM reactivities.
According to another embodiment, the increased IgM reactivity is of
at least one antigen selected from the group consisting of SEQ ID
NOs: 1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing. According to another embodiment, the increased IgG
reactivity is of at least one antigen selected from the group
consisting of SEQ ID NOs: 1-115, isoforms thereof,
post-translationally modified forms thereof, fragments thereof, or
combinations of any of the foregoing. Each possibility represents a
separate embodiment of the invention.
[0031] According to additional embodiments of the methods of the
present invention, the sample obtained from the subject is a
biological fluid. According to some embodiments, the sample is
selected from the group consisting of plasma, serum, blood,
cerebrospinal fluid, synovial fluid, sputum, urine, saliva, tears,
lymph specimen, or any other biological fluid known in the art.
Each possibility represents a separate embodiment of the invention.
According to certain embodiments, the sample obtained from the
subject is selected from the group consisting of serum, plasma and
blood. According to one embodiment, the sample is a serum sample.
In certain embodiments, the sample is obtained or derived from the
subject by non-invasive means or methods.
[0032] According to certain embodiments of the methods of the
present invention, the control is selected from the group
consisting of a sample from at least one healthy individual, a
baseline sample from same subject, a panel of control samples from
a set of healthy individuals, and a stored set of data from healthy
individuals. Each possibility represents a separate embodiment of
the invention. Typically, a healthy individual is a subject not
afflicted with brain injury. In another embodiment, a healthy
individual is a subject not afflicted with neurodegenerative
disease.
[0033] According to another embodiment, the method comprises
determining the reactivity of antibodies in the sample to a
plurality of antigens.
[0034] According to another embodiment, the method comprises
determining the reactivity of antibodies in the sample to at least
one antigen selected from the group consisting of SEQ ID NOs:
1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing. According to another embodiment, the method comprises
determining the reactivity of antibodies in the sample to at least
two antigens selected from the group consisting of SEQ ID NOs:
1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing.
[0035] According to another embodiment, the plurality of antigens
is used in the form of an antigen probe set, an antigen array, or
an antigen chip.
[0036] According to another aspect, the present invention provides
an antigen probe set comprising a plurality of antigen probes
selected from the group consisting of SEQ ID NOs: 1-115, isoforms
thereof, post-translationally modified forms thereof, fragments
thereof, or combinations of any of the foregoing. In another
embodiment, the antigen probe set comprises the antigen probes of
SEQ ID NOs: 1-115, isoforms thereof, post-translationally modified
forms thereof, fragments thereof, or combinations of any of the
foregoing.
[0037] According to another aspect, the present invention provides
an article of manufacture comprising the antigen probe set
described above.
[0038] In certain embodiments, the article of manufacture, further
comprising one or more biomarkers selected from the group
consisting of glial fibrillary acidic protein (GFAP), Synuclein
beta (Sncb), Metallothionein-3 (MT3), Neurogranin (NRGN),
intercellular adhesion molecule-5 (ICAM5) and Brain derived
neurotrophic factor (BDNF), or citrullinated forms thereof.
[0039] In certain embodiments, the article of manufacture is in the
form of an antigen probe array or in the form of an antigen chip or
in the form of a dipstick or in the form of a lateral flow test or
in the form of an ELISA plate or in the form of a Quanterix system,
an Agilent Plate reader, a Meso Scale Diagnostics platform, or any
other platform known to those skilled in the art. In certain
embodiments, the article of manufacture is in the form of a
kit.
[0040] According to certain embodiments, the kit further comprises
means for determining the reactivity of antibodies in a sample to
at least one antigen of the plurality of antigens. According to
another embodiment, the kit further comprises means for comparing
reactivity of antibody in different samples to at least one antigen
of the plurality of antigens. According to another embodiment, the
kit further comprises instructions for use of the kit for
diagnosing brain injury.
[0041] According to another aspect, there is provided use of the at
least one antigen selected from the group consisting of: SEQ ID
NOs: 1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing; for the preparation of a diagnostic kit for diagnosing
brain injury in a subject. Each possibility represents a separate
embodiment of the invention. The diagnostic kit is, in some
embodiments, useful for determining the reactivity of antibodies in
a sample, thereby determining the reactivity pattern of the sample
to the at least one antigen. In some embodiments, a significant
difference (e.g., increase) between the reactivity pattern of the
sample compared to a reactivity pattern of a control sample is an
indication for brain injury.
[0042] According to another aspect, there is provided a method for
qualifying brain injury status in a subject the method comprising
the steps of: obtaining a sample from the subject; determining the
reactivity of antibodies in the sample to at least one antigen
selected from the group consisting of SEQ ID NOs: 1-115, isoforms
thereof, post-translationally modified forms thereof, fragments
thereof, or combinations of any of the foregoing; and comparing the
reactivity of antibodies in the sample to a predefined reactivity
that correlate to one or more brain injury statuses selected from
the group consisting of having brain injury, not having brain
injury, predisposition to brain injury, sub-acute brain injury,
acute brain injury, post-acute brain injury, progressing brain
injury, regressing brain injury, subclinical brain injury, mild
brain injury, moderate brain injury, severe brain injury and
chronic brain injury, wherein a correlation to one of the
predefined reactivities determines the brain injury status of the
subject.
[0043] In certain embodiments, said method further comprising
measuring the levels of one or more biomarkers in the sample; and
comparing the levels of the one or more biomarkers with predefined
levels of the same biomarkers that correlate to one or more brain
injury statuses, wherein a correlation to one of the predefined
levels determines the brain injury status of the subject.
[0044] According to another aspect, there is provided a method of
detecting recovery from brain injury in a subject, the method
comprising the steps of: obtaining a sample from the subject;
determining the reactivity of antibodies in the sample to at least
one antigen selected from the group consisting of SEQ ID NOS: 10,
44, 61, 66, 102, 104, isoforms thereof, post-translationally
modified forms thereof, fragments thereof, or any combinations
thereof; and comparing the reactivity of antibodies in the sample
to a predefined reactivity threshold; wherein a significantly
different reactivity of the antibodies in the sample compared to
the predefined reactivity threshold is indicative of recovery from
brain injury in said subject.
[0045] In certain embodiments, said method further comprising
measuring the levels of one or more biomarkers in the sample; and
comparing the levels of the one or more biomarkers with predefined
levels of the same biomarkers that correlates with recovery from
brain injury, wherein a correlation to one of the predefined levels
is indicative of recovery from brain injury of the subject.
[0046] In certain embodiments, the one or more biomarkers is
selected from the group consisting of glial fibrillary acidic
protein (GFAP), Synuclein beta (Sncb), Metallothionein-3 (MT3),
Neurogranin (NRGN), intercellular adhesion molecule-5 (ICAM5) and
Brain derived neurotrophic factor (BDNF), or citrullinated forms
thereof.
[0047] In certain embodiments, a combination of one or more
antibodies and one or more biomarkers are used.
[0048] According to certain embodiments, the comparison is
conducted by using at least one classifier algorithm.
[0049] According to certain embodiments, the at least one
classifier algorithm is selected from the group consisting of a
decision tree classifier, logistic regression classifier, nearest
neighbor classifier, neural network classifier, Gaussian mixture
model (GMM), Support Vector Machine (SVM) classifier, nearest
centroid classifier, linear regression classifier, linear
discriminant analysis (LDA) classifier, quadratic discriminant
analysis (QDA) classifier and random forest classifier.
[0050] Other objects, features and advantages of the present
invention will become clear from the following description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 illustrates anti-Fatty acid-binding protein (FABP-3,
SEQ ID No: 61) IgM autoantibody levels at day 30 post brain injury.
TBI patients with Glasgow Outcome Scale Extended (GOSE) score <8
(cross labeled) represent lower IgM levels than patients with GOSE
score=8 (circle labeled).
[0052] FIG. 2 illustrates anti-Myelin basic protein (MBPR149, SEQ
ID No: 10) derived BSA conjugated peptide IgM autoantibody levels
at day 30 post brain injury. TBI patients with Glasgow Outcome
Scale Extended (GOSE) score <8 (cross labeled) represent higher
IgM levels than patients with GOSE score=8 (circle labeled).
[0053] FIG. 3 illustrates anti-Myeloperoxidase (MPO, SEQ ID No: 85)
IgM autoantibody levels in serum samples obtained from TBI patients
(circle labeled) in comparison with healthy controls (cross
labeled).
[0054] FIG. 4A illustrates anti-CMV (SEQ ID No: 86) IgG
autoantibody levels in serum samples obtained from TBI patients
(circle labeled) at day 30 and day 90 post injury (N=142) in
comparison with healthy controls (cross labeled) (N=21).
[0055] FIG. 4B shows the above separation performance by receivers
operating characteristic (ROC) curves of anti-Cytomegalovirus (CMV)
IgG autoantibody levels. T test P value for separation: 3.746E-07,
after FDR correction: 5.02E-05. Kruskal-Wallis test P value for
separation: 4.567E-05, after FDR correction: 0.0081593.
[0056] FIG. 5A demonstrates the prediction of the clinical status
of TBI patients at day 90 post injury, based on the anti-TNFRSF12A
(SEQ ID No: 104) IgM autoantibody levels in serum samples obtained
from TBI patients at day 30 post injury. TBI patients, with GOSE
<8 at day 90 post injury (circle labeled) (N=52) were compared
with TBI patients, with GOSE=8 at day 90 post injury (cross
labeled) (N=15).
[0057] FIG. 5B shows the above separation performance by receivers
operating characteristic (ROC) curves of anti-TNFRSF12A IgM
autoantibody levels. T test P value for separation: 6.808E-06,
after FDR correction: 0.0036493. Kruskal-Wallis test P value for
separation: 0.0004082, after FDR correction: 0.1541973
[0058] FIG. 6 shows the area under the Receiver Operating
Characteristics (ROC) curves of six classification methods (SVM,
LR, QDA, CART, RF and LDA) based on 100 iterations of 70:30 cross
validation. Features were ranked according to their median scoring
or frequency of model inclusion, depending on the method. Serum
samples obtained from TBI patients at time 0 (t0, N=85) were
compared with serum samples obtained from healthy control (HC,
N=21). The analysis was based on 464 iChip features (232 antigen,
IgM and IgG) and four ELISA features. iChip data is based on
average of two block replicates, following correction procedure.
ELISA features were selected based on data availability; only
features with data available for >80% of the iChip samples were
used. Samples with missing ELISA data were removed from the
analysis.
[0059] FIG. 7 shows ROC curves of six classification methods (SVM,
LR, QDA, CART, RF and LDA), based on 100 iterations of 70:30 cross
validation. Features were ranked according to their median scoring
or frequency of model inclusion, depending on the method. Serum
samples obtained from TBI patients at time 0 (t0) with abnormal CT
were compared with samples obtained from TBI patients at time 0
(t0) with Normal CT. Analysis was based on 464 iChip features (232
antigen, IgM and IgG) and four ELISA features. iChip data is based
on average of two block replicates, following correction procedure.
ELISA features were selected based on data availability; only
features with data available for >80% of the iChip samples were
used. Samples with missing ELISA data were removed from the
analysis.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention provides methods of diagnosing brain
injury or a neurodegenerative disorder in a subject. The present
invention further provides antigen probe sets or arrays for
practicing such a diagnosis, and identifies specific antigen probe
sets for generating such sets or arrays.
[0061] Without wishing to be bound by any particular theory or
mechanism of action, the invention is based, in part, on the
finding of unique antigens highly distinctive between healthy
subjects and patients suffering from brain injury. The invention is
further based on the finding that the antibody reactivity profile
in serum of patients suffering from brain injury was clearly
distinct from healthy control individuals. Although protein
biomarkers of brain injury patients have been extensively
investigated, the unique antibody immune signatures as described
herein have not been described before. Advantageously, the unique
antibody signatures of the present disclosure provide highly
sensitive and specific assays for diagnosing brain injury.
[0062] The present invention provides, in some embodiments, unique
antigen-antibody reactivity patterns particularly relevant to brain
injury. In the course of investigating specific antibodies, the
inventors examined the reactivity of IgM and IgG antibodies in the
sera of healthy persons and those diagnosed with brain injury to a
variety of antigens, using antigen microarray and informatics
analysis.
Definitions
[0063] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only and is
not intended to be limiting. It must be noted that, as used in the
specification and the appended claims, the singular forms "a," "an"
and "the" include plural referents unless the context clearly
dictates otherwise.
[0064] The term "about" as used herein means in quantitative terms
plus or minus 5%, or in another embodiment plus or minus 10%, or in
another embodiment plus or minus 15%, or in another embodiment plus
or minus 20%.
[0065] As used herein, the term "autoantibodies" refers to
antibodies that are capable of reacting against an antigenic
constituent of an individual's own tissue or cells (e.g., the
antibodies recognize and bind to "self-antigens").
[0066] The term "brain injury" refers to a condition in which the
brain is damaged by injury caused by an event. As used herein, an
"injury" is an alteration in cellular or molecular integrity,
activity, level, robustness, state, or other alteration that is
traceable to an event. For example, an injury includes a physical,
mechanical, chemical, biological, functional, infectious, or other
modulator of cellular or molecular characteristics. An event can
include a physical trauma such as an impact (percussive or
concussive) or a biological abnormality such as a stroke resulting
from either blockade or leakage of a blood vessel. An event is
optionally an infection by an infectious agent. A person of skill
in the art recognizes numerous equivalent events that are
encompassed by the terms injury or event.
[0067] According to some embodiments of the method of the present
invention, a healthy subject's predisposition to future onset of
brain injury is also diagnosed. According to some embodiments said
predisposition is due to previous injury or due to family
history.
[0068] More specifically, the term "brain injury" refers to a
condition that results in central nervous system damage,
irrespective of its pathophysiological basis. Among the most
frequent origins of a "brain injury" are stroke and traumatic brain
injury (TBI). A "stroke" is classified into hemorrhagic and
non-hemorrhagic. Examples of hemorrhagic stroke include cerebral
hemorrhage, subarachnoid hemorrhage, and intracranial hemorrhage
secondary to cerebral arterial malformation, while examples of
non-hemorrhagic stroke include cerebral infarction.
[0069] The term "brain injury" also refers to subclinical brain
injury, spinal cord injury, and anoxic-ischemic brain injury. The
term "subclinical brain injury" (SCI) refers to brain injury
without overt clinical evidence of brain injury. A lack of clinical
evidence of brain injury when brain injury actually exists could
result from degree of injury, type of injury, level of
consciousness, medications particularly sedation and anesthesia.
Many of these origins can lead to Chronic Traumatic Encephalopathy
(CTE).
[0070] As employed herein, the term "traumatic brain injury" shall
mean a brain injury resulting from direct or indirect shock load or
loads applied to the brain causing it to move rapidly and
unnaturally within a patient's skull and shall expressly include,
but not be limited to, brain injuries caused by: (a) objects
penetrating the skull, such as, bullets, arrows, and other physical
objects which pass through the skull and enter the brain, (b)
impact loads applied to the head or other portions of the patient's
body, (c) surgically induced trauma, (d) explosions, such as might
exist in warfare, through impacting of grenades, bombs, and other
explosives, which cause substantial tremors in the earth in
relatively-close proximity to where an individual is standing, as
well as similar tremors created by nonexplosive means, such as
sports injuries, vehicular accidents, collapse of buildings and
earthquakes, for example. The results of traumatic brain injury may
be of various types, but in each instance, will involve temporary
or permanent reduction in the ability of the brain to function
normally and may cause death.
[0071] One of the consequences of a traumatic brain injury
frequently is the generation of inflammation within the brain as
the shock to the brain serves to increase the permeability of the
endothelial cells, thereby permitting loss of fluids from the
vascular structure into the brain. Such a leakage frequently occurs
due to the increased porosity of the blood vessels resulting from
the trauma, thereby causing blood serum to leak through the vessels
into the brain area. As this builds up, this can generate
inflammation and swelling of the brain, which may require surgical
intervention.
[0072] Clinically, traumatic brain injury can be rated as mild,
moderate or severe based on TBI variables that include duration of
loss of consciousness (LOC), Glasgow Coma Score (GCS) and
post-traumatic stress amnesia.
[0073] As used herein, "secondary brain trauma" refers to damage to
the brain of a patient post-acute brain injury, i.e., during the
secondary injury phase of a TBI.
[0074] "Chronic traumatic encephalopathy (CTE)" is a
neurodegenerative disease that is most often identified in
postmortem autopsies of individuals exposed to repetitive head
impacts, such as boxers and football players. The neuropathology of
CTE is characterized by the accumulation of hyperphosphorylated tau
protein in a pattern that is unique from that of other
neurodegenerative diseases, including Alzheimer's disease. The
clinical features of CTE are often progressive, leading to dramatic
changes in mood, behavior, and cognition, frequently resulting in
debilitating dementia. In some cases, motor features, including
Parkinsonism, can also be present.
[0075] A "non-traumatic brain injury" refers to brain injuries that
do not involve ischemia or external mechanical force (e.g., stroke,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
multiple sclerosis, amyotrophic lateral sclerosis, brain
hemorrhage, brain infections, brain tumor, among others).
[0076] "Stroke" refers to the destruction of brain tissue as a
result of intracerebral hemorrhage or infarction. Stroke is a
leading cause of death in the developed world. It may be caused by
reduced blood flow and death of tissues in one area of the brain
(infarction). Causes of strokes include blood clots that form in
the blood vessels in the brain (thrombus) and blood clots or pieces
of atherosclerotic plaque or other material that travel to the
brain from another location (emboli). Bleeding (hemorrhage) within
the brain may also cause symptoms that mimic stroke.
[0077] "Alzheimer's disease (AD)" is a very common yet
irreversible, progressive brain disease that slowly destroys memory
and thinking skills, and eventually the ability to carry out the
simplest tasks. AD is the most common cause of dementia among older
people causing the loss of cognitive functioning thinking,
remembering, and reasoning to such an extent that it interferes
with a person's daily life and activities. Estimates vary, but
experts suggest that as many as 5.1 million Americans may have AD.
Currently brain imaging of people with, and those with a family
history, of AD or its earlier stage, amnesic mild cognitive
impairment (MCI), are beginning to detect changes in the brain. The
clinical dementia of AD is coupled with a distinct pathology of
senile plaques. AD is characterized by abnormal amyloid beta
accumulation and deposition in brain parenchyma and cerebral
capillaries, which leads to blood-brain barrier (BBB)
disruption.
[0078] As used herein, "chronic brain injury" refers to a subject
who has suffered a brain injury from three days post injury until
at least 12 months previously yet continues to present symptoms of
brain injury.
[0079] As used herein, "sub-acute brain injury" refers to a subject
who has suffered a brain injury from about 2-5 days post
injury.
[0080] "Conscious", as used herein, has the conventional meaning,
as set forth in Plum, et al., The Diagnosis of Stupor and Coma, CNS
Series, Philadelphia:Davis (1982), which is hereby incorporated by
reference. Conscious patients include those who have a capacity for
reliable, reproducible, interactive behavior evidencing awareness
of self or the environment. Conscious patients include patients who
recover consciousness with less severe brain injury but who,
because of their impaired cognitive function, do not reach
independent living. Conscious patients do not include those who
exhibit wakefulness but lack interaction (e.g., those deemed to be
in a persistent vegetative state).
[0081] The subject who is conscious after exposure to a head trauma
may be asymptomatic of any visible symptoms of traumatic brain
injury. Conversely, the subject may exhibit various symptoms of
brain injury and cognitive dysfunction.
[0082] This is in contrast to a subject who is unconscious at the
time of the obtaining, as indicated by conditions such as a
concussion or intracranial hemorrhage (e.g. intra-axial hematoma,
epidural hematoma, and subdural hematoma).
[0083] The phrase "brain injury status" includes any
distinguishable manifestation of the condition, including not
having brain injury. For example, brain injury status includes,
without limitation, the presence or absence of brain injury in a
patient, the risk of developing brain injury, the stage or severity
of brain injury, the progress of brain injury (e.g., progress of
brain injury over time) and the effectiveness or response to
treatment of brain injury (e.g., clinical follow up and
surveillance of brain injury after treatment). Based on this
status, further procedures may be indicated, including additional
diagnostic tests or therapeutic procedures or regimens.
[0084] The "spinal cord injury" refers to a condition in which the
spinal cord receives compression/detrition due to a vertebral
fracture or dislocation to cause dysfunction. As used herein, the
term "anoxic-ischemic brain injury" refers to deprivation of oxygen
supply to brain tissue resulting in compromised brain function and
includes cerebral hypoxia. For example, anoxic-ischemic brain
injury includes focal cerebral ischemia, global cerebral ischemia,
hypoxic hypoxia (i.e., limited oxygen in the environment causes
reduced brain function, such as with divers, aviators, mountain
climbers, and fire fighters, all of whom are at risk for this kind
of cerebral hypoxia), obstructions in the lungs (e.g., hypoxia
resulting from choking, strangulation, the crushing of the
windpipe).
[0085] The term "brain injury biomarker" (BIB), "brain injury
biomarker protein", "brain injury biomarker peptide", brain injury
biomarker polypeptide" and the like refer to a protein, including
those described herein, that can be used in a method of the present
invention, e.g., to diagnose brain injury in a patient. Brain
injury biomarker proteins include, but are not limited to, SNCB,
GFAP, S100B, MT3, ICAM5, BDNF, and/or NSE.
[0086] The term also includes other brain injury biomarker proteins
known in the art including neurogranin (NRGN), myelin basic protein
(MBP), PAD-2, tubulin beta-4B chain, tubulin alpha-IB chain,
CNPase, PPIA, Septin-7, Elongation factor 1-alpha2, TPPP, TPPP3,
Ermin Isoform 2, NDRG2 Isoform 2, astrotactin 1 (ASTN1), brain
angiogenesis inhibitor 3 (BAD); carnosine dipeptidase 1 (CNDP 1);
ERMTN; glutamate receptor metabotropic 3 (GRM3); kelch-like protein
32 (KLH32); melanoma antigen family E,2 (MAGE2); neuregulin 3
(NRG3); oligodendrocyte myelin glycoprotein (OMG); solute carrier
family 39 (zinc transporter); reticulon 1 (RTN1); and
peptidylarginine deiminase (types 1-4 and 6) (PAD).
[0087] In addition, the term "brain injury biomarkers" also
includes the isoforms and/or post-translationally modified forms of
any of the foregoing. The present invention contemplates the
detection, measurement, quantification, determination and the like
of both unmodified and modified (e.g., citrullination or other
post-translational modification) proteins/polypeptides/peptides as
well as autoantibodies to any of the foregoing. In certain
embodiments, it is understood that reference to the detection,
measurement, determination, and the like, of a biomarker refers
detection of the protein/polypeptide/peptide (modified and/or
unmodified). In other embodiments, reference to the detection,
measurement, determination, and the like, of a biomarker refers
detection of autoantibodies of the protein/polypeptide/peptide.
[0088] As used herein, the term "comparing" refers to making an
assessment of how the reactivity of antibodies in a sample from a
patient relates to the reactivity of the corresponding antibodies
in a standard or control sample. For example, "comparing" may refer
to assessing whether the reactivity of antibodies from a sample of
a patient to one or more antigens is the same as, more or less
than, or different from the corresponding reactivity of antibodies
from the standard or control sample. More specifically, the term
may refer to assessing whether the reactivity of antibodies of a
sample from a patient to one or more antigens is the same as, more
or less than, different from or otherwise corresponds (or not) to a
predefined reactivity of antibodies that correspond to, for
example, a patient having subclinical brain injury (SCI), not
having SCI, is responding to treatment for SCI, is not responding
to treatment for SCI, is/is not likely to respond to a particular
SCI treatment, or having/not having another disease or
condition.
[0089] As used herein, the terms "indicates" or "correlates" (or
"indicating" or "correlating," or "indication" or "correlation,"
depending on the context) in reference to a parameter, e.g., a
modulated reactivity of antibodies of a sample from a patient, may
mean that the patient has brain injury. In specific embodiments,
the parameter may comprise the reactivity of antibodies to one or
more antigens of the present invention. A particular reactivity of
antibodies to one or more antigens may indicate that a patient has
brain injury (i.e., correlates to a patient having brain injury).
In other embodiments, a particular reactivity of antibodies to one
or more antigens may be correlated to a patient being unaffected
(i.e., indicates a patient does not have brain injury. In certain
embodiments, "indicating," or "correlating," as used according to
the present invention, may be by any linear or non-linear method of
quantifying the relationship between levels of reactivity of
antibodies to a standard, control or comparative value for the
assessment of the diagnosis, prediction of brain injury or brain
injury progression, assessment of efficacy of clinical treatment,
identification of a patient that may respond to a particular
treatment regime or pharmaceutical agent, monitoring of the
progress of treatment, and in the context of a screening assay, for
the identification of an anti-brain injury therapeutic.
[0090] According to some embodiments, monitoring the progression of
brain injury is conducted at 7-20 day time point post injury, where
the neural circulatory reconnections begin to occur. According to
some embodiments, the risk of damage to the neural circulatory
system is predicted.
[0091] The terms "patient," "individual," or "subject" are used
interchangeably herein, and refer to a mammal, particularly, a
human. The patient may have mild, intermediate or severe disease.
The patient may be treatment naive, responding to any form of
treatment, or refractory. The patient may be an individual in need
of treatment or in need of diagnosis based on particular symptoms
or family history. In some cases, the terms may refer to treatment
in experimental animals, in veterinary application, and in the
development of animal models for disease, including, but not
limited to, rodents including mice, rats, and hamsters; and
primates.
[0092] The term "healthy control" as used herein refers to a
healthy individual; a baseline from the same individual, a
plurality of healthy individuals, a data set or value corresponding
to or obtained from a healthy individual or a plurality of healthy
individuals.
[0093] The term "Extended Glasgow Outcome Scale (GOSE)" as used
herein categorizes functional disability after TBI on a scale of
1-8, where 1=Dead and 8=Upper Good Recovery. The functional
disability is defined as a GOSE score of <8.
[0094] The terms "measuring", "detecting" and "determining" are
used interchangeably throughout, and refer to methods which include
obtaining a patient sample and detecting reactivity of antibodies
in a sample. In some embodiments, the terms refer to obtaining a
patient sample and detecting the reactivity of antibodies in the
sample to one or more antigens. Measuring can be accomplished by
methods known in the art and those further described herein.
[0095] The terms "sample," "patient sample," "biological sample,"
and the like, encompass a variety of sample types obtained from a
patient, individual, or subject and can be used in a diagnostic or
monitoring assay. The patient sample may be obtained from a healthy
subject, a diseased patient or a patient having associated symptoms
of brain injury. Moreover, a sample obtained from a patient can be
divided and only a portion may be used for diagnosis. Further, the
sample, or a portion thereof, can be stored under conditions to
maintain sample for later analysis. The definition specifically
encompasses blood and other liquid samples of biological origin
(including, but not limited to, peripheral blood, serum, plasma,
cerebrospinal fluid, urine, saliva, stool and synovial fluid). In a
specific embodiment, a sample comprises a blood sample. In another
embodiment, a serum sample is used. The definition also includes
samples that have been manipulated in any way after their
procurement, such as by centrifugation, filtration, precipitation,
dialysis, chromatography, treatment with reagents, washed, or
enriched for certain cell populations. The terms further encompass
a clinical sample, and also include cells in culture, cell
supernatants, tissue samples, organs, and the like. Samples may
also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded
tissue blocks, such as blocks prepared from clinical or
pathological biopsies, prepared for pathological analysis or study
by immunohistochemistry.
[0096] The samples may be tested immediately after collection,
after storage at 4 degrees, -20 degrees, or -80 degrees Celsius.
After storage for 24 hours, 1 week, 1 month, 1 year, 10 years or up
to 30 years.
[0097] Various methodologies of the instant invention include a
step that involves comparing a value, level, feature,
characteristic, property, etc. to a "suitable control," referred to
interchangeably herein as an "appropriate control" or a "control
sample." A "suitable control," "appropriate control" or a "control
sample" is any control or standard familiar to one of ordinary
skill in the art useful for comparison purposes. In one embodiment,
a "suitable control" or "appropriate control" is a value, level,
feature, characteristic, property, etc., determined in a cell,
organ, or patient, e.g., a control or normal cell, organ, or
patient, exhibiting, for example, normal traits. For example, the
reactivity of antibodies in a sample from an unaffected individual
(UI) or a normal control individual (NC) (both terms are used
interchangeably herein). In another embodiment, a "suitable
control" or "appropriate control" is a value, level, feature,
characteristic, property, etc. determined prior to performing a
therapy (e.g., an brain injury treatment) on a patient. In yet
another embodiment, a transcription rate, mRNA level, translation
rate, protein level, biological activity, cellular characteristic
or property, genotype, phenotype, etc., can be determined prior to,
during, or after administering a therapy into a cell, organ, or
patient. In a further embodiment, a "suitable control" or
"appropriate control" is a predefined value, level, feature,
characteristic, property, etc. A "suitable control" can be a
profile or pattern of reactivity of antibodies to at least one
antigen that correlates to brain injury, to which a patient sample
can be compared. The patient sample can also be compared to a
negative control, i.e., a profile that correlates to not having
brain injury.
[0098] Antigen probes to be used in the assays of the invention may
be purified or synthesized using methods well known in the art. For
example, an antigenic protein or peptide may be produced using
known recombinant or synthetic methods, including, but not limited
to, solid phase (e.g. Boc or f-Moc chemistry) and solution phase
synthesis methods (Stewart and Young, 1963; Meienhofer, 1973;
Schroder and Lupke, 1965; Sambrook et al., 2001). One of skill in
the art will possess the required expertise to obtain or synthesize
the antigen probes of the invention. Some of the antigen probes are
also commercially available, e.g. from Sigma (St. Louis, Mo., USA),
Prospec (Ness-Ziona, Israel), Abnova (Taipei City, Taiwan), Matreya
LLC (Pleasant Gap, Pa., USA), Avanti Polar Lipids (Alabaster, Ala.,
USA), Calbiochem (San Diego, Calif., USA), Chemicon (Temecula,
Calif., USA), GeneTex (San Antonio, Tex., USA), Novus Biologicals
(Littleton, Colo., USA) Assay Designs (Ann Arbor, Mich., USA),
ProSci Inc. (Poway, Calif., USA), EMD Biosciences (San Diego,
Calif., USA), Cayman Chemical (Ann Arbor, Mich., USA), HyTest
(Turku, Finland), Meridian Life Science (Memphis, Tenn. USA) and
Biodesign International (Saco, Me., USA), as detailed herein
below.
[0099] It should be noted, that the invention utilizes antigen
probes as well as homologs, fragments, partial sequences, mutant
forms, modified forms and derivatives thereof, as long as these
homologs, fragments, partial sequences, mutant forms, modified
forms and derivatives are immunologically cross-reactive with these
antigen probes. The term "immunologically cross-reactive" as used
herein refers to two or more antigens that are specifically bound
by the same antibody. The term "homolog" as used herein refers to a
peptide which having at least 70%, at least 75%, at least 80%, at
least 85% or at least 90% identity to the antigen's amino acid
sequence. Cross-reactivity can be determined by any of a number of
immunoassay techniques, such as a competition assay (measuring the
ability of a test antigen to competitively inhibit the binding of
an antibody to its known antigen).
[0100] The term "peptide" typically refers to a polypeptide of up
to about 50 amino acid residues in length. According to particular
embodiments, the antigenic peptides of the invention may be 10-50
amino acids in length and are typically about 10-30 or about 15-25
amino acids in length.
[0101] The term encompasses native peptides (either degradation
products, synthetically synthesized peptides, or recombinant
peptides), peptidomimetics (typically, synthetically synthesized
peptides), and the peptide analogues peptoids and semipeptoids, and
may have, for example, modifications rendering the peptides more
stable while in a body or more capable of penetrating into cells.
Such modifications include, but are not limited to: N-terminus
modifications; C-terminus modifications; peptide bond
modifications, backbone modifications; and residue
modifications.
[0102] The peptide antigens of the invention may be used having a
terminal carboxy acid, as a carboxy amide, as a reduced terminal
alcohol or as any pharmaceutically acceptable salt, e.g., as metal
salt, including sodium, potassium, lithium or calcium salt, or as a
salt with an organic base, or as a salt with a mineral acid,
including sulfuric acid, hydrochloric acid or phosphoric acid, or
with an organic acid e.g., acetic acid or maleic acid. According to
some embodiments, the peptide antigens of the invention are
BSA-conjugated peptides.
[0103] Functional derivatives consist of chemical modifications to
amino acid side chains and/or the carboxyl and/or amino moieties of
said peptides. Such derivatized molecules include, for example,
those molecules in which free amino groups have been derivatized to
form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups may be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine may be derivatized
to form N-im-benzylhistidine. Also included as chemical derivatives
are those polypeptides, which contain one or more naturally
occurring or modified amino acid derivatives of the twenty standard
amino acid residues. For example: 4-hydroxyproline may be
substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted or serine; and ornithine may be
substituted for lysine.
[0104] The amino acid residues described herein are in the "L"
isomeric form, unless otherwise indicated. However, residues in the
"D" isomeric form can be substituted for any L-amino acid residue,
as long as the peptide substantially retains the desired antibody
specificity.
[0105] Suitable analogs may be readily synthesized by now-standard
peptide synthesis methods and apparatus or recombinant methods. All
such analogs will essentially be based on the antigens of the
invention as regards their amino acid sequence but will have one or
more amino acid residues deleted, substituted or added. When amino
acid residues are substituted, such conservative replacements which
are envisaged are those which do not significantly alter the
structure or antigenicity of the polypeptide. For example basic
amino acids will be replaced with other basic amino acids, acidic
ones with acidic ones and neutral ones with neutral ones. In
addition to analogs comprising conservative substitutions as
detailed above, analogs comprising non-conservative amino acid
substitutions are further contemplated, as long as these analogs
are immunologically cross reactive with a peptide antigen of the
invention.
[0106] In other aspects, there are provided nucleic acids encoding
these peptides, vectors comprising these nucleic acids and host
cells containing them. These nucleic acids, vectors and host cells
are readily produced by recombinant methods known in the art (see,
e.g., Sambrook et al., 2001). For example, an isolated nucleic acid
sequence encoding an antigen of the invention can be obtained from
its natural source, either as an entire (i.e., complete) gene or a
portion thereof. A nucleic acid molecule can also be produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Nucleic acid
sequences include natural nucleic acid sequences and homologs
thereof, including, but not limited to, natural allelic variants
and modified nucleic acid sequences in which nucleotides have been
inserted, deleted, substituted, and/or inverted in such a manner
that such modifications do not substantially interfere with the
nucleic acid molecule's ability to encode a functional peptide of
the present invention.
[0107] "Functionally equivalent variants" of the polypeptide or
peptide antigens of the invention as used herein are polypeptides
or peptides with partial sequence homology, polypeptides or
peptides having one or more specific conservative and/or
non-conservative amino acid changes and polypeptide or peptide
conjugates which do not alter the biological or structural
properties of the polypeptide or peptide.
[0108] In terms of "functional analogues", it is well understood by
those skilled in the art, that inherent in the definition of a
biologically functional polypeptide or peptide analogue is the
concept that there is a limit to the number of changes that may be
made within a defined portion of the molecule and still result in a
molecule with an acceptable level of equivalent biological
activity. A plurality of distinct polypeptides or peptides with
different substitutions may easily be made and used in accordance
with the invention. It is also understood that certain residues are
particularly important to the biological or structural properties
of a polypeptide, and such residues may not generally be
exchanged.
[0109] Functional analogues can be generated by conservative or
non-conservative amino acid substitutions. Amino acid substitutions
are generally based on the relative similarity of the amino acid
side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size and the like. Thus, within the scope
of the invention, conservative amino acid changes means, an amino
acid change at a particular position which is of the same type as
originally present; i.e. a hydrophobic amino acid exchanged for a
hydrophobic amino acid, a basic amino acid for a basic amino acid,
etc. Examples of conservative substitutions include the
substitution of non-polar (hydrophobic) residues such as
isoleucine, valine, leucine or methionine for another, the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between glycine and serine, the substitution of one basic residue
such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid for another, the substitution of a branched chain
amino acid, such as isoleucine, leucine, or valine for another, the
substitution of one aromatic amino acid, such as phenylalanine,
tyrosine or tryptophan for another. Such amino acid changes result
in functional analogues in that they do not significantly alter the
overall charge and/or configuration of the polypeptide. Examples of
such conservative changes are well-known to the skilled artisan and
are within the scope of the present invention. Conservative
substitution also includes the use of a chemically derivatized
residue in place of a non-derivatized residue provided that the
resulting polypeptide is a biologically functional equivalent to
the polypeptide antigens.
[0110] Therefore, the "citrullinated polypeptides" encompass a
polypeptide having an amino acid sequence that differs from the
sequences provided herein by one or more conservative amino acid
substitutions. The citrullinated polypeptides also encompass a
polypeptide having an amino acid sequence that differs from the
sequences provided herein by a single mutation, where the single
mutation represents a single amino acid deletion, insertion or
substitution.
[0111] The citrullinated peptides may be made by methods known to
those of skill in the art most notably and preferably by chemical
synthesis using techniques well known in the chemistry of proteins
such as solid phase synthesis (Merrifield et al, 65 J. AM. CHEM.
ASSOC. 2149 (1964); Merrifield et al, 85 J. AMER. CHEM. SOC. 2149
(1963); and Merrifield et al, 35 INT. J. PEPTIDE PROTEIN RES.
161-214 (1990)) or synthesis in homogenous solution (METHODS OF
ORGANIC CHEMISTRY, E. Wansch (Ed.) Vol. 15, pts. I and II, Thieme,
Stuttgart (1987)) to generate synthetic peptides. Citrulline is a
post-translationally modified arginine that is created through the
process of deimination which is catalyzed by the enzyme
peptidylarginine deiminase 4 (PAD-4) that removes a positive charge
from arginine and makes the resulting citrulline polar in
nature.
[0112] In one embodiment, citrullinated peptides can be made from
known commercially available sources. In this aspect, the
lyophilized protein is reconstituted in an appropriate buffer to
which the enzyme peptidylarginine deiminase 4 is added.
Alternatively, Ca.sup.2+ is added to PAD-4 in solution. The
solution is allowed to stand at an appropriate temperature for a
time sufficient to cause modification of arginine residues to
citrulline and thus create a citrullinated protein. The
citrullinated protein is then isolated by the removal of the enzyme
using a high molecular weight membrane to separate the enzyme or
other methods of chromatography. One of skill in the art will
understand that the temperature of incubation, buffer condition and
time of incubation may vary depending on the protein that is being
deiminated (Masson-Bessiere et al, 166 J. IMMUNOL. 4177-4184
(2001)).
[0113] The citrullinated proteins may be further isolated and
purified by methods selected on the basis of properties revealed by
its sequence. Purification can be achieved by protein purification
procedures such as chromatography methods (gel-filtration,
ion-exchange and immunoaffinity), by high-performance liquid
chromatography (HPLC, RP-HPLC, ion-exchange HPLC, size-exclusion
HPLC, high-performance chromatofocusing and hydrophobic interaction
chromatography) or by precipitation (immunoprecipitation).
[0114] Polyacrylamide gel electrophoresis can also be used to
isolate the citrullinated proteins based on the molecular weight of
the protein, charge properties and hydrophobicity. The purified
citrullinated proteins can be used in further biochemical analyses
to establish secondary and tertiary structure which may aid in the
design of pharmaceuticals to interact with the protein, alter the
protein charge configuration or charge interaction with other
proteins or alter its function.
[0115] The term "oligonucleotide antigen" as used herein refer to a
stretch of contiguous nucleotides of a certain length. Unless
otherwise indicated, the term "oligonucleotide antigen" as used
herein relates to a nucleotide sequence of between 15 and 40
nucleotides in length, alternatively between 17 and 28 nucleotides
in length, or between 18-25 nucleotides in length. In certain
embodiments, an oligonucleotide antigen consists of at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 16, or more contiguous nucleotides. Each possibility
represents a separate embodiment of the invention. In certain
embodiments, an antigen consists of not more than 50, not more than
45, not more than 40, not more than 35, not more than 30, not more
than 25, not more than 20, not more than 16, or less contiguous
nucleotides. Each possibility represents a separate embodiment of
the invention. In certain embodiments, an antigen consists of
10-30, 15-25 or 17-20 contiguous nucleotides. In certain
embodiments, an antigen consists of 17, 18, 19 or 20 contiguous
nucleotides.
[0116] As used herein, the "reactivity of antibodies in a sample"
or "reactivity of an antibody in a sample" to "an antigen" or to "a
plurality of antigens" refers to the immune reactivity of at least
one antibody in the sample to at least one specific antigen
selected from the plurality of antigens. The immune reactivity of
the antibody to the antigen, i.e. its ability to specifically bind
the antigen, may be used to determine the amount of the antibody in
the sample. The calculated levels of each one of the tested
antibodies in the sample are collectively referred to as the
reactivity pattern of the sample to these antigens. The reactivity
pattern of the sample reflects the levels of each one of the tested
antibodies in the sample, thereby providing a quantitative assay.
In a preferred embodiment, the antibodies are quantitatively
determined.
[0117] A "significant difference" between reactivity patterns
refers, in different embodiments, to a statistically significant
difference, or in other embodiments to a significant difference as
recognized by a skilled artisan. In another embodiment, a
significant difference between the reactivity pattern of the sample
obtained from the subject compared to the control reactivity
pattern is an indication that the subject is afflicted with brain
injury. In specific embodiments, up-regulation or higher reactivity
of the reactivity of an antibody in a sample to an antigen refers
to an increase (i.e., elevation) of about at least two, about at
least three, about at least four, or about at least five times
higher (i.e., greater) than the reactivity levels of the antibody
to the antigen in the control. In another embodiment,
down-regulation or lower reactivity of the reactivity of an
antibody in a sample to an antigen refers to a decrease (i.e.,
reduction) of about at least two, about at least three, about at
least four, or about at least five times lower than the reactivity
levels of the antibody to the antigen in the control.
[0118] According to some embodiments, the at least one
oligonucleotide antigen is an oligonucleotide sequence comprising
at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 contiguous adenine nucleotides. According to another embodiment,
the oligonucleotide sequence comprises at most 20 contiguous
adenine nucleotides. According to additional embodiments, the at
least one oligonucleotide antigen is an oligonucleotide sequence
comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 contiguous thymine nucleotides. According to another
embodiment, the oligonucleotide sequence comprises at most 20
contiguous thymine nucleotides.
[0119] According to additional embodiments, the at least one
oligonucleotide antigen is an oligonucleotide sequence comprising
at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 contiguous cytosine nucleotides. According to another
embodiment, the oligonucleotide sequence comprises at most 20
contiguous cytosine nucleotides. According to additional
embodiments, the at least one oligonucleotide antigen is an
oligonucleotide sequence comprising 5-17, 6-17, 7-17, 8-17, 9-17,
10-17, 11-17, 12-17, 13-17, 14-17, 15-17, 16-17, or at most 17
contiguous guanine nucleotides.
[0120] According to some embodiments, the at least one antigen is
selected from the group consisting of SEQ ID NOs: 1-115, isoforms
thereof, post-translationally modified forms thereof, fragments
thereof; or combinations of any of the foregoing.
[0121] According to some embodiments, the antigens are selected
from proteins, peptides, oligonucleotide antigens, or any
combinations thereof.
[0122] It should be understood each antigen according to the
present invention may be bound by IgM antibodies and/or IgG
antibodies found or isolated from a sample obtained or derived from
the tested subject. Since the relative amounts of IgM antibodies
and IgG antibodies against a certain epitope or antigen naturally
change over the course of time, each antigen according to the
present invention may be bound by IgM antibodies, IgG antibodies or
both. In certain embodiments, the reactivity of antibodies means
the reactivity of IgG antibodies. In certain embodiments, the
reactivity of antibodies means the reactivity of IgM antibodies.
According to another embodiment, the significantly higher
reactivity of the antibodies in the sample means increased IgG
reactivity. According to another embodiment, the significantly
higher reactivity of the antibodies in the sample comprises
increased IgM reactivity.
[0123] According to another embodiment, the increased IgM
reactivity is of at least one antigen selected from the group
consisting of SEQ ID NOs: 1-115, isoforms thereof,
post-translationally modified forms thereof, or combinations of any
of the foregoing.
[0124] According to another embodiment, the increased IgG
reactivity is of at least one antigen selected from the group
consisting of SEQ ID NOs: 1-115, isoforms thereof,
post-translationally modified forms thereof, or combinations of any
of the foregoing.
[0125] In certain embodiments, the increased IgM and IgG reactivity
is of at least one antigen selected from the group consisting of
SEQ ID NOs: 1-115, isoforms thereof, post-translationally modified
forms thereof, or combinations of any of the foregoing. In certain
embodiments, the increased IgM reactivity is of at least one
antigen selected from the group consisting of SEQ ID NOs: 1-115,
isoforms thereof, post-translationally modified forms thereof,
fragments thereof, or combinations of any of the foregoing. Each
possibility represents a separate embodiment of the invention.
[0126] It should be understood that in order to perform the methods
of the present invention, samples obtained or derived from subjects
must comprise antibodies produced by the subject himself.
Therefore, samples may be obtained or derived from any tissue,
organ or liquid naturally comprising at least a subset of the
subject's antibodies. In certain embodiments, the sample obtained
from the subject is a biological fluid. According to some
embodiments, the sample is selected from the group consisting of
plasma, serum, blood, cerebrospinal fluid, synovial fluid, sputum,
urine, saliva, tears, lymph specimen, or any other biological fluid
known in the art. Each possibility represents a separate embodiment
of the invention. According to certain embodiments, the sample
obtained from the subject is selected from the group consisting of
serum, plasma and blood. According to one embodiment, the sample is
a serum sample. Methods for obtaining and isolating appropriate
samples are well within the purview of the skilled artisan.
[0127] According to certain embodiments of the methods of the
present invention, the control is selected from the group
consisting of a sample from at least one healthy individual, base
line of the same subject, a panel of control samples from a set of
healthy individuals, and a stored set of data from healthy
individuals. Each possibility represents a separate embodiment of
the invention. Typically, a healthy individual is a subject not
afflicted with brain injury.
[0128] In particular embodiments, the significant difference is
determined using a cutoff of a positive predictive value (PPV) of
at least 85%, preferably at least 90%. Determining a PPV for a
selected marker (e.g., an antigen) is well known to the ordinarily
skilled artisan and is exemplified in the methods described below.
Typically, positivity for an antigen is determined if it detected
above 10% of the subjects in a specific study subgroup using a
selected cutoff value, such as PPV .gtoreq.90%. For example,
antigen i is determined to specifically characterize group A if it
detected at least 10% of the subjects in group A with a PPV
.gtoreq.90% when compared to a different test group B. Subjects in
group A that are above the cutoff of PPV .gtoreq.90% for antigen i
are considered to be positive for antigen i.
[0129] An antibody "directed to" an antigen, as used herein is an
antibody which is capable of specific binding to the antigen.
Determining the levels of antibodies directed to a plurality of
antigens includes measuring the level of each antibody in the
sample, wherein each antibody is directed to a specific antigen of
the invention. This step is typically performed using an
immunoassay, as detailed herein.
[0130] In other embodiments, determining the reactivity of
antibodies in the sample to the at least one antigen (and the
levels of each one of the tested antibodies in the sample) is
performed by a process comprising contacting the sample, under
conditions such that a specific antigen-antibody complex may be
formed, with at least one antigen (or when a plurality of antigens
is used, to an antigen probe set comprising the plurality of
antigens), and quantifying the amount of antigen-antibody complex
formed for each antigen probe. The amount of antigen-antibody
complex is indicative of the level of the tested antibody in the
sample (or the reactivity of the sample with the antigen).
[0131] In another embodiment the method comprises determining the
reactivity of at least one IgG antibody and at least one IgM
antibody in the sample to the plurality of antigens. In another
embodiment, the method comprises determining the reactivity of a
plurality of IgG antibodies and at least one IgM antibody in the
sample to the plurality of antigens. In another embodiment, the
method comprises determining the reactivity of at least one IgG
antibody and a plurality of IgM antibodies in the sample to the
plurality of antigens. According to another embodiment, the method
comprises determining the reactivity of antibodies in the sample to
a plurality of antigens.
[0132] Typically, determining the reactivity of antibodies in the
sample to at least one antigen is performed using an immunoassay.
Advantageously, when a plurality of antigens is used, the plurality
of antigens may be used in the form of an antigen array.
[0133] Antigen Probes and Antigen Probe Sets
[0134] According to further embodiments, the invention provides
antigen probes and antigen probe sets useful for diagnosing brain
injury, as detailed herein.
[0135] The invention further provides a plurality of antigens also
referred to herein as antigen probe sets. These antigen probe sets
comprise a plurality of antigens which are reactive specifically
with the sera of subjects having brain injury. According to the
principles of the invention, the plurality of antigens may
advantageously be used in the form of an antigen array. According
to some embodiments the antigen array is conveniently arranged in
the form of an antigen chip.
[0136] A "probe" as used herein means any compound capable of
specific binding to a component. According to one aspect, the
present invention provides an antigen probe set comprising a
plurality of antigens selected from the group consisting of: SEQ ID
NOs: 1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing. According to certain embodiments, the antigen probe set
comprises a subset of the antigens of the present invention. In a
particular embodiment, the subset of antigens consists of: SEQ ID
NOs: 1-24, 27-30, 42, 75, 76, isoforms thereof,
post-translationally modified forms thereof, fragments thereof, or
combinations of any of the foregoing.
[0137] In some embodiments, antigen probe set consists of up to 300
antigens. In some embodiments, the antigen probe set consists of
2-5 antigens.
[0138] According to another embodiment, the methods of the present
invention comprise determining the reactivity of antibodies in the
sample to at least one antigen selected from the group consisting
of SEQ ID NO: 1-115, isoforms thereof, post-translationally
modified forms thereof, fragments thereof, or combinations of any
of the foregoing.
[0139] The reactivity of antibodies to the plurality of antigens of
the invention may be determined according to techniques known in
the art.
[0140] Preferably, the plurality of antigens of the methods and
kits of the invention comprises a set of the antigens as disclosed
herein. Yet in other embodiments, the plurality of antigens (or the
antigen probe set) comprises or consists of a subset thereof, e.g.
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 or 115 different
antigens, each selected from the antigens of the present invention,
wherein each possibility represents a separate embodiment of the
invention. Such subsets may be selected so as to result in optimal
sensitivity and/or specificity of the diagnostic assay.
[0141] Antigen probes to be used in the assays of the invention may
be synthesized or purified using methods well known in the art.
[0142] It should be noted, that the invention utilizes antigen
probes as well as homologs, fragments and derivatives thereof, as
long as these homologs, fragments and derivatives are
immunologically cross-reactive with these antigen probes. The term
"f" as used herein refers to two or more antigens that are
specifically bound by the same antibody. The term "homolog" as used
herein refers to an antigen probes having at least 80%, at least
85% or at least 90% identity to the antigen's sequence or
structure. Cross-reactivity can be determined by any of a number of
immunoassay techniques, such as a competition assay (measuring the
ability of a test antigen to competitively inhibit the binding of
an antibody to its known antigen).
[0143] The term "fragment" as used herein refers to a portion of an
antigen, or antigen analog which remains immunologically
cross-reactive with the antigen probes, e.g., to immunospecifically
recognize the target antigen. The fragment may have the length of
about 80%, about 85%, about 90% or about 95% of the respective
antigen.
[0144] According to another aspect, the present invention provides
an antigen probe set comprising a plurality of antigen probes
selected from the group consisting of SEQ ID NOs: 1-115, isoforms
thereof, post-translationally modified forms thereof, fragments
thereof, or combinations of any of the foregoing.
[0145] According to another related aspect, the present invention
provides an antigen probe set comprising at least one antigen probe
selected from the group consisting of SEQ ID NOs: 1-115, isoforms
thereof, post-translationally modified forms thereof, fragments
thereof, or combinations of any of the foregoing.
[0146] According to another aspect, the present invention provides
an article of manufacture comprising the at least one of the
antigen probe sets described above.
[0147] In certain embodiments, the article of manufacture is in the
form of an antigen probe array or in the form of an antigen chip or
in the form of a dipstick or in the form of a lateral flow test or
any other platform known to those skilled in the art. An "antigen
probe array" generally refers to a plurality of antigen probes,
either mixed in a single container or arranges in to or more
containers. An "antigen chip" generally refers to a substantially
two dimensional surface, onto which a plurality of antigens are
attached or adhered. A "dipstick" generally refers to an object,
onto which one or a plurality of antigens are attached or adhered,
which is dipped into a liquid to perform a chemical test or to
provide a measure of quantity found in the liquid. A "lateral flow
test" generally refers to devices intended to detect the presence
(or absence) of a target analyte in sample (matrix) without the
need for specialized and costly equipment. In certain embodiments,
the article of manufacture is in the form of a kit.
[0148] According to certain embodiments, the kit further comprises
means for determining the reactivity of antibodies in a sample to
at least one antigen of the plurality of antigens. According to
another embodiment, the kit further comprises means for comparing
reactivity of antibody in different samples to at least one antigen
of the plurality of antigens. According to another embodiment, the
kit further comprises instructions for use. For example, the
aforementioned means may include reagents, detectable labels and/or
containers which may be used for measuring specific binding of
antibodies to the antigen probes of the invention. "Means" as used
herein may also refer to devices, reagents and chemicals, such as
vials, buffers and written protocols or instructions, used to
perform biological or chemical assays.
[0149] According to another aspect, there is provided use of the at
least one antigen selected from the group consisting of: SEQ ID
NOs: 1-115, isoforms thereof, post-translationally modified forms
thereof, fragments thereof, or combinations of any of the
foregoing; for the preparation of a diagnostic kit for diagnosing
brain injury in a subject. The diagnostic kit is, in some
embodiments, useful for determining the reactivity of antibodies in
a sample, thereby determining the reactivity pattern of the sample
to the at least one antigen. In some embodiments, a significant
difference (e.g., increase) between the reactivity pattern of the
sample compared to a reactivity pattern of a control sample is an
indication for brain injury.
[0150] In other embodiments, the plurality of antigens comprised in
the antigen probe set comprises or consists up to 50, 55, 60, 70,
80, 90 or 100 different antigens. In other embodiments, the
plurality of antigens comprised in the antigen probe set comprises
or consists at least 50, 100, 150, 200 or 500 different
antigens.
[0151] In other aspects, there are provided nucleic-acid vectors
comprising the oligonucleotides of the invention and host cells
containing them. These nucleic acids, vectors and host cells are
readily produced by recombinant methods known in the art. A
poly-nucleic acid molecule can also be produced using recombinant
DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Nucleic acid
sequences include natural nucleic acid sequences and homologs
thereof, including, but not limited to, natural allelic variants
and modified nucleic acid sequences in which nucleotides have been
inserted, deleted, substituted, and/or inverted in such a manner
that such modifications do not substantially interfere with the
nucleic acid molecule's ability to perform the methods of the
present invention.
[0152] According to the invention, the kits comprise a plurality of
antigens also referred to herein as antigen probe sets. These
antigen probe sets comprising a plurality of antigens are reactive
specifically with the sera of subjects having brain injury. In some
embodiments, the antigen probe sets can differentiate between sera
of subjects having brain injury and normal subject. According to
the principles of the invention, the plurality of antigens may
advantageously be used in the form of an antigen array. According
to some embodiments the antigen array is conveniently arranged in
the form of an antigen chip.
[0153] In other embodiments, the kit may further comprise means for
determining the reactivity of antibodies in a sample to the
plurality of antigens. For example, the kit may contain reagents,
detectable labels and/or containers which may be used for measuring
specific binding of antibodies to the antigen probes of the
invention. In a particular embodiment, the kit is in the form of an
antigen array.
[0154] In some embodiments, the kit comprises means for comparing
reactivity patterns of antibodies in different samples to the
plurality of antigens. In other embodiments, the kit may further
comprise negative and/or positive control samples. For example, a
negative control sample may contain a sample from at least one
healthy individual (e.g., an individual not-afflicted with brain
injury). A positive control may contain a sample from at least one
individual afflicted with brain injury, or a subtype of brain
injury which is being diagnosed. Other non-limiting examples are a
panel of control samples from a set of healthy individuals or
diseased individuals, or a stored set of data from control
individuals.
[0155] Antibodies, Samples and Immunoassays
[0156] Antibodies, or immunoglobulins, comprise two heavy chains
linked together by disulfide bonds and two light chains, each light
chain being linked to a respective heavy chain by disulfide bonds
in a "Y" shaped configuration. Each heavy chain has at one end a
variable domain (VH) followed by a number of constant domains (CH).
Each light chain has a variable domain (VL) at one end and a
constant domain (CL) at its other end, the light chain variable
domain being aligned with the variable domain of the heavy chain
and the light chain constant domain being aligned with the first
constant domain of the heavy chain (CH1). The variable domains of
each pair of light and heavy chains form the antigen binding
site.
[0157] The isotype of the heavy chain (gamma, alpha, delta, epsilon
or mu) determines immunoglobulin class (IgG, IgA, IgD, IgE or IgM,
respectively). The light chain is either of two isotypes (kappa,
.kappa. or lambda, .lamda.) found in all antibody classes.
[0158] It should be understood that when the terms "antibody" or
"antibodies" are used, this is intended to include intact
antibodies, such as polyclonal antibodies or monoclonal antibodies
(mAbs), as well as proteolytic fragments thereof such as the Fab or
F(ab').sub.2 fragments. Further included within the scope of the
invention (for example as immunoassay reagents, as detailed herein)
are chimeric antibodies; recombinant and engineered antibodies, and
fragments thereof.
[0159] Exemplary functional antibody fragments comprising whole or
essentially whole variable regions of both light and heavy chains
are defined as follows: (i) Fv, defined as a genetically engineered
fragment consisting of the variable region of the light chain and
the variable region of the heavy chain expressed as two chains;
(ii) single-chain Fv ("scFv"), a genetically engineered
single-chain molecule including the variable region of the light
chain and the variable region of the heavy chain, linked by a
suitable polypeptide linker; (iii) Fab, a fragment of an antibody
molecule containing a monovalent antigen-binding portion of an
antibody molecule, obtained by treating whole antibody with the
enzyme papain to yield the intact light chain and the Fd fragment
of the heavy chain, which consists of the variable and CH1 domains
thereof: (iv) Fab', a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule,
obtained by treating whole antibody with the enzyme pepsin,
followed by reduction (two Fab' fragments are obtained per antibody
molecule); and (v) F(ab') 2, a fragment of an antibody molecule
containing a monovalent antigen-binding portion of an antibody
molecule, obtained by treating whole antibody with the enzyme
pepsin (i.e., a dimer of Fab' fragments held together by two
disulfide bonds).
[0160] The term "antigen" as used herein is a molecule or a portion
of a molecule capable of being bound by an antibody. The antigen is
typically capable of inducing an animal to produce antibody capable
of binding to an epitope of that antigen. An antigen may have one
or more epitopes. The specific reaction referred to above is meant
to indicate that the antigen will react, in a highly selective
manner, with its corresponding antibody and not with the multitude
of other antibodies which may be evoked by other antigens. An
"antigenic peptide" is a peptide which is capable of specifically
binding an antibody.
[0161] In another embodiment, detection of the capacity of an
antibody to specifically bind an antigen probe may be performed by
quantifying specific antigen-antibody complex formation. The term
"specifically bind" as used herein means that the binding of an
antibody to a specific antigen probe is not affected by the
presence of non-related molecules.
[0162] In certain embodiments, the method of the present invention
is performed by determining the capacity of an antigen of the
invention to specifically bind antibodies of the IgG isotype, or,
in other embodiments, antibodies of the IgM, isolated from a
subject.
[0163] Methods for obtaining suitable antibody-containing
biological samples from a subject are well within the ability of
those of skill in the art. Typically, suitable samples comprise
whole blood and products derived therefrom, such as plasma and
serum. In other embodiments, other antibody-containing samples may
be used, e.g. CSF, urine and saliva samples.
[0164] Numerous well known fluid collection methods can be utilized
to collect the biological sample from the subject in order to
perform the methods of the invention.
[0165] In accordance with the present invention, any suitable
immunoassay can be used with the subject antigens. Such techniques
are well known to the ordinarily skilled artisan and have been
described in many standard immunology manuals and texts. In certain
preferable embodiments, determining the capacity of the antibodies
to specifically bind the antigen probes is performed using an
antigen probe array-based method. Preferably, the array is
incubated with suitably diluted serum of the subject so as to allow
specific binding between antibodies contained in the serum and the
immobilized antigen probes, washing out unbound serum from the
array, incubating the washed array with a detectable
label-conjugated ligand of antibodies of the desired isotype,
washing out unbound label from the array, and measuring levels of
the label bound to each antigen probe.
[0166] In various embodiments, the method of the present invention
further comprises diluting the sample before performing the
determining step. In one embodiment, the sample is diluted 1:2, for
instance, using PBS. In another embodiment, the sample is diluted
1:4, 1:6, 1:8, 1:15, 1:20, 1:50, or preferably 1:10. Each
possibility represents a separate embodiment of the present
invention. In another embodiment, the sample is diluted in the
range of times 2-times 10. In another embodiment, the sample is
diluted in the range of times 4-times 10. In another embodiment,
the sample is diluted in the range of times 6-times 10. In another
embodiment, the sample is diluted in the range of times 8-times
10.
[0167] The Antigen Chip
[0168] Antigen microarrays are used for the high-throughput
characterization of the immune response (Robinson et al., 2002, Nat
Med 8, 295-301), and have been used to analyze immune responses in
vaccination and in autoimmune disorders (Robinson et al., 2002;
Robinson et al., 2003, Nat Biotechnol. 21, 1033-9; Quintana et al.,
2004; Kanter et al., 2006, Nat Med 12, 138-43). It has been
hypothesized, that patterns of multiple reactivities may be more
revealing than single antigen-antibody relationships (Quintana et
al., 2006, Lupus 15, 428-30) as shown in previous analyses of
autoimmune repertoires of mice (Quintana et al., 2004; Quintana et
al., 2001, J Autoimmun 17, 191-7) and humans (Merbl et al., 2007, J
Clin Invest 117, 712-8; Quintana et al., 2003, J Autoimmun 21,
65-75) in health and disease. Thus, autoantibody repertoires have
the potential to provide both new insights into the pathogenesis of
the disease and to serve as immune biomarkers (Cohen, 2007, Nat Rev
Immunol. 7, 569-74) of the disease process.
[0169] According to some aspects the methods of the present
invention may be practiced using antigen arrays as disclosed in WO
02/08755 and U.S. 2005/0260770, the contents of which are
incorporated herein by reference. WO 02/08755 is directed to a
system and an article of manufacture for clustering and thereby
identifying predefined antigens reactive with undetermined
immunoglobulins of sera derived from patient subjects in need of
diagnosis of disease or monitoring of treatment. Further disclosed
are diagnostic methods, and systems useful in these methods,
employing the step of clustering a subset of antigens of a
plurality of antigens, the subset of antigens being reactive with a
plurality of antibodies being derived from a plurality of patients,
and associating or disassociating the antibodies of a subject with
the resulting cluster.
[0170] U.S. Pat. App. Pub. No. 2005/0260770 discloses an antigen
array system and diagnostic uses thereof. The application provides
a method of diagnosing an immune disease, particularly diabetes
type 1, or a predisposition thereto in a subject, comprising
determining a capacity of immunoglobulins of the subject to
specifically bind each antigen probe of an antigen probe set. The
teachings of the disclosures are incorporated in their entirety as
if fully set forth herein.
[0171] In other embodiments, various other immunoassays may be
used, including, without limitation, enzyme-linked immunosorbent
assay (ELIS A), flow cytometry with multiplex beads (such as the
system made by Luminex), surface plasmon resonance (SPR),
elipsometry, and various other immunoassays which employ, for
example, laser scanning, light detecting, photon detecting via a
photo-multiplier, photographing with a digital camera based system
or video system, radiation counting, fluorescence detecting,
electronic, magnetic detecting and any other system that allows
quantitative measurement of antigen-antibody binding.
[0172] Various methods have been developed for preparing arrays
suitable for the methods of the present invention. State-of-the-art
methods involves using a robotic apparatus to apply or "spot"
distinct solutions containing antigen probes to closely spaced
specific addressable locations on the surface of a planar support,
typically a glass support, such as a microscope slide, which is
subsequently processed by suitable thermal and/or chemical
treatment to attach antigen probes to the surface of the support.
First, the glass surface is activated by a chemical treatment that
leaves a layer of reactive groups such as epoxy groups on the
surface, which bind covalently any molecule containing free amine
or thiol groups. Suitable supports may also include silicon,
nitrocellulose, paper, cellulosic supports and the like.
[0173] Preferably, each antigen probe, or distinct subset of
antigen probes of the present invention, which is attached to a
specific addressable location of the array is attached
independently to at least two, more preferably to at least three
separate specific addressable locations of the array in order to
enable generation of statistically robust data.
[0174] According to additional embodiments, the antigen probe set
comprises at least 5, at least 25, at least 100, at least 150, at
least 200, at least 250, at least 300 or more antigens, including
one or a plurality of the antigens provided by the present
invention.
[0175] In addition to antigen probes of the invention, the array
may advantageously include control antigen probes or other standard
chemicals. Such control antigen probes may include normalization
control probes. The signals obtained from the normalization control
probes provide a control for variations in binding conditions,
label intensity, "reading" efficiency and other factors that may
cause the signal of a given binding antibody-probe ligand
interaction to vary. For example, signals, such as fluorescence
intensity, read from all other antigen probes of the antigen probe
array are divided by the signal (e.g., fluorescence intensity) from
the normalization control probes thereby normalizing the
measurements. Normalization control probes can be bound to various
addressable locations on the antigen probe array to control for
spatial variation in antibody-ligand probe efficiency. Preferably,
normalization control probes are located at the corners or edges of
the array to control for edge effects, as well as in the middle of
the array.
[0176] The labeled antibody ligands may be of any of various
suitable types of antibody ligand. Preferably, the antibody ligand
is an antibody which is capable of specifically binding the Fc
portion of the antibodies of the subject used. For example, where
the antibodies of the subject are of the IgM isotype, the antibody
ligand is preferably an antibody capable of specifically binding to
the Fc region of IgM antibodies of the subject.
[0177] The ligand of the antibodies of the subject may be
conjugated to any of various types of detectable labels. Preferably
the label is a fluorophore, most preferably Cy3. Alternately, the
fluorophore may be any of various fluorophores, including Cy5, Dy5,
fluorescein isothiocyanate (FITC), phycoerythrin (PE), rhodamine,
Texas red, and the like. Suitable fluorophore-conjugated antibodies
specific for antibodies of a specific isotype are widely available
from commercial suppliers and methods of their production are well
established.
[0178] Antibodies of the subject may be isolated for analysis of
their antigen probe binding capacity in any of various ways,
depending on the application and purpose. While the subject's
antibodies may be suitably and conveniently in the form of blood
serum or plasma or a dilution thereof (e.g. 1:10 dilution), the
antibodies may be subjected to any desired degree of purification
prior to being tested for their capacity to specifically bind
antigen probes. The method of the present invention may be
practiced using whole antibodies of the subject, or antibody
fragments of the subject which comprises an antibody variable
region.
[0179] Combination Measurement of the Levels of One or More
Antibodies and One or More Biomarkers in the Sample Obtained from
the Subject
[0180] The present invention is based, at least in part, on the
discovery that a combination measurement of the levels of one or
more antibodies and one or more biomarkers in the sample obtained
from the subject can measure both the real-time background
physiology of the subject and the status of the acute event.
[0181] In a patient with Brain Injury, the response to injury and
the recovery process from the injury is dependent upon a
combination of the nature of the injury and the state of the
individual prior to injury. Patients that are injured on top of a
`healthy` background will likely have a better (faster, more
complete) recovery profile than patients that are injured on a
`sick` or `previously injured` background.
[0182] Determination of the autoantibody profile of a patient can
be used as a surrogate measurement of the state of the patient
prior to brain injury and determination of the levels of
circulating antigen shortly after injury can be used as a surrogate
measurement of the nature/degree of injury. Algorithms that combine
the information about the state of the patient prior to injury and
the nature/degree of the injury can be used in order to predict
outcomes.
[0183] Determination of the autoantibody profile can be performed
using any platform where antigens are bound to a surface,
circulating antibodies bind to the antigen and are detected with a
tagged secondary antibody. Determination of the circulating antigen
profile can be done in any ELISA type sandwich assay format which
includes a capture antibody and a detection antibody.
[0184] The platforms used for antibody and antigen detection may be
independent (eg. iCHIP for autoantibody, MSD ELISA for antigens or
any relevant ELISA based platform) or may be combined into a single
platform to simultaneously measure both circulating autoantibody
and antigen. This can be done by printing an iCHIP with both
relevant antigens and capture antibodies, contacting serum with the
printed surface such that circulating antibodies will bind to the
surface bound antigen, and circulating antigens will bind to the
surface bound capture antibodies. Detection can be with a cocktail
of secondary and detection antibodies.
[0185] In the case where there is a need to measure autoantibodies
to the same antigen that is informative about the disease state,
these measurements can we done in two separate chambers. The data
from multiple tests can be combined for the purpose of an
algorithmic analysis to finally predict the status of the
patient.
[0186] Kits for the Detection of Biomarkers
[0187] In another aspect, the present invention provides kits for
qualifying brain injury status, which kits are used to detect the
biomarkers described herein. In a specific embodiment, the kit is
provided as an ELISA kit comprising antibodies to the biomarkers of
the present invention including, but not limited to, glial
fibrillary acidic protein (GFAP) and Synuclein beta (Sncb).
[0188] In an alternative embodiment, the panel of biomarkers
comprises BDNF, GFAP, MT3 and SNCB. In another embodiment, the
panel of biomarkers comprises BDNF, GFAP, NRGN and SNCB. In a
further embodiment, the panel of biomarkers comprises BDNF, ICAM5,
MT3 and SNCB.
[0189] The ELISA kit may comprise a solid support, such as a chip,
microtiter plate (e.g., a 96-well plate), bead, or resin having
biomarker capture reagents attached thereon.
[0190] The kit may further comprise a means for detecting the
biomarkers, such as antibodies, and a secondary antibody-signal
complex such as horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG antibody and tetramethyl benzidine (TMB) as a
substrate for HRP.
[0191] The kit may be provided as an immuno-chromatography strip
comprising a membrane on which the antibodies are immobilized, and
a means for detecting, e.g., gold particle bound antibodies, where
the membrane, includes NC membrane and PVDF membrane. The kit may
comprise a plastic plate on which a sample application pad, gold
particle bound antibodies temporally immobilized on a glass fiber
filter, a nitrocellulose membrane on which antibody bands and a
secondary antibody band are immobilized and an absorbent pad are
positioned in a serial manner, so as to keep continuous capillary
flow of blood serum.
[0192] Data Analysis
[0193] Advantageously, the methods of the invention may employ the
use of learning and pattern recognition analyzers, clustering
algorithms and the like, in order to discriminate between
reactivity patterns of healthy control subjects to those of
patients having brain injury. As such, this term specifically
includes a difference measured by, for example, determining the
reactivity of antibodies in a test sample to a plurality of
antigens, and comparing the resulting reactivity pattern to the
reactivity patterns of negative and positive control samples (e.g.
samples obtained from control subjects which are not afflicted with
brain injury or patients afflicted with brain injury, respectively)
using such algorithms and/or analyzers. The difference may also be
measured by comparing the reactivity pattern of the test sample to
a predetermined classification rule obtained in such manner.
[0194] In some embodiments, the methods of the invention may employ
the use of learning and pattern recognition analyzers, clustering
algorithms and the like, in order to discriminate between
reactivity patterns of subjects having a subtype of brain injury to
control subjects. For example, the methods may include determining
the reactivity of antibodies in a test sample to a plurality of
antigens, and comparing the resulting pattern to the reactivity
patterns of negative and positive control samples using such
algorithms and/or analyzers.
[0195] Thus, in another embodiment, a significant difference
between the reactivity patterns of a test sample compared to a
reactivity pattern of a control sample, wherein the difference is
computed using a learning and pattern recognition algorithm,
indicates that the subject is afflicted with brain injury. For
example, the algorithm may include, without limitation, supervised
or non-supervised classifiers including statistical algorithms
including, but not limited to, principal component analysis (PCA),
partial least squares (PLS), multiple linear regression (MLR),
principal component regression (PCR), discriminant function
analysis (DFA) including linear discriminant analysis (LDA), and
cluster analysis including nearest neighbor, artificial neural
networks, coupled two-way clustering algorithms, multi-layer
perceptrons (MLP), generalized regression neural network (GRNN),
fuzzy inference systems (FIS), self-organizing map (SOM), genetic
algorithms (GAS), neuro-fuzzy systems (NFS), adaptive resonance
theory (ART).
[0196] In certain embodiments, one or more algorithms or computer
programs may be used for comparing the amount of each antibody
quantified in the test sample against a predetermined cutoff (or
against a number of predetermined cutoffs). Alternatively, one or
more instructions for manually performing the necessary steps by a
human can be provided.
[0197] Algorithms for determining and comparing pattern analysis
include, but are not limited to, principal component analysis,
Fischer linear analysis, neural network algorithms, genetic
algorithms, fuzzy logic pattern recognition, and the like. After
analysis is completed, the resulting information can, for example,
be displayed on display, transmitted to a host computer, or stored
on a storage device for subsequent retrieval.
[0198] Many of the algorithms are neural network based algorithms.
A neural network has an input layer, processing layers and an
output layer. The information in a neural network is distributed
throughout the processing layers. The processing layers are made up
of nodes that simulate the neurons by the interconnection to their
nodes. Similar to statistical analysis revealing underlying
patterns in a collection of data, neural networks locate consistent
patterns in a collection of data, based on predetermined
criteria.
[0199] Suitable pattern recognition algorithms include, but are not
limited to, principal component analysis (PCA), Fisher linear
discriminant analysis (FLDA), soft independent modeling of class
analogy (SIMCA), K-nearest neighbors (KNN), neural networks,
genetic algorithms, fuzzy logic, and other pattern recognition
algorithms. In some embodiments, the Fisher linear discriminant
analysis (FLDA) and canonical discriminant analysis (CDA) as well
as combinations thereof are used to compare the output signature
and the available data from the database.
[0200] In other embodiments, principal component analysis is used.
Principal component analysis (PCA) involves a mathematical
technique that transforms a number of correlated variables into a
smaller number of uncorrelated variables. The smaller number of
uncorrelated variables is known as principal components. The first
principal component or eigenvector accounts for as much of the
variability in the data as possible, and each succeeding component
accounts for as much of the remaining variability as possible. The
main objective of PCA is to reduce the dimensionality of the data
set and to identify new underlying variables.
[0201] Principal component analysis compares the structure of two
or more covariance matrices in a hierarchical fashion. For
instance, one matrix might be identical to another except that each
element of the matrix is multiplied by a single constant. The
matrices are thus proportional to one another. More particularly,
the matrices share identical eigenvectors (or principal
components), but their eigenvalues differ by a constant. Another
relationship between matrices is that they share principal
components in common, but their eigenvalues differ. The
mathematical technique used in principal component analysis is
called eigenanalysis. The eigenvector associated with the largest
eigenvalue has the same direction as the first principal component.
The eigenvector associated with the second largest eigenvalue
determines the direction of the second principal component. The sum
of the eigenvalues equals the trace of the square matrix and the
maximum number of eigenvectors equals the number of rows of this
matrix.
[0202] In another embodiment, the algorithm is a classifier. One
type of classifier is created by "training" the algorithm with data
from the training set and whose performance is evaluated with the
test set data. Examples of classifiers used in conjunction with the
invention are discriminant analysis, decision tree analysis,
receiver operator curves or split and score analysis.
[0203] The term "decision tree" refers to a classifier with a
flow-chart-like tree structure employed for classification.
Decision trees consist of repeated splits of a data set into
subsets. Each split consists of a simple rule applied to one
variable, e.g., "if value of "variable 1" larger than "threshold
1"; then go left, else go right". Accordingly, the given feature
space is partitioned into a set of rectangles with each rectangle
assigned to one class.
[0204] The terms" test set" or "unknown" or "validation set" refer
to a subset of the entire available data set consisting of those
entries not included in the training set. Test data is applied to
evaluate classifier performance.
[0205] The terms "training set" or "known set" or "reference set"
refer to a subset of the respective entire available data set. This
subset is typically randomly selected, and is solely used for the
purpose of classifier construction.
[0206] Diagnostic Methods
[0207] As used herein the term "diagnosing" or "diagnosis" refers
to the process of identifying a medical condition or disease (e.g.,
brain injury) by its signs, symptoms, and in particular from the
results of various diagnostic procedures, including e.g. detecting
the reactivity, or reactivity pattern, of antibodies in a
biological sample (e.g. serum) obtained from an individual, to one
or more antigens. Furthermore, as used herein the term "diagnosing"
or "diagnosis" encompasses screening for a disease, detecting a
presence or a severity of a disease, distinguishing a disease from
other diseases including those diseases that may feature one or
more similar or identical symptoms, providing prognosis of a
disease, monitoring disease progression or relapse, as well as
assessment of treatment efficacy and/or relapse of a disease,
disorder or condition, as well as selecting a therapy and/or a
treatment for a disease, optimization of a given therapy
(dose/schedule) for a disease, monitoring the treatment of a
disease, and/or predicting the suitability of a therapy for
specific patients or subpopulations or determining the appropriate
dosing of a therapeutic product in patients or subpopulations.
[0208] In one embodiment, diagnosing brain injury further permits
assessing a risk of said brain injury evolving to brain damage and
leading to long-term dysfunction. In another embodiment, assessment
of a risk of said brain injury evolving to long-term dysfunction
permits therapeutic intervention at an early stage.
[0209] The immediate issue facing an individual that has suffered a
TBI is determining when it is safe to return to high risk
activities after a concussive injury without risking permanent
brain damage that occurs at a cellular level. According to some
embodiments, the present invention provides a broad immune system
test to monitor, assess chronic outcomes and verify safety to
return to work or play.
[0210] Assessment of pathology and neurological impairment
immediately after TBI is crucial for determination of appropriate
clinical management and for predicting long-term outcome. The
outcome measures most often used in head injuries are the Glasgow
Coma Scale (GCS), the Glasgow Outcome Scale (GOS), computed
tomography, and magnetic resonance imaging (MRI) to detect
intracranial pathology. However, despite dramatically improved
emergency triage systems based on these outcome measures, most TBI
suffer long term impairment and a large number of TBI survivors are
severely affected despite predictions of "good recovery" on the
GOS. In addition, CT and MRI are expensive and cannot be rapidly
employed in an emergency room environment. Moreover, in austere
medical environments associated with combat, accurate diagnosis of
TBI would be an essential prerequisite for appropriate triage of
casualties.
[0211] In one embodiment, the type of brain damage associated with
brain injury is a white matter structural abnormality. In another
embodiment, the white matter structural abnormality or damage is in
the corpus callosum region. In another embodiment, the abnormality
or damage is in the uncinate fasciculus. In another embodiment, the
abnormality or damage is in the right brain frontal lobe. In
another embodiment, the abnormality or damage is in the left
frontal lobe. In another embodiment, the abnormality or damage is
diffuse axonal injury (DAI). In another embodiment, the abnormality
or damage is diffuse vascular injury.
[0212] In some embodiments, the brain injury is a mild TBI, in one
embodiment a concussion is a mild TBI. In another embodiment, mild
TBI is caused by a head injury, where the head injury is, in
another embodiment, blunt trauma, acceleration, or deceleration
forces. It will be appreciated that such head injuries can be
characterized by having one or more of the following conditions:
(1) observed or self-reported contusion, disorientation, or
impaired consciousness, dysfunction of memory at the time of the
injury, loss of consciousness lasting less than 30 minutes; and,
(2) symptoms such as headache, dizziness, fatigue, irritability,
and poor concentration soon after the injury. Head injuries are
also categorized as mild based on clinical examinations using the
Glasgow Coma Scale. In one embodiment, the head injury has a
Glasgow Coma Scale score (GCS) of 13-15 upon examination at an
emergency center, with no abnormal findings on head CT, duration of
loss of consciousness for no more than 30 minutes, post-traumatic
amnesia for less than 24 hours, and an Abbreviated Injury Score
(AIS) S3 and an ISS of <12 modified to exclude the head
region.
[0213] Diagnostic methods differ in their sensitivity and
specificity. The "sensitivity" of a diagnostic assay is the
percentage of diseased individuals who test positive (percent of
"true positives"). Diseased individuals not detected by the assay
are "false negatives." Subjects who are not diseased and who test
negative in the assay, are termed "true negatives." The
"specificity" of a diagnostic assay is 1 minus the false positive
rate, where the "false positive" rate is defined as the proportion
of those without the disease who test positive. While a particular
diagnostic method may not provide a definitive diagnosis of a
condition, it suffices if the method provides a positive indication
that aids in diagnosis. The "accuracy" of a diagnostic assay is the
proximity of measurement results to the true value. The "p value"
of a diagnostic assay is the probability of obtaining the observed
sample results (or a more extreme result) when the null hypothesis
is actually true.
[0214] In certain embodiments, the use of an antigen probe set
provided by the present invention, or an antigen probe array
provided by the present invention, results in an antibody
reactivity profile which is brain injury-indicative (p value
.ltoreq.1.00E-08), sensitive (.gtoreq.0.600), specific
(.gtoreq.0.700) and accurate (.gtoreq.0.600). In certain
embodiments, the use results in an antibody reactivity profile
which is more brain injury-indicative (p value .ltoreq.1.00E-10),
sensitive (.gtoreq.0.700), specific (.gtoreq.0.800) and accurate
(.gtoreq.0.700). In certain embodiments, the use results in an
antibody reactivity profile which is even more brain
injury-indicative (p value .ltoreq.1.00E-12), sensitive
(.gtoreq.0.800), specific (.gtoreq.0.900) and accurate
(.gtoreq.0.800). In certain embodiments, the use results in an
antibody reactivity profile which is yet even more brain
injury-indicative (p value .ltoreq.1.00E-14), sensitive
(.gtoreq.0.900), specific (.gtoreq.0.950) and accurate
(.gtoreq.0.900). In certain embodiments, the use results in an
antibody reactivity profile which highly brain injury-indicative (p
value .ltoreq.1.00E-16), sensitive (.gtoreq.0.950), specific
(.gtoreq.0.990) and accurate (.gtoreq.0.950). Each possibility
represents a separate embodiment of the invention.
[0215] In certain embodiments, the antigens provided by the present
invention, or the antigen patterns provided by the present
invention, are brain injury-indicative (p value .ltoreq.1.87E-08),
sensitive (.gtoreq.0.609), specific (.gtoreq.0.769) and accurate
(.gtoreq.0.687). In certain embodiments, the antigens provided by
the present invention, or the antigen patterns provided by the
present invention, are advantageously brain injury-indicative (p
value .ltoreq.2.81E-12), sensitive (>0.657), specific
(.gtoreq.0.798) and accurate (.gtoreq.0.725). In certain
embodiments, the antigens provided by the present invention, or the
antigen patterns provided by the present invention, are further
advantageously brain injury-indicative (p value .ltoreq.8.00E-14),
sensitive (.gtoreq.0.663), specific (.gtoreq.0.814) and accurate
(.gtoreq.0.738).
[0216] In another embodiment, the methods may result in determining
a level of brain injury progression. In a further embodiment, the
methods may result in providing the comparison to an entity for
monitoring brain injury progression. In these embodiments, the
methods can be used, for example, to differentiate between subjects
with progressing brain injury, and subjects with regressing brain
injury.
[0217] In one embodiment, the subject being diagnosed according to
the methods of the invention is symptomatic. In other embodiments,
the subject is asymptomatic. In certain embodiments the subject
shows immediate symptoms. In certain embodiments the subject shows
delayed symptoms. In certain embodiments, the subject is not or was
not receiving a treatment.
[0218] As used herein, the term "treating" may encompass curing,
preventing, reducing the incidence of, ameliorating symptoms of, to
inducing remission of, or slowing the progression of a disease. The
terms "reducing", "suppressing" and "inhibiting" refer to lessening
or decreasing.
[0219] The diagnostic procedure can be performed in vivo or in
vitro, preferably in vitro. In certain embodiments of the methods
of the present invention, the diagnostic procedure is performed by
non-invasive means or methods.
[0220] The diagnostic procedure and platform of the present
invention may be suitable for use as point of care device or point
of service in clinic, in physician's office, in hospital
laboratories, or in commercial diagnostic laboratories.
[0221] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed, however, as limiting the broad scope of the
invention.
EXAMPLES
Materials and Methods
[0222] Human Subjects
[0223] The study was approved by the Institutional Review Boards of
the participating clinical unit; informed consent was obtained from
all participants. In an initial study, sera derived from blood
samples obtained from healthy subjects, and subjects suffering from
brain injury at varying times post injury, and with varying GOSE
scores, were tested using an antigen microarray that included 228
antigens (see Table 1).
[0224] Blood samples and clinical data were collected from patients
in the HeadSMART trial, arriving at the emergency departments (ED)
of Johns Hopkins Hospital (JHH, Baltimore; n=61) or at one of the
participating centers of the COBRIT clinical trial (n=31; as
described in JAMA. 2012; 308(19):1993-2000).
[0225] Defined human serum samples were used for this study.
Samples from adult TBI patients were analyzed retrospectively. The
healthy control cohort of patients, evaluated for non-TBI
complaints was obtained from Baylor College of Medicine (Houston,
Tex.; n=21).
[0226] To be considered a TBI patient for the HeadSMART trial, the
following criteria had to be met: 18 years old or greater, blunt
TBI presenting within 24 hours of injury, met the American College
of Emergency Physicians (ACEP) criteria for obtaining head CT scans
in TBI. Patients having brain tumor, brain surgery, pregnant,
non-English speakers, were excluded. Serial serum samples were
collected from enrollment to up to 6 months from 61 TBI patients.
Three samples per patient at eight different time points after
brain injury were collected, a selection of which were used in the
analysis For COBRIT trial samples, the following criteria were
used: Inclusion Criteria were that the patient had a
non-penetrating traumatic brain injury, age 18 (19 in Alabama)--70
years, GCS criteria on/off paralytics as specified in protocol,
reasonable expectation of completion of outcome measures at a
network center at six months post-injury, reasonable expectation of
enrollment within 24-hour time window, and English-speaking.
Exclusion criteria included: Intubated patients with GCS motor
score=6 and not meeting CT criteria, bilaterally fixed and dilated
pupils, positive pregnancy test, known pregnancy, or currently
breast feeding, evidence of diseases that interfere with outcome
assessment, current acetylcholinesterase inhibitor use, imminent
death or current life-threatening disease, currently enrolment in
another study, or prisoners. For healthy controls, 21 non-TBI
individuals at least 18 years of age were recruited under informed
consent at Baylor College of Medicine. One blood sample was
collected per control individual and processed to obtain replicate
vials of serum and plasma, which were stored at -80.degree. Celsius
until use. All patient identifiers were kept confidential.
[0227] Antigens and Serum Testing
[0228] 228 different antigens were spotted on in-house produced
epoxyhexyltriethoxysilane (EHTES) activated epoxy slides using a
Scienion S-11 non-contact microarray printer (Scienion AG,
Germany). The microarrays were then blocked for 1 hour at room
temperature with 1% casein. Test serum samples in 1% casein
blocking buffer (1:20 dilution) were incubated under a coverslip
for 1 hour at 37.degree.. The arrays were then washed and incubated
for 1 hour at 37.degree. with a 1:500 dilution of two detection
antibodies, mixed together: a goat anti-human IgG Cy3-conjugated
antibody, and a goat anti-human IgM AF647-conjugated antibody (both
purchased from Jackson ImmunoResearch Laboratories Inc., West
Grove, Pa.). Image acquisition was performed by laser at two
wavelengths 530 nm and 630 nm (Agilent Technologies, Santa Clara,
Calif.) and the results were analyzed using Genepix pro 7 software
(Molecular devices, Sunnyvale, Calif.). The quantitative range of
signal intensity of binding to each antigen spot was 0-65,000; this
range of detection made it possible to obtain reliable data at a
1:20 dilution of test serum samples.
[0229] Image Analysis and Data Processing
[0230] Each spot's intensity is represented by its pixels' mean
after subtraction of its local background median, followed by Log 2
transform. Negative spots (following background subtraction) are
imputed with background-like intensity. Background intensity was
subtracted for each spot, to obtain net signals. For every antigen
in every slide, outlier spots were removed. Outliers spots are
defined as having Z score >2 or <-2. The intensity of
multiple spots was combined through median, following removal of
outlier spots. The foreground and background intensities of
multiple spots of each antigen were averaged, and the difference
between the foreground and the background was calculated. The
resulting value was taken as the antigen reactivity of the
antibodies binding to that spotted antigen. All antigens showed
meaningful reactivity in a significant number of slides; thus no
antigen was excluded.
[0231] Statistical Analysis of Antibody Results
[0232] Antigens whose reactivity was higher or lower in a specific
study subgroup compared to other subgroups were identified.
Univariare analysis was used for separating antigens in a T test.
Antigens that allowed for setting a classification threshold such
as positive predictive value (PPV) .gtoreq.90% and sensitivity
.gtoreq.20% were achieved and determined to significantly
characterize a specific subgroup. For added restriction, only
antigens whose p value for a two sided t-test (after
Benjamini-Hochberg correction for multiple hypothesis) was smaller
than 0.05 were selected.
[0233] ELISA Plate Assay Methods
[0234] Biomarkers are tested by either the colorimetric,
fluorescence, chemiluminescent, or electrochemiluminescent
detection methodologies. For the colorimetric detection methods,
Maxisorb 96 well plates are used. For fluorescence assays, black
opaque-walled plates are used. For luminescence based-assays,
microtiter plates suitable for luminescence are used. Plates are
prepared as follows. Plates are rinsed once with coating buffer
specific to each plate type. Capture antibodies are added to each
well at an optimized concentration for an optimal time period.
Generally coating is performed over 12 hours at 4.degree. C. in
optimal coating buffer. Following the coating period, the excess
antibody is removed and the plates are blocked in an optimized
blocking buffer consisting of buffered saline with one of the
following: Casein, bovine serum Albumin, species-specific whole
serum, or filtered non-fat dry milk powder, or other blocking
agent, and/or non-ionic detergent. A series of sequential
incubations of optimal length are used to allow: 1) masking of
non-specific binding sites (i.e., blocking), 2), capture
antibody-antigen binding, 3) binding of antigen followed by washing
for removal of excess and non-bound antigens, 4) incubation of
anti-antigen detection antibody solution and detection tag, 5)
washing for removal of excess non-bound detection antibody and tag,
and 6) addition of detection substrate (ELISA) or optimal detection
solution (fluorescence or luminescence). Colorimetric detection is
performed on a microtiter plate reader, or similar technology, by
measuring absorbance of a colored substrate at an appropriate
wavelength of light. Fluorescence assays are performed using a
fluorescence based plate reader. Luminescence is detected on a
luminescence based reader. Data are collected and biomarker
concentrations are determined using a standard curve of recombinant
protein of known concentration.
Example 1: Association Between FABP (SEQ ID No: 61) and MBPR149
(SEQ ID No: 10) with TBI Outcomes
[0235] Each patient was profiled with its own measured time-points
in order to explore its autoantibodies profile change with time
post injury. Samples from TBI patients with Extended Glasgow
Outcome Scale (GOSE) equals 8 were compared to samples from TBI
patients with GOSE lower than 8 at a specific time-point (3
months/1 month).
[0236] Antibodies' Binding
[0237] Sera samples from healthy subjects and brain injury patients
at varying times post injury, and with varying GOSE scores were
tested for binding of serum IgG and/or IgM antibodies to the
various antigens disclosed in Table 1.
TABLE-US-00001 TABLE 1 List of brain injury related antigens. SEQ
ID Antigen Amino acid sequence or manufacture (Catalog number) NO:
MBP MASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIG 1 (myelin
RFFGGDRGAPKRGSGKVPWLKPGRSPL basic
PSHARSQPGLCNMYKDSHHPARTAHYGSLPQKSHGRTQDENP protein)
VVHFFKNIVTPRTPPPSQGKGRGLSLSR
FSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFK LGGRDSRSGSPMARR Enzo LS
(ALX-200-606-M001) MBP-in Post translational citullination of
arginine(s) 2 vitro in Enzo LS (ALX-200-606-M001) citrullinated MBP
R26 Ac-TMDHA(Cit)HGFLPC-amide 3 MBP R32,
Ac-GFLP(Cit)H(Cit)DTGIC-amide 4 R34 MBP R44
Ac-CILDSIG(Cit)FFGG-amide 5 MBP R50 Ac-FGGD(Cit)GAPKRGC-amide 6 MBP
R92 Ac-CDSHHPA(Cit)TAHYG-amide 7 MBP Ac-CQKSHG(Cit)TQDEN-amide 8
R106 MBP Ac-CFKNIVTP(Cit)TP-amide 9 R124 MBP
Ac-GAEGQ(Cit)PGFGYC-amide 10 R149 MBP Ac-CGYGG(Cit)ASDYKS-amide 11
R157 MBP Ac-CKLGG(Cit)DS(Cit)SG-amide 12 R186, R189 MBP
Ac-C(Ahx)SGSPMA(Cit)(Cit)-OH 13 R196, R197 GFAP
MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARM 14 (glial
PPPLPTRVDFSLAGALNAGFKETRASER fibrillary
AEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKL acid
ADVYQAELRELRLRLDQLTANSARLEVE protein)
RDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATL
ARLDLERKIESLEEEIRFLRKIHEEEVRE
LQEQLARQQVHVELDVAKPDLTAALKEIRTQYEAMASSNMHE
AEEWYRSKFADLTDAAARNAELLRQAKH
EANDYRRQLQSLTCDLESLRGTNESLERQMREQEERHVREAA
SYQEALARLEEEGQSLKDEMARHLQEYQ
DLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLD
TKSVSEGHLKRNIVVKTVEMRDGEV IKESKQEHKDVM Calbiochem (345996) GFAP-in
Post translational citullination of arginine(s) 15 vitro in
Calbiochem (345996) citrullinated GFAP R30
Ac-LAPGR(Cit)LGPGTC-amide 16 GFAP R36 Ac-CLGPGT(Cit)LSLAR-amide 17
GFAP Ac-AA(Cit)NAELLRQC-amide 18 R270 GFAP
Ac-CEGHLK(Cit)NIVVK-amide 19 R406 GFAP Ac-CVKTVEM(Cit)DGEVI-amide
20 R416 NRGN MDCCTENACSKPDDDILDIPLDDPGANAAAAKIQASFRGHMA 21
(neurogranin) RKKIKSGERGRKGPGPGGPGGAGVARGG AGGGPSGD NRGN-in Post
translational citullination of arginine(s) 22 vitro in NRGN
citrullinated NRGN Ac-CKSGE(Cit)G(Cit)KGPG-amide 23 R51, R53 NRGN
Ac-CGGAGVA(Cit)GGAG-amide 24 R68 ERMIN
MKTLSPDRIQPHIMTDVPATFTQAECNGDKPPENGQQTITKISE 25
ELTDVDSPLPHYRVEPSLEGALTKGS
QEERRKLQGNMLLNSSMEDKMLKENPEEKLFIVHKAITDLSLQ
ETSADEMTFREGHQWEKIPLSGSNQEI
RRQKERITEQPLKEEEDEDRKNKGHQAAEIEWLGFRKPSQAD
MLHSKHDEEQKVWDEEIDDDDDDNCNND
EDEVRVIEFKKKHEEVSQFKEEGDASEDSPLSSASSQAVTPDEQ
PTLGKKSDISRNAYSRYNTISYRKIR KGNTKQRIDEFESMMHL ERMIN- Post
translational citullination of arginine(s) 26 in vitro in ERMIN
citrullinated Ermin R57 Ac-DSPLPHY(Cit)VEPSLEC-amide 27 ICAM5
MPGPSPGLRRALLGLWAALGLGLFGLSAVSQEPFWADLQPRV 28
AFVERGGSLWLNCSTNCPRPERGGLETS
RRNGTQRGLRWLARQLVDIREPETQPVCFFRCARRTLQARGL
IRTFQRPDRVELMPLPPWQPVGENFTL
SCRVPGAGPRASLTLTLLRGAQELIRRSFAGEPPRARGAVLTAT
VLARREDHGANFSCRAELDLRPHGLG
LFENSSAPRELRTFSLSPDAPRLAAPRLLEVGSERPVSCTLDGLF
PASEARVYLALGDQNLSPDVTLEGD
AFVATATATASAEQEGARQLVCNVTLGGENRETRENVTIYSFP
APLLTLSEPSVSEGQMVTVTCAAGAQA
LVTLEGVPAAVPGQPAQLQLNATENDDRRSFFCDATLDVDGE
TLIKNRSAELRVLYAPRLDDSDCPRSWT
WPEGPEQTLRCEARGNPEPSVHCARSDGGAVLALGLLGPVTR
ALSGTYRCKAANDQGEAVKDVTLTVEYA
PALDSVGCPERITWLEGTEASLSCVAHGVPPPDVICVRSGELGA
VIEGLLRVAREHAGTYRCEATNPRGS
AAKNVAVTVEYGPRFEEPSCPSNWTWVEGSGRLFSCEVDGKP
QPSVKCVGSGGATEGVLLPLAPPDPSPR
APRIPRVLAPGIYVCNATNRHGSVAKTVVVSAESPPEMDESTC
PSHQTWLEGAEASALACAARGRPSPGV
RCSREGIPWPEQQRVSREDAGTYHCVATNAHGTDSRTVTVGV
EYRPVVAELAASPPGGVRPGGNFTLTCR
AEAWPPAQISWRAPPGALNIGLSSNNSTLSVAGAMGSHGGEYE
CAATNAHGRHARRITVRVAGPWLWVAV
GGAAGGAALLAAGAGLAFYVQSTACKKGEYNVQEAESSGEA
VCLNGAGGGAGGAAGAEGGPEAAGGAAES PAEGEVFAIQLTSA R&D (1950-M5) SNCB
MDVFMKGLSMAKEGVVAAAEKTKQGVTEAAEKTKEGVLYV 29 (Beta-
GSKTREGVVQGVASVAEKTKEQASHLGGAV synuclein)
FSGAGNIAAATGLVKREEFPTDLKPEEVAQEAAEEPLIEPLMEP EGESYEDPPQEEYQEYEPEA
OriGene (TP315165) MT3 MDPETCPCPSGGSCTCADSCKCEGCKCTSCKKSCCSCCPAECEK
30 (Metallo- CAKDCVCKGGEAAEAEAEKCSCCQ thionein III) OMG
MEYQILKMSLCLFILLFLTPGILCICPLQCICTERHRHVDCSGRN 31 (Oligo-
LSTLPSGLQENIIHLNLSYNHFTDL denrocyte
HNQLTQYTNLRTLDISNNRLESLPAHLPRSLWNMSAANNNIKL Myelin
LDKSDTAYQWNLKYLDVSKNMLEKVVL Glycoprotein)
IKNTLRSLEVLNLSSNKLWTVPTNMPSKLHIVDLSNNSLTQILP
GTLINLTNLTHLYLHNNKFTFIPDQS
FDQLFQLQEITLYNNRWSCDHKQNITYLLKWMMETKAHVIGT
PCSTQISSLKEHNMYPTPSGFTSSLFTV
SGMQTVDTINSLSVVTQPKVTKIPKQYRTKETTFGATLSKDTTF
TSTDKAFVPYPEDTSTETINSHEAAA
ATLTIHLQDGMVTNTSLTSSTKSSPTPMTLSITSGMPNNFSEMP
QQSTTLNLWREETTTNVKTPLPSVAN AWKVNASFLLLLNVVVMLAV CNDP1 32 (Carnosine
dipeptidase 1) Reticulon MAAPGDPQDELLPLAGPGSQWLRHRGEGENEAVTPKGATPAP
33 1 QAGEPSPGLGARAREAASREAGSGPARQ
SPVAMETASTGVAGVSSAMDHTFSTTSKDGEGSCYTSLISDICY
PPQEDSTYFTGILQKENGHVTISESP
EELGTPGPSLPDVPGIESRGLFSSDSGIEMTPAESTEVNKILADP
LDQMKAEAYKYIDITRPEEVKHQEQ
HHPELEDKDLDFKNKDTDISIKPEGVREPDKPAPVEGKIIKDHL
LEESTFAPYIDDLSEEQRRAPQITTP
VKITLTEIEPSVETTTQEKTPEKQDICLKPSPDTVPTVTVSEPED
DSPGSITPPSSGTEPSAAESQGKGS
ISEDELITAIKEAKGLSYETAENPRPVGQLADRPEVKARSGPPTI
PSPLDHEASSAESGDSEIELVSEDP
MAAEDALPSGYVSFGHVGGPPPSPASPSIQYSILREEREAELDS
ELIIESCDASSASEESPKREQDSPPM
KPSALDAIREETGVRAEERAPSRRGLAEPGSFLDYPSTEPQPGP
ELPPGDGALEPETPMLPRKPEEDSSS
NQSPAATKGPGPLGPGAPPPLLFLNKQKAIDLLYWRDIKQTGIV
FGSFLLLLFSLTQFSVVSVVAYLALA
ALSATISFRIYKSVLQAVQKTDEGHPFKAYLELEITLSQEQIQKY
TDCLQFYVNSTLKELRRLFLVQDLV DSLKFAVLMWLLTYVGALFNGLTLLLMAVVSMFTLPVVYVK
HQAQIDQYLGLVRTHINAVVAKIQAKIPG AKRHAE Astrotactin
MALAGLCALLACCWGPAAVLATAAGDVDPSKELECKLKSITV 34 1
SALPFLRENDLSIMHSPSASEPKLLFSV
RNDFPGEMVVVDDLENTELPYFVLEISGNTEDIPLVRWRQQWL
ENGTLLFHIHHQDGAPSLPGQDPTEEP
QHESAEEELRILHISVMGGMIALLLSILCLVMILYTRRRWCKRR
RVPQPQKSASAEAANEIHYIPSVLIG
GHGRESLRNARVQGHNSSGTLSIRETPILDGYEYDITDLRHHLQ
RECMNGGEDFASQVTRTLDSLQGCNE
KSGMDLTPGSDNAKLSLMNKYKDNIIATSPVDSNHQQATLLSH
TSSSQRKRINNKARAGSAFLNPEGDSG
TEAENDPQLTFYTDPSRSRRRSRVGSPRSPVNKTTLTLISITSCVI
GLVCSSHVNCPLVVKITLHVPEHL
IADGSRFILLEGSQLDASDWLNPAQVVLFSQQNSSGPWAMDLC
ARRLLDPCEHQCDPETGECLCYEGYMK
DPVHKHLCIRNEWGTNQGPWPYTIFQRGFDLVLGEQPSDKIFR
FTYTLGEGMWLPLSKSFVIPPAELAIN
PSAKCKTDMTVMEDAVEVREELMTSSSFDSLEVLLDSFGPVRD
CSKDNGGCSKNFRCISDRKLDSTGCVC
PSGLSPMKDSSGCYDRHIGVDCSDGFNGGCEQLCLQQMAPFP
DDPTLYNILMFCGCIEDYKLGVDGRSCQ
LITETCPEGSDCGESRELPMNQTLFGEMFFGYNNHSKEVAAGQ
VLKGTFRQNNFARGLDQQLPDGLVVAT
VPLENQCLEEISEPTPDPDFLTGMVNFSEVSGYPVLQHWKVRS
VMYHIKLNQVAISQALSNALHSLDGAT
SRADFVALLDQFGNHYIQEAIYGFEESCSIWYPNKQVQRRLWL
EYEDISKGNSPSDESEERERDPKVLTF
PEYITSLSDSGTKHMAAGVRMECHSKGRCPSSCPLCHVTSSPD
TPAEPVLLEVTKAAPIYELVTNNQTQR
LLQEATMSSLWCSGTGDVIEDWCRCDSTAFGADGLPTCAPLP
QPVLRLSTVHEPSSTLVVLEWEHSEPPI
GVQIVDYLLRQEKVTDRMDHSKVETETVLSFVDDIISGAKSPC
AMPSQVPDKQLTTISLIIRCLEPDTIY
MFTLWGVDNTGRRSRPSDVIVKTPCPVVDDVKAQEIADKIYNL
FNGYTSGKEQQTAYNTLLDLGSPTLHR
VLYHYNQHYESFGEFTWRCEDELGPRKAGLILSQLGDLSSWC
NGLLQEPKISLRRSSLKYLGCRYSEIKP YGLDWAELSRDLRKTCEEQTLSIPYNDYGDSKEI
Brain uniprot# O60242 35 Angio- genesis Inhibitor 3 Glutamate
uniprot# Q14832 36 Receptor, Metabo- trophic 3 Kelch like uniprot#
Q96NJ5 37 32 Matrix uniprot# P14780 38 metallo- proteinase-9
Melanoma uniprot# Q8TD90 39 Antigen
Family E, 2 Neuregulin uniprot# P56975 40 3 SLIT and uniprot#
O94933 41 NTRK-Like Family, Member 3 BDNF
MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGT 42 (Brain
LESVNGPKAGSRGLTSLADTFEHVIEEL derived
LDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEY neurotrophic
KNYLDAANMSMRVRRHSDPARRGELSV factor)
CDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFY
ETKCNPMGYTKEGCRGIDKRHWNSQCRT TQSYVRALTMDSKKRIGWRHRIDTSCVCTLTIKRGR
R&D (248BD005) UBIQUITIN uniprot# P09936 43 CTERMINAL HYDROLASE
L1 Oligo24 T16G1: TTT TTT TTT TTT TTT TG 44 Tubulin uniprot# P68371
45 beta-4B chain in vitro citrullinated Tubulin (K)IREEYPDrIMNTF(S)
46 beta-4B chain Tubulin uniprot# P68363 47 alpha-1B chain Tubulin
48 alpha-1B chain in vitro citrullinated Tubulin
(K)YMAccLLYrGDVVPK(D) 49 alpha-1B chain Tubulin (E)VrTGTYrQLFHPE(Q)
50 alpha-1B chain synaptotag uniprot# P21579 51 min AB1-42 52
CNPase (K)STLArVIVDK(Y) 53 CNPase (K)ITPGArGAFSEEYK(R) 54 Laminin
uniprot# Q13753 55 PPIA in uniprot# P62937 56 vitro citrullinated
PPIA (K)TAENFrALSTGEK(G) 57 S100A10 Uniprot# P60903 58 Septin-7 in
59 vitro citrullinated Septin-7 (R)ILEQQNSSrTLEK(N) 60 Fatty acid
Prospec (PRO-340) 61 binding Protein (FABP-3) Elongation Uniprot#
Q05639 62 factor 1-alpha 2 in vitro citrullinated Elongation
(K)PLrLPLQDVYK(I) 63 factor 1-alpha 2 Elongation
(D)VYKIGGIGTVPVGrVE(T) 64 factor 1-alpha 2 ICNPase uniprot# P09543
65 (2', 3 cyclic nucleotide 3'-phospho- diesterase) Collagen-
uniprot# P02462 66 IV TPPP (K)AISSPTVSrLTDTTK(F) 67 Phospho-
uniprot# P05412 68 c-Jun TPPP3 in (K)TGGAVD(Cit)LTDTSrYTGSHK(E) 69
vitro citrullinated TPPP3 (K)TGGAVDRLTDTSrYTGSHK(E) 70 TPPP3
(K)GIAGrQDILDDSGYVSAYK(N) 71 vesicular uniprot# Q8IZ57 72 membrane
protein neurensin-1 (p24 NDRG2, uniprot# Q9UN36 73 Isoform 2 in
vitro citrullinated NDRG2, (R)TASLTSAASVDGNrSR(S) 74 Isoform 2 S100
MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSH 75 calcium
FLEEIKEQEVVDKVMETLDNDGDGECD binding FQEFMAFVAMVTTACHEFFEHE protein
B Sigma (S6677) (S100B) NSE
MSIEKIWAREILDSRGNPTVEVDLYTAKGLFRAAVPSGASTGIY 76 (neuron
EALELRDGDKQRYLGKGVLKAVDHIN specific
STIAPALISSGLSVVEQEKLDNLMLELDGTENKSKFGANAILGV enolase
SLAVCKAGAAERELPLYRHIAQLAGN aka
SDLILPVPAFNVINGGSHAGNKLAMQEFMILPVGAESFRDAMR ENO2)
LGAEVYHTLKGVIKDKYGKDATNVGDE
GGFAPNILENSEALELVKEAIDKAGYTEKIVIGMDVAASEFYRD
GKYDLDFKSPTDPSRYITGDQLGALY
QDFVRDYPVVSIEDPFDQDDWAAWSKFTANVGIQIVGDDLTV
TNPKRIERAVEEKACNCLLLKVNQIGSV
TEAIQACKLAQENGWGVMVSHRSGETEDTFIADLVVGLCTGQI
KTGAPCRSERLAKYNQLMRIEEELGDE ARFAGHNFRNPSVL Abnova (H00002026-P01)
MCP1 Pro spec (CHM-271) 77 (monocyte chemotactic protein- 1) Tau,
total Sigma (T9392) 78 Neurofila 79 ment light polypeptide
Neurofila 80 ment heavy polypeptide y-Enolase 81 Prothrombin- 82
FactorII EXOSC10 uniprot# Q01780 83 Spectrin, Sigma (S3644) 84
breakdown products Myelopero Sigma (M6908) 85 xidase (MPO) CMV
Prospec (CMV Pp150) 86 ICAM uniprot# Q8N6I2 87 SLC39A11 uniprot#
Q8N1S5 88 MAP2 89 (Microtubule- associated protein 2 MAPT 90
(microtubule- associated protein tau gene) HTR1A 91 (Serotonin
receptor 1A gene) PLXNA4 92 (PlexinsA4) Interleukin- PVPPGEDSKD
VAAPHRQPLT SSERIDKQIR YILDGISALR 93 6 KETCNKSNMC ESSKEALAEN
NLNLPKMAEK DGCFQSGFNE ETCLVKIITG LLEFEVYLEY LQNRFESSEE QARAVQMSTK
VLIQFLQKKA KNLDAITTPD PTTNASLLTK LQAQNQWLQD MTTHLILRSF KEFLQSSLRA
LRQM Peprotech (200-06) Interleukin- p40 Subunit: IWELKK DVYVVELDWY
PDAPGEMVVL 94 12 TCDTPEEDGI TWTLDQSSEV LGSGKTLTIQ VKEFGDAGQY
TCHKGGEVLS HSLLLLHKKE DGIWSTDILK DQKEPKNKTF LRCEAKNYSG RFTCWWLTTI
STDLTFSVKS SRGSSDPQGV TCGAATLSAE RVRGDNKEYE YSVECQEDSA CPAAEESLPI
EVMVDAVHKL KYENYTSSFF IRDIIKPDPP KNLQLKPLKN SRQVEVSWEY PDTWSTPHSY
FSLTFCVQVQ GKSKREKKDR VFTDKTSATV ICRKNASISV RAQDRYYSSS WSEWASVPCS
Peprotech (200-12) Interleukin- MNWVNVISDL KKIEDLIQSM HIDATLYTES
DVHPSCKVTA 95 15 MKCFLLELQV ISLESGDASI HDTVENLIIL ANNSLSSNGN
VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS Peprotech (200-15)
Interleukin- MIVKAGITIP RNPGCPNSED KNFPRTVMVN LNIHNRNTNT 96 17
NPKRSSDYYN RSTSPWNLHR NEDPERYPSV IWEAKCRHLG CINADGNVDY HMNSVPIQQE
ILVLRREPPH CPNSFRLEKI LVSVGCTCVT PIVHHVA Peprotech (200-17)
Interleukin- MRPSGRKSSK MQAFRIWDVN QKTFYLRNNQ LVAGYLQGPN 97 1ra
VNLEEKIDVV PIEPHALFLG IHGGKMCLSC VKSGDETRLQ LEAVNITDLS ENRKQDKRFA
FIRSDSGPTT SFESAACPGW FLCTAMEADQ PVSLTNMPDE GVMVTKFYFQ EDE
Peprotech (200-01RA) TNFRI MDSVCPQGKY IHPQNNSICC TKCHKGTYLY
NDCPGPGQDT 98 DCRECESGSF TASENHLRHC LSCSKCRKEM GQVEISSCTV
DRDTVCGCRK NQYRHYWSEN LFQCFNCSLC LNGTVHLSCQ EKQNTVCTCH AGFFLRENEC
VSCSNCKKSL ECTKLCLPQI EN Peprotech (310-07) VEGF YQRSYCHPIE
TLVDIFQEYP 99
DEIEYIFKPS CVPLMRCGGC CNDEGLECVP TEESNITMQI MRIKPHQGQH IGEMSFLQHN
KCECRPKKDR ARQENPCGPC SERRKHLFVQ DPQTCKCSCK NTDSRCKARQ LELNERTCRC
DKPRR Peprotech (100-20) VCAM1 FKIETTPESR YLAQIGDSVS LTCSTTGCES
PFFSWRTQID 100 SPLNGKVTNE GTTSTLTMNP VSFGNEHSYL CTATCESRKL
EKGIQVEIYS FPKDPEIHLS GPLEAGKPIT VKCSVADVYP FDRLEIDLLK GDHLMKSQEF
LEDADRKSLE TKSLEVTFTP VIEDIGKVLV CRAKLHIDEM DSVPTVRQAV KELQVYISPK
NTVISVNPST KLQEGGSVTM TCSSEGLPAP EIFWSKKLDN GNLQHLSGNA TLTLIAMRME
DSGIYVCEGV NLIGKNRKEV ELIVQEKPFT VEISPGPRIA AQIGDSVMLT CSVMGCESPS
FSWRTQlDSP LSGKVRSEGT NSTLTLSPVS FENEHSYLCT VTCGHKKLEK GIQVELYSFP
RDPEIEMSGG LVNGSSVTVS CKVPSVYPLD RLEIELLKGE TILENIEFLE DTDMKSLENK
SLEMTFIPTI EDTGKALVCQ AKLHIDDMEF EPKQRQSTQT LYVNVAPRDT TVLVSPSSIL
EEGSSVNMTC LSQGFPAPKI LWSRQLPNGE LQPLSENATL TLISTKMEDS GVYLCEGINQ
AGRSRKEVEL IIQVTPKDIK LTAFPSESVK EGDTVIISCT CGNVPETWII LKKKAETGDT
VLKSIDGAYT IRKAQLKDAG VYECESKNKV GSQLRSLTLD VQGRENNKDY FSP
Peprotech (150-04) FactorVIIa AKRONbiotech (AK9916) 101 Collagen
102 II Microglobulin- Sigma ( M4890) 103 b2 TNFRSF12A EQAPGTAPCS
RGSSWSADLD KCMDCASCRA RPHSDFCLGC 104 AAAPPAPFRL LWP Peprotech
(310-21) TNFRII MAPEPGSTCR LREYYDQTAQ MCCSKCSPGQ HAKVFCTKTS 105
DTVCDSCEDS TYTQLWNWVP ECLSCGSRCS SDQVETQACT REQNRICTCR PGWYCALSKQ
EGCRLCAPLR KCRPGFGVAR PGTETSDVVC KPCAPGTFSN TTSSTDICRP HQICNVVAIP
GNASMDAVCT STSP Peprotech (310-12) CRP Sigma (C4063) 106 BAFF-R
MRRGPRSLRG RDAPAPTPCV PAECFDLLVR HCVACGLLRT 107 PRPKPAGASS
PAPRTALQPQ ESVGAGAGEA ALPLPG Peprotech (310-13R) BAFF AVQGPEETVT
QDCLQLIADS ETPTIQKGSY TFVPWLLSFK 108 RGSALEEKEN KILVKETGYF
FIYGQVLYTD KTYAMGHLIQ RKKVHVFGDE LSLVTLFRCI QNMPETLPNN SCYSAGIAKL
EEGDELQLAI PRENAQISLD GDVTFFGALK LL Peprotech (310-13) GLP1
HAEGTFTSDV SSYLEGQAAK EFIAWLVKGR G 109 Peprotech (130-08) HSP90
Sigma (H6774) 110 EGFP Prospec (cyt-332) 111 C4 Sigma (C8195) 112
C3 Sigma (C2910) 113 C1q Prospec (pro-554) 114 Fibrinogen
AKRONbiotech (AK9026) 115
[0238] As shown in FIG. 1, the levels of anti-Fatty acid-binding
protein (FABP, SEQ ID No: 61) IgM autoantibodies in serum samples
obtained from TBI patients at day 30 post injury, with Glasgow
Outcome Scale Extended (GOSE) score <8 (cross labeled) are lower
in comparison to patients with GOSE score=8 (circle labeled).
[0239] As shown in FIG. 2, the levels of anti-Myelin basic protein
(MBPR149, SEQ ID No: 10, MBP derived BSA conjugated peptide) IgM
autoantibodies in serum samples obtained from TBI patients at day
30 post injury, with Glasgow Outcome Scale Extended (GOSE) score
<8 (cross labeled) are higher in comparison to patients with
GOSE score=8 (circle labeled). These results demonstrate for the
first time that increased levels of anti-FABP IgM autoantibodies in
serum samples obtained from a TBI patient are indicative of
recovery from brain injury of said TBI patient. Furthermore,
decreased levels of anti-MBPR149 IgM autoantibodies in serum
samples obtained from a TBI patient are indicative of recovery from
brain injury of said TBI patient. Thus the present invention
disclosed specific antigen antibody reactivities that can be used
for monitoring, and/or prognosis of brain injury.
Example 2: Elevated Levels of Anti-Myeloperoxidase (MPO, SEQ ID No:
85) IgM Autoantibodies in Serum Samples Obtained from TBI Patients
as Compared to Healthy Controls
[0240] As shown in FIG. 3, the levels of anti-MPO IgM
autoantibodies in serum samples obtained from TBI patients (circle
labeled) are higher in comparison with healthy controls (cross
labeled). These results demonstrate for the first time that
increased levels of anti-MPO autoantibodies are indicative of brain
injury.
Example 3: Decreased Levels of Anti-CMV (SEQ ID No: 86) IgG
Autoantibody in Serum Samples Obtained from TBI Patients as
Compared to Healthy Controls
[0241] As shown in FIG. 4A, the levels of anti-CMV (SEQ ID No: 86)
IgG autoantibody levels in serum samples obtained from TBI patients
(circle labeled) at day 30 and day 90 post injury (N=142) are lower
in comparison with healthy controls (cross labeled) (N=21). FIG. 4B
shows the separation performance by receivers operating
characteristic (ROC) curves of anti-CMV IgG autoantibody
levels.
Example 4: The Prediction of the Clinical Status of TBI Patients at
Day 90 Post Injury, Based on the Anti-TNFRSF12A (SEQ ID No: 104)
IgM Autoantibody Levels in Serum Samples Obtained from TBI Patients
at Day 30 Post Injury
[0242] As shown in FIG. 5A, the levels of anti-TNFRSF12A (SEQ ID
No:104) IgM autoantibody in serum samples obtained from TBI
patients at day 30 post injury can be used for the prediction of
the clinical status (GOSE <8 or GOSE=8) of TBI patients at day
90 post injury. FIG. 5B shows the separation performance by
receivers operating characteristic (ROC) curves of anti-TNFRSF12A
IgM autoantibody levels.
Example 5: Combination Measurement of the Levels of Autoantibodies
and Biomarkers in Serum Samples Obtained from TBI Patients as
Compared to Healthy Controls
[0243] To determine whether combination measurement of the levels
of antibodies and biomarkers in serum samples can differentiate
between TBI patients and healthy controls, a combined analysis was
conducted. Serum samples obtained from TBI patients at time 0 (t0,
N=85) were compared with serum samples obtained from healthy
control (HC, N=21). The analysis was based on 464 iChip features
(232 antigens, IgM and IgG) and four ELISA features. iChip data is
based on average of two block replicates, following correction
procedure. ELISA features were selected based on data availability;
only features with data available for >80% of the iChip samples
were used. Samples with missing ELISA data were removed from the
analysis.
[0244] FIG. 6 shows the area under the Receiver Operating
Characteristics (ROC) curves of six classification methods (SVM,
LR, QDA, CART, RF and LDA) based on 100 iteration; of 70:30 cross
validation. Features were ranked according to their median scoring
or frequency of model inclusion, depending on the method.
[0245] Using the LDA classification method revealed that the top
six features above the random background level are the biomarkers:
GFAP and SNCB in combination with the autoantibodies: anti-MBP in
vitro citrullinated (SEQ ID No: 2) IgM, anti-GFAP (SEQ ID No: 14)
IgM, anti-ICAM5 (SEQ ID No: 28) IgM, and anti-BDNF (SEQ ID No: 42)
IgM.
[0246] Using the QDA classification method revealed that the top
three features above the random background level are the
biomarkers: GFAP and SNCB in combination with the autoantibodies:
anti-MBP in vitro citrullinated (SEQ ID No: 2).
Example 6: Combination Measurement of the Levels of Antibodies and
Biomarkers in Serum Samples Obtained from TBI Patients with
Intracranial Hemorrhage on Head CT as Compared to TBI Patients with
Normal CT
[0247] To determine whether combination measurement of the levels
of antibodies and biomarkers in serum samples can differentiate
between TBI patients with intracranial hemorrhage on head CT and
those with normal CT, a combined analysis was conducted. Serum
samples obtained from TBI patients at time 0 (t0) with abnormal CT
were compared with samples obtained from TBI patients at time 0
(t0) with normal CT. Analysis was based on 464 iChip features (232
antigen, IgM and IgG) and four ELISA features. iChip data is based
on average of two block replicates, following correction procedure.
ELISA features were selected based on data availability; only
features with data available for >80% of the iChip samples were
used. Samples with missing ELISA data were removed from the
analysis.
[0248] FIG. 7 shows ROC curves of six classification methods (SVM,
LR, QDA, CART, RF and LDA), based on 100 iterations of 70:30 cross
validation. Features were ranked according to their median scoring
or frequency of model inclusion, depending on the method.
[0249] Using the LDA classification method revealed that the top
five features above the random background level are the biomarker:
SNCB in combination with the autoantibodies: anti-Collagen IV (SEQ
ID No: 66) IgG, anti-Oligo24 (SEQ ID No: 44) IgM, anti-EBV IgM and
anti-Collagen II (SEQ ID No: 102) IgG.
[0250] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means, materials,
and steps for carrying out various disclosed functions may take a
variety of alternative forms without departing from the
invention.
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