U.S. patent application number 17/560144 was filed with the patent office on 2022-04-14 for neural proteins as biomarkers for nervous system injury and other neural disorders.
This patent application is currently assigned to University of Florida Research Foundation, Inc.. The applicant listed for this patent is Banyan Biomarkers, Inc., University of Florida Research Foundation, Inc.. Invention is credited to Ming-Cheng LIU, Monika OLI, Kevin Ka-Wang WANG.
Application Number | 20220113321 17/560144 |
Document ID | / |
Family ID | 1000006039685 |
Filed Date | 2022-04-14 |
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United States Patent
Application |
20220113321 |
Kind Code |
A1 |
WANG; Kevin Ka-Wang ; et
al. |
April 14, 2022 |
NEURAL PROTEINS AS BIOMARKERS FOR NERVOUS SYSTEM INJURY AND OTHER
NEURAL DISORDERS
Abstract
The present invention identifies biomarkers that are diagnostic
of nerve cell injury and/or neuronal disorders. Detection of
different biomarkers of the invention are also diagnostic of the
degree of severity of nerve injury, the cell(s) involved in the
injury, and the subcellular localization of the injury.
Inventors: |
WANG; Kevin Ka-Wang;
(Gainesville, FL) ; OLI; Monika; (Gainesville,
FL) ; LIU; Ming-Cheng; (Gainesville, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Florida Research Foundation, Inc.
Banyan Biomarkers, Inc. |
Gainsville
San Diego |
FL
CA |
US
US |
|
|
Assignee: |
University of Florida Research
Foundation, Inc.
Gainesville
FL
Banyan Biomarkers, Inc.
San Diego
CA
|
Family ID: |
1000006039685 |
Appl. No.: |
17/560144 |
Filed: |
December 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16449096 |
Jun 21, 2019 |
11221342 |
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17560144 |
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15802489 |
Nov 3, 2017 |
10330689 |
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16449096 |
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15340002 |
Nov 1, 2016 |
9810698 |
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15802489 |
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12950142 |
Nov 19, 2010 |
9664694 |
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15340002 |
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12822560 |
Jun 24, 2010 |
8492107 |
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12950142 |
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12137194 |
Jun 11, 2008 |
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12822560 |
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11107248 |
Apr 15, 2005 |
7396654 |
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12137194 |
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60562944 |
Apr 15, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/28 20130101;
G01N 33/6896 20130101; G01N 2800/52 20130101; C12Q 1/6883 20130101;
C07K 16/18 20130101; A61B 5/4064 20130101; C12Y 304/19012
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 16/18 20060101 C07K016/18; C12Q 1/6883 20060101
C12Q001/6883 |
Goverment Interests
[0002] The invention was made with government support under Grant
NS039091 awarded by the National Institutes of Health and Grant
NS040182 awarded by the National Institutes of Health and Grants
DAMD 17-99-1-9565 and DAMD 17-01-1-0765 awarded by the United
States Army. The government has certain rights in the invention.
Claims
1. A kit comprising: (a) a substrate for holding a sample isolated
from a subject; (b) an agent that specifically interacts with
ubiquitin C-terminal hydrolase L1 (UCH-L1); (c) an optional
additional agent that specifically interacts with at least one
additional protein biomarkers upon contact with said sample; and
(d) printed instructions for reacting the agent and the optional
additional agent with the sample or a portion of the sample for
diagnosing a neural injury or neuronal disorder in the subject.
2. The kit of claim 1, wherein said optional additional agent is
present, and said optional additional agent interacts with said one
additional protein biomarker upon contact with said sample, wherein
said additional protein biomarkers is selected from the group
consisting of: vesicular membrane protein p-24, synuclein,
microtubule-associated protein, synaptophysin, Vimentin,
Synaptotagmin, Synaptojanin-2, Synapsin2, CRMP1, 2, Amphiphysin-1,
PSD95, PSD-93, Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, Myelin basic protein (MBP), Myelin
proteolipid protein (PLP), Myelin Oligodendrocyte specific protein
(MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), NF-H, NF-L, NF-M, BIII-tubulin-1 and
combinations thereof.
3. The kit of claim 1, wherein said additional protein biomarkers
is selected from the group consisting of: vesicular membrane
protein p-24, synuclein, synaptophysin and combinations
thereof.
4. The kit of claim 1, further comprising reagents for the reacting
the agent with the sample as an immunoassay.
5. The kit of claim 4, wherein the immunoassay is an ELISA.
6. The kit of claim 1, wherein the agent is an antibody that binds
to UCH-L1.
7. The kit of claim 1, wherein the optional agent is an antibody
that binds with said at least one additional protein biomarker, the
said at least one additional protein biomarker selected from the
group consisting of: vesicular membrane protein p-24, synuclein,
microtubule-associated protein, synaptophysin, Vimentin,
Synaptotagmin, Synaptojanin-2, Synapsin2, CRMP1, 2, Amphiphysin-1,
PSD95, PSD-93, Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, Myelin basic protein (MBP), Myelin
proteolipid protein (PLP), Myelin Oligodendrocyte specific protein
(MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), NF--H, NF-L, NF-M, BIII-tubulin-1 and
combinations thereof.
8. The kit of claim 7, wherein said additional protein biomarkers
is selected from the group consisting of: vesicular membrane
protein p-24, synuclein, synaptophysin and combinations
thereof.
9. The kit of claim 7, further comprising a second antibody that
binds with a second protein biomarker of said at least one
additional protein biomarker.
10. The kit of claim 7, further comprising a third antibody that
binds with a third protein biomarker of said at least one
additional protein biomarker.
11. The kit of claim 1 wherein said substrate is a biochip
array.
12. The kit of claim 11 wherein the biochip array is a protein chip
array.
13. The kit of claim 11 wherein the biochip array is a nucleic acid
array.
14. The kit of claim 11 wherein the biochip array has a surface
comprising a substance selected from the group consisting of: an
antibody, nucleic acid, protein, peptides, amino acid probes, and a
phage display library.
15. A method of detecting a neural injury or neuronal disorder in a
subject comprising: collecting a sample of a bodily fluid or a
tissue in contact with neural tissue from the subject; and
analyzing said sample or a fraction thereof for an amount of
ubiquitin C-terminal hydrolase L1 (UCH-L1) associated with the
neural injury or neuronal disorder; optionally analyzing a sample
of the fluid or the tissue for at least one additional protein
biomarker associated with the neural injury and/or neuronal
disorder.
16. The method of claim 15, wherein said at least one additional
protein biomarker is selected from the group consisting of:
vesicular membrane protein p-24, synuclein, microtubule-associated
protein, synaptophysin, Vimentin, Synaptotagmin, Synaptojanin-2,
Synapsin2, CRMP1, 2, Amphiphysin-1, PSD95, PSD-93, Calmodulin
dependent protein kinase II (CAMPK)-alpha, beta, gamma, Myelin
basic protein (MBP), Myelin proteolipid protein (PLP), Myelin
Oligodendrocyte specific protein (MOSP), Myelin Oligodendrocyte
glycoprotein (MOG), myelin associated protein (MAG), NF--H, NF-L,
NF-M, BIII-tubulin-1 and combinations thereof.
17. The method of claim 15, wherein said at least one additional
protein biomarkers is selected from the group consisting of:
vesicular membrane protein p-24, synuclein, synaptophysin and
combinations thereof.
18. The method of claim 15, wherein the amount decreases with
recovery of the subject from the neural injury and/or neuronal
disorder.
19. The method of claim 15, wherein the amount of UCH-L1 is related
to severity of the neural injury or neuronal disorder.
20. The method of claim 15, further comprising analyzing a second
sample of the bodily fluid or tissue in contact with neural tissue
from the subject for a second sample amount of UCH-L1.
21. The method of claim 20 wherein said second sample amount is
less than the sample amount.
22. The method of claim 20 wherein said second sample amount is
more than the sample amount.
23. The method of claim 15, wherein the neural injury or neuronal
disorder is subcellular neural cell injury.
24. The method of claim 15, wherein the neural injury or neuronal
disorder is traumatic brain injury (TBI).
25. The method of claim 15 wherein said bodily fluid in contact
with neural tissue is selected from the group consisting of blood,
blood plasma, serum and urine.
26. The method of claim 15 wherein said UCH-L1 or one of said at
least one additional protein biomarkers are detected using an
immunoassay.
27. The method of claim 26 wherein the immunoassay is an ELISA.
28. The method of claim 15 wherein said at least one additional
protein biomarker is two or three or four or five protein
biomarkers.
29. The method of claim 15 further comprising immobilizing said at
least one protein biomarker on a biochip array and laser ionizing
to detect a biomarker molecular weight.
30. The method of claim 29 further comprising comparing the
molecular weight against a threshold intensity that is normalized
against total ion current.
31. The method of claim 29 wherein the biochip array surface
comprises a substance selected from the group consisting of: an
antibody, nucleic acid, protein, peptides, amino acid probes, and a
phage display library.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
16/449,096, filed Jun. 21, 2019, which is a continuation of U.S.
Ser. No. 15/802,489, filed Nov. 3, 2017, now U.S. Pat. No.
10,330,689, which is a continuation of U.S. Ser. No. 15/340,002,
filed Nov. 1, 2016, now U.S. Pat. No. 9,810,698, which is a
continuation of U.S. Ser. No. 12/950,142, filed Nov. 19, 2010, now
U.S. Pat. No. 9,664,694, which is a continuation of U.S. Ser. No.
12/822,560, filed Jun. 24, 2010, now U.S. Pat. No. 8,492,107, which
is a continuation-in-part of U.S. Ser. No. 12/137,194, filed Jun.
11, 2008, now abandoned, which is a divisional of U.S. Ser. No.
11/107,248, filed Apr. 15, 2005, now U.S. Pat. No. 7,396,654, which
claims the benefit of U.S. Provisional Application Ser. No.
60/562,944, filed Apr. 15, 2004, the disclosures of which are
hereby incorporated by reference in their entirety, including all
figures, tables and amino acid or nucleic acid sequences.
FIELD OF THE INVENTION
[0003] The invention provides for the reliable detection and
identification of biomarkers, important for the diagnosis and
prognosis of damage to the nervous system (central nervous system
(CNS) and peripheral nervous system (PNS)), brain injury and neural
disorders. The protein/peptide profile in patients with damage to
nerves and brain cells are distinguished from normal individuals
using inexpensive techniques. These techniques provide simple yet
sensitive approaches to diagnosing damage to the central nervous
system, brain injury and neuronal disorders using biological
fluids.
BACKGROUND OF THE INVENTION
[0004] The incidence of traumatic brain injury (TBI) in the United
States is conservatively estimated to be more than 2 million
persons annually with approximately 500,000 hospitalizations. Of
these, about 70,000 to 90,000 head injury survivors are permanently
disabled. The annual economic cost to society for care of
head-injured patients is estimated at $25 billion. These figures
are for the civilian population only and the incidence is much
greater when combat casualties are included. In modern warfare
(1993-2000), TBI is the leading cause of death (53%) among wounded
who have reached medical care facilities.
[0005] 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.
[0006] The mammalian nervous system comprises a peripheral nervous
system (PNS) and a central nervous system (CNS, comprising the
brain and spinal cord), and is composed of two principal classes of
cells: neurons and glial cells. The glial cells fill the spaces
between neurons, nourishing them and modulating their function.
Certain glial cells, such as Schwann cells in the PNS and
oligodendrocytes in the CNS, also provide a protective myelin
sheath that surrounds and protects neuronal axons, which are the
processes that extend from the neuron cell body and through which
the electric impulses of the neuron are transported. In the
peripheral nervous system, the long axons of multiple neurons are
bundled together to form a nerve or nerve fiber. These, in turn,
may be combined into fascicles, wherein the nerve fibers form
bundles embedded, together with the intraneural vascular supply, in
a loose collagenous matrix bounded by a protective multilamellar
sheath. In the central nervous system, the neuron cell bodies are
visually distinguishable from their myelin-ensheathed processes,
and are referenced in the art as gray and white matter,
respectively.
[0007] During development, differentiating neurons from the central
and peripheral nervous systems send out axons that must grow and
make contact with specific target cells. In some cases, growing
axons must cover enormous distances; some grow into the periphery,
whereas others stay confined within the central nervous system. In
mammals, this stage of neurogenesis is complete during the
embryonic phase of life and neuronal cells do not multiply once
they have fully differentiated.
[0008] Accordingly, the neural pathways of a mammal are
particularly at risk if neurons are subjected to mechanical or
chemical trauma or to neuropathic degeneration sufficient to put
the neurons that define the pathway at risk of dying. A host of
neuropathies, some of which affect only a subpopulation or a system
of neurons in the peripheral or central nervous systems have been
identified to date. The neuropathies, which may affect the neurons
themselves or the associated glial cells, may result from cellular
metabolic dysfunction, infection, exposure to toxic agents,
autoimmunity dysfunction, malnutrition or ischemia. In some cases
the cellular dysfunction is thought to induce cell death directly.
In other cases, the neuropathy may induce sufficient tissue
necrosis to stimulate the body's immune/inflammatory system and the
mechanisms of the body's immune response to the initial neural
injury then destroys the neurons and the pathway defined by these
neurons.
[0009] Another common injury to the CNS is stroke, 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 or ischemia that
results in deficient blood supply and death of tissues in one area
of the brain (infarction). Causes of ischemic 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. The ability to detect such injury is lacking in the prior
art.
[0010] Mammalian neural pathways also are at risk due to damage
caused by neoplastic lesions. Neoplasias of both the neurons and
glial cells have been identified. Transformed cells of neural
origin generally lose their ability to behave as normal
differentiated cells and can destroy neural pathways by loss of
function. In addition, the proliferating tumors may induce lesions
by distorting normal nerve tissue structure, inhibiting pathways by
compressing nerves, inhibiting cerebrospinal fluid or blood supply
flow, and/or by stimulating the body's immune response. Metastatic
tumors, which are a significant cause of neoplastic lesions in the
brain and spinal cord, also similarly may damage neural pathways
and induce neuronal cell death.
[0011] There is thus, a need in the art appropriate, specific,
inexpensive and simple diagnostic clinical assessments of nervous
system injury severity and therapeutic treatment efficacy. Thus
identification of neurochemical markers that are specific to or
predominantly found in the nervous system (CNS (brain and spinal
cord) and PNS), would prove immensely beneficial for both
prediction of outcome and for guidance of targeted therapeutic
delivery.
SUMMARY
[0012] The present invention provides neuronal protein markers that
are differentially present in the samples of patients suffering
from neural injury and/or neuronal disorders as compared to samples
of control subjects. The present invention also provides sensitive
and quick methods and kits that can be used as an aid for diagnosis
of neural injury and/or neuronal disorders by detecting these
markers. The measurement of these markers, alone or in combination,
in patient samples provides information that a diagnostician can
correlate with a probable diagnosis of the extent of neural injury
such as in traumatic brain injury (TBI) and stroke.
[0013] In a preferred embodiment, the invention provides biomarkers
that are indicative of traumatic brain injury, neuronal damage,
neural disorders, brain damage, neural damage due to drug or
alcohol addiction, diseases associated with the brain or nervous
system, such as the central nervous system. Preferably, the
biomarkers are proteins, fragments or derivatives thereof, and are
associated with neuronal cells, brain cells or any cell that is
present in the brain and central nervous system.
[0014] In a preferred embodiment the biomarkers are preferably
neural proteins, peptides, fragments or derivatives thereof.
Examples of neural proteins, include, but are not limited to axonal
proteins, amyloid precursor protein, dendritic proteins, somal
proteins, presynaptic proteins, post-synaptic proteins and neural
nuclear proteins.
[0015] In another preferred embodiment the biomarkers are selected
from at least one protein, peptide, variant or fragment thereof,
such as those proteins listed in Table 1 below. For example, Axonal
Proteins: .alpha. II spectrin (and SPDB)-1, NF-68 (NF-L)--2, Tau-3,
.alpha. II, III spectrin, NF-200 (NF--H), NF-160 (NF-M), Amyloid
precursor protein, .alpha. internexin; Dendritic Proteins: beta
III-tubulin-1, p24 microtubule-associated protein-2, alpha-Tubulin
(P02551), beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4,
Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000),
Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0016] In another preferred embodiment the biomarkers are from at
least two or more proteins, peptides, variants or fragments
thereof, such as those proteins listed in Table 1 below. For
example, Axonal Proteins: .alpha. II spectrin (and SPDB)-1, NF-68
(NF-L)-2, Tau-3, .alpha. II, III spectrin, NF-200 (NF-H), NF-160
(NF-M), Amyloid precursor protein, .alpha. internexin; Dendritic
Proteins: beta III-tubulin-1, p24 microtubule-associated protein-2,
alpha-Tubulin (P02551), beta-Tubulin (P04691), MAP-2A/B--3,
MAP-2C-3, Stathmin-4, Dynamin-1 (P21575), Phocein, Dynactin
(Q13561), Vimentin (P31000), Dynamin, Profilin, Cofilin 1,2; Somal
Proteins: UCH-L1 (Q00981)-1, Glycogen phosphorylase-BB--2, PEBP
(P31044), NSE (P07323), CK-BB (P07335), Thy 1.1, Prion protein,
Huntingtin, 14-3-3 proteins (e.g. 14-3-3-epsolon (P42655)),
SM22-.alpha., Calgranulin AB, alpha-Synuclein (P37377),
beta-Synuclein (Q63754), HNP 22; Neural nuclear proteins: NeuN-1,
S/G(2) nuclear autoantigen (SG2NA), Huntingtin; Presynaptic
Proteins: Synaptophysin-1, Synaptotagmin (P21707), Synaptojanin-1
(Q62910), Synaptojanin-2, Synapsin1 (Synapsin-Ia), Synapsin2
(Q63537), Synapsin3, GAP43, Bassoon(NP_003449), Piccolo (aczonin)
(NP_149015), Syntaxin, CRMP1, 2, Amphiphysin-1 (NP_001626),
Amphiphysin-2 (NP_647477); Post-Synaptic Proteins: PSD95-1,
NMDA-receptor (and all subtypes)-2, PSD93, AMPA-kainate receptor
(all subtypes), mGluR (all subtypes), Calmodulin dependent protein
kinase II (CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0017] In another preferred embodiment, the biomarkers comprise at
least one biomarker from each neural cell type. The composition of
biomarkers is diagnostic of neural injury, damage and/or neural
disorders. The composition comprises: .alpha. II spectrin, SPDB-1,
NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0018] In another preferred embodiment an expanded panel of
biomarkers are used to provide highly enriched information of
mechanism of injury, modes of cell death (necrosis versus
apoptosis), sites of injury, sites and status of different cell
types in the nervous system and enhanced diagnosis (better
selectivity and specificity). This invention is an important and
significant improvement over existing technologies focused on small
panel (e.g. a four-marker panel:-MBP-Thrombomodulin-S100B-NSE from
Syn X Pharma (Mississauga, Canada)- or single markers (e.g. S 100B
from DiaSorin (Sweden)).
[0019] In another preferred embodiment the biomarkers are selected
to distinguish between different host anatomical regions. For
example, at least one biomarker can be selected from neural
subcellular protein biomarkers, nervous system anatomical markers
such as hippocampus protein biomarkers and cerebellum protein
biomarkers. Examples of neural subcellular protein biomarkers are
NF-200, NF-160, NF-68. Examples of hippocampus protein biomarkers
are SCG10, stathmin. An example of a cerebellum protein biomarker
is Purkinje cell protein-2 (Pcp2).
[0020] In another preferred embodiment the biomarkers are selected
to distinguish between injury at the cellular level, thereby
detecting which cell type has been injured. For example at least
one biomarker protein is selected from a representative panel of
protein biomarkers specific for that cell type. Examples for
biomarkers specific for cell types include myelin-oligodendrocyte
biomarkers such as myelin basic protein (MBP), myelin proteolipid
protein (PLP), myelin oligodendrocyte specific protein (MOSP),
oligodendrocyte NS-1 protein, myelin oligodendrocyte glycoprotein
(MOG). Examples of biomarkers specific for Schwann cells include,
but not limited to Schwann cell myelin protein. Examples of Glial
cell protein biomarkers include, but not limited to GFAP (protein
accession number P47819), protein disulfide isomerase (PDI)-P04785.
Thus, by detecting one or more specific biomarkers the specific
cell types that have been injured can be determined.
[0021] In another preferred embodiment, biomarkers specific for
different subcellular structures of a cell can be used to determine
the subcellular level of injury. Examples include but not limited
to neural subcellular protein biomarkers such as, NF-200, NF-160,
NF-68; dendritic biomarkers such as for example, alpha-tubulin
(P02551), beta-tubulin (P04691), MAP-2A/B, MAP-2C, Tau, Dynamin-1
(P212575), Phoecin, Dynactin (Q13561), p24 microtubule-associated
protein, vimentin (P31000); somal proteins such as for example,
UCH-L1 (Q00981), PEBP (P31044), NSE (P07323), CK-BB (P07335), Thy
1.1, prion protein, 14-3-3 proteins; neural nuclear proteins, such
as for example S/G(2) nuclear autoantigen (SG2NA), NeuN. Thus,
detection of specific biomarkers will determine the extent and
subcellular location of injury.
[0022] In another preferred embodiment, biomarkers specific for
different anatomical regions, different cell types, and/or
different subcellular structures of a cell are selected to provide
information as to the location of anatomical injury, the location
of the injured cell type, and the location of injury at a
subcellular level. Any number of biomarkers from each set can be
used to provide highly enriched and detailed information of
mechanism, mode and subcellular sites of injury, anatomical
locations of injury and status of different cell types in the
nervous system (neuronal subtypes, neural stem cells, astro-glia,
oligodendrocyte and microglia cell).
[0023] In a preferred embodiment at least one biomarker specific
different locations such as for an anatomical region, different
cell types and/or different subcellular structures of a cell are
used to determine the mechanism, mode, subcellular sites of injury,
anatomical locations of injury and status of different cell types
in the nervous system, more preferably a panel of at least 2
biomarkers are selected from each desired location, more preferably
at least 3, 4, 5, 6, 7, 8, 9, 10 up to about 100 biomarkers are
selected from each location.
[0024] In a preferred embodiment, subcellular neuronal biomarkers
for diagnosis and detection of brain and/or CNS injury and/or
neural disorders, preferably are at least one of axonal proteins,
dendritic proteins, somal proteins, neural nuclear proteins,
presynaptic proteins, post-synaptic proteins.
[0025] In a preferred embodiment, axonal proteins identified as
biomarkers for diagnosis and detection of brain and/or CNS injury
or neural disorders, preferably are: .alpha. II spectrin (and
SPDB)-1, NF-68 (NF-L)-2, Tau-3, .alpha. II, III spectrin, NF-200
(NF-H), NF-160 (NF-M), Amyloid precursor protein, .alpha.
internexin, peptides, fragments or derivatives thereof.
[0026] In a preferred embodiment, dendritic proteins identified as
biomarkers for diagnosis and detection of brain and/or CNS injury
or neural disorders, preferably are: beta III-tubulin-1, p24
microtubule-associated protein-2, alpha-Tubulin (P02551),
beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4, Dynamin-1
(P21575), Phocein, Dynactin (Q13561), Vimentin (P31000), Dynamin,
Profilin, Cofilin 1, 2, peptides, fragments or derivatives
thereof.
[0027] In another preferred embodiment, neural nuclear proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: NeuN-1,
S/G(2) nuclear autoantigen (SG2NA), Huntingtin, peptides or
fragments thereof.
[0028] In another preferred embodiment, somal proteins identified
as biomarkers for diagnosis and detection of brain and/or CNS
injury or neural disorders, preferably are: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22,
peptides, fragments or derivatives thereof.
[0029] In another preferred embodiment, presynaptic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are:
Synaptophysin-1, Synaptotagmin (P21707), Synaptojanin-1 (Q62910),
Synaptojanin-2, Synapsin1 (Synapsin-Ia), Synapsin2 (Q63537),
Synapsin3, GAP43, Bassoon(NP_003449), Piccolo (aczonin)
(NP_149015), Syntaxin, CRMP1, 2, Amphiphysin-1 (NP_001626),
Amphiphysin-2 (NP_647477), peptides, fragments or derivatives
thereof.
[0030] In another preferred embodiment, post-synaptic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: PSD95-1,
NMDA-receptor (and all subtypes)-2, PSD93, AMPA-kainate receptor
(all subtypes), mGluR (all subtypes), Calmodulin dependent protein
kinase II (CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP,
peptides, fragments or derivatives thereof.
[0031] In another preferred embodiment, identified biomarkers
distinguish the damaged neural cell subtype such as, for example,
myelin-oligodendrocytes, glial, microglial, Schwann cells, glial
scar.
[0032] In a preferred embodiment, Myelin-Oligodendrocyte biomarkers
are: Myelin basic protein (MBP) and fragments, Myelin proteolipid
protein (PLP), Myelin Oligodendrocyte specific protein (MOSP),
Myelin Oligodendrocyte glycoprotein (MOG), myelin associated
protein (MAG), Oligodendrocyte NS-1 protein; Glial Protein
Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin.
[0033] In another preferred embodiment, biomarkers identifying the
anatomical location of neural injury and/or neural damage, include,
but not limited to: Hippocampus: Stathmin, Hippocalcin, SCG10;
Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); PH8 (S Serotonergic
Dopaminergic), PEP-19, Neurocalcin (NC), a neuron-specific EF-hand
Ca.sup.2+-binding protein, Encephalopsin, Striatin, SG2NA, Zinedin,
Recoverin, and Visinin.
[0034] In another preferred embodiment, biomarkers identifying
damaged neural subtypes include, but not limited to:
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0035] Demyelination proteins identified as biomarkers for
diagnosis and detection of brain and/or CNS injury or neural
disorders, preferably are: myelin basic protein (MBP), myelin
proteolipid protein, peptides, fragments or derivatives
thereof.
[0036] In another preferred embodiment, glial proteins identified
as biomarkers for diagnosis and detection of brain and/or CNS
injury or neural disorders, preferably are: GFAP (P47819), protein
disulfide isomerase (PDI--P04785), peptides, fragments and
derivatives thereof.
[0037] In another preferred embodiment, cholinergic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are:
acetylcholine esterase, choline acetyltransferase, peptides,
fragments or derivatives thereof.
[0038] In another preferred embodiment, dopaminergic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: tyrosine
hydroxylase (TH), phospho-TH, DARPP32, peptides, fragments or
derivatives thereof.
[0039] In another preferred embodiment, noradrenergic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: dopamine
beta-hydroxylase (DbH), peptides, fragments or derivatives
thereof.
[0040] In another preferred embodiment, serotonergic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: tryptophan
hydroxylase (TrH), peptides, fragments or derivatives thereof.
[0041] In another preferred embodiment, glutamatergic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: glutaminase,
glutamine synthetase, peptides, fragments or derivatives
thereof.
[0042] In another preferred embodiment, GABAergic proteins
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are: GABA
transaminase (4-aminobutyrate-2-ketoglutarate transaminase
[GABAT]), glutamic acid decarboxylase (GAD25, 44, 65, 67),
peptides, fragments and derivatives thereof.
[0043] In another preferred embodiment, neurotransmitter receptors
identified as biomarkers for diagnosis and detection of brain
and/or CNS injury or neural disorders, preferably are:
beta-adrenoreceptor subtypes, (e.g. beta (2)), alpha-adrenoreceptor
subtypes, (e.g. (alpha (2c)), GABA receptors (e.g. GABA(B)),
metabotropic glutamate receptor (e.g. mGluR3), NMDA receptor
subunits (e.g. NR1A2B), Glutamate receptor subunits (e.g. GluR4),
5-HT serotonin receptors (e.g. 5-HT(3)), dopamine receptors (e.g.
D4), muscarinic Ach receptors (e.g. M1), nicotinic acetylcholine
receptor (e.g. alpha-7), peptides, fragments or derivatives
thereof.
[0044] In another preferred embodiment, neurotransmitter
transporters identified as biomarkers for diagnosis and detection
of brain and/or CNS injury or neural disorders, preferably are:
norepinephrine transporter (NET), dopamine transporter (DAT),
serotonin transporter (SERT), vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), glutamate transporter (e.g. GLT1),
vesicular acetylcholine transporter, choline transporter (e.g.
CHT1), peptides, fragments, or derivatives thereof.
[0045] In another preferred embodiment, other proteins identified
as biomarkers for diagnosis and detection of brain and/or CNS
injury or neural disorders, include, but are not limited to
vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2,
MMP9, peptides, fragments or derivatives thereof.
[0046] The markers are characterized by molecular weight, enzyme
digested fingerprints and by their known protein identities. The
markers can be resolved from other proteins in a sample by using a
variety of fractionation techniques, e.g., chromatographic
separation coupled with mass spectrometry, or by traditional
immunoassays. In preferred embodiments, the method of resolution
involves Surface-Enhanced Laser Desorption/Ionization ("SELDI")
mass spectrometry, in which the surface of the mass spectrometry
probe comprises adsorbents that bind the markers.
[0047] In other preferred embodiments, a plurality of the
biomarkers are detected, preferably at least two of the biomarkers
are detected, more preferably at least three of the biomarkers are
detected, most preferably at least four of the biomarkers are
detected.
[0048] In one aspect, the amount of each biomarker is measured in
the subject sample and the ratio of the amounts between the markers
is determined. Preferably, the amount of each biomarker in the
subject sample and the ratio of the amounts between the biomarkers
and compared to normal healthy individuals. The increase in ratio
of amounts of biomarkers between healthy individuals and
individuals suffering from injury is indicative of the injury
magnitude, disorder progression as compared to clinically relevant
data.
[0049] Preferably, biomarkers that are detected at different stages
of injury and clinical disease are correlated to assess anatomical
injury, type of cellular injury, subcellular localization of
injury. Monitoring of which biomarkers are detected at which stage,
degree of injury in disease or physical injury will provide panels
of biomarkers that provide specific information on mechanisms of
injury, identify multiple subcellular sites of injury, identify
multiple cell types involved in disease related injury and identify
the anatomical location of injury.
[0050] In another aspect, preferably a single biomarker is used in
combination with one or more biomarkers from normal, healthy
individuals for diagnosing injury, location of injury and
progression of disease and/or neural injury, more preferably a
plurality of the markers are used in combination with one or more
biomarkers from normal, healthy individuals for diagnosing injury,
location of injury and progression of disease and/or neural injury.
It is preferred that one or more protein biomarkers are used in
comparing protein profiles from patients susceptible to, or
suffering from disease and/or neural injury, with normal
subjects.
[0051] Preferred detection methods include use of a biochip array.
Biochip arrays useful in the invention include protein and nucleic
acid arrays. One or more markers are immobilized on the biochip
array and subjected to laser ionization to detect the molecular
weight of the markers. Analysis of the markers is, for example, by
molecular weight of the one or more markers against a threshold
intensity that is normalized against total ion current. Preferably,
logarithmic transformation is used for reducing peak intensity
ranges to limit the number of markers detected.
[0052] In another preferred method, data is generated on
immobilized subject samples on a biochip array, by subjecting said
biochip array to laser ionization and detecting intensity of signal
for mass/charge ratio; and, transforming the data into computer
readable form; and executing an algorithm that classifies the data
according to user input parameters, for detecting signals that
represent markers present in injured and/or diseased patients and
are lacking in non-injured and/or diseased subject controls.
[0053] Preferably the biochip surfaces are, for example, ionic,
anionic, comprised of immobilized nickel ions. comprised of a
mixture of positive and negative ions, comprises one or more
antibodies, single or double stranded nucleic acids, comprises
proteins, peptides or fragments thereof, amino acid probes,
comprises phage display libraries.
[0054] In other preferred methods one or more of the markers are
detected using laser desorption/ionization mass spectrometry,
comprising, providing a probe adapted for use with a mass
spectrometer comprising an adsorbent attached thereto, and;
contacting the subject sample with the adsorbent, and; desorbing
and ionizing the marker or markers from the probe and detecting the
deionized/ionized markers with the mass spectrometer.
[0055] Preferably, the laser desorption/ionization mass
spectrometry comprises, providing a substrate comprising an
adsorbent attached thereto; contacting the subject sample with the
adsorbent; placing the substrate on a probe adapted for use with a
mass spectrometer comprising an adsorbent attached thereto; and,
desorbing and ionizing the marker or markers from the probe and
detecting the desorbed/ionized marker or markers with the mass
spectrometer.
[0056] The adsorbent can for example be, hydrophobic, hydrophilic,
ionic or metal chelate adsorbent, such as, nickel or an antibody,
single- or double stranded oligonucleotide, amino acid, protein,
peptide or fragments thereof.
[0057] In another embodiment, a process for purification of a
biomarker, comprising fractioning a sample comprising one or more
protein biomarkers by size-exclusion chromatography and collecting
a fraction that includes the one or more biomarker; and/or
fractionating a sample comprising the one or more biomarkers by
anion exchange chromatography and collecting a fraction that
includes the one or more biomarkers. Fractionation is monitored for
purity on normal phase and immobilized nickel arrays. Generating
data on immobilized marker fractions on an array, is accomplished
by subjecting said array to laser ionization and detecting
intensity of signal for mass/charge ratio; and, transforming the
data into computer readable form; and executing an algorithm that
classifies the data according to user input parameters, for
detecting signals that represent markers present in injured and/or
diseased patients and are lacking in non-injured and/or diseased
subject controls. Preferably fractions are subjected to gel
electrophoresis and correlated with data generated by mass
spectrometry. In one aspect, gel bands representative of potential
markers are excised and subjected to enzymatic treatment and are
applied to biochip arrays for peptide mapping.
[0058] In another preferred embodiment, the presence of certain
biomarkers is indicative of the extent of CNS and/or brain injury.
For example, detection of one or more dendritic damage markers,
soma injury markers, demyelination markers, axonal injury markers
would be indicative of CNS injury and the presence of one or more
would be indicative of the extent of nerve injury.
[0059] In another preferred embodiment, the presence of certain
biomarkers is indicative of a neurological disorder. i.e. dendritic
damage markers, soma injury markers, demyelination markers, axonal
injury markers, synaptic terminal markers, post-synaptic
markers.
[0060] Preferred methods for detection and diagnosis of CNS/PNS
and/or brain injury comprise detecting at least one or more protein
biomarkers in a subject sample, and; correlating the detection of
one or more protein biomarkers with a diagnosis of CNS and/or brain
injury, wherein the correlation takes into account the detection of
one or more biomarker in each diagnosis, as compared to normal
subjects, wherein the one or more protein markers are selected
from: neural proteins, such as for example, Axonal Proteins:
.alpha. II spectrin (and SPDB)-1, NF-68 (NF-L)-2, Tau-3, .alpha.
II, III spectrin, NF-200 (NF-H), NF-160 (NF-M), Amyloid precursor
protein, .alpha. internexin; Dendritic Proteins: beta
III-tubulin-1, p24 microtubule-associated protein-2, alpha-Tubulin
(P02551), beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4,
Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000),
Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0061] In another preferred embodiment, the invention provides a
kit for analyzing cell damage in a subject. The kit, preferably
includes: (a) one or more biomarkers (b) a substrate for holding a
biological sample isolated from a human subject suspected of having
a damaged nerve cell, (c) an agent that specifically binds at least
one or more of the neural proteins; and (d) printed instructions
for reacting the agent with the biological sample or a portion of
the biological sample to detect the presence or amount of at least
one marker in the biological sample. The biomarkers include but not
limited to: Axonal Proteins: .alpha. II spectrin (and SPDB)-1,
NF-68 (NF-L)-2, Tau-3, .alpha. II, III spectrin, NF-200 (NF-H),
NF-160 (NF-M), Amyloid precursor protein, .alpha. internexin;
Dendritic Proteins: beta III-tubulin-1, p24 microtubule-associated
protein-2, alpha-Tubulin (P02551), beta-Tubulin (P04691),
MAP-2A/B--3, MAP-2C-3, Stathmin-4, Dynamin-1 (P21575), Phocein,
Dynactin (Q13561), Vimentin (P31000), Dynamin, Profilin, Cofilin
1,2; Somal Proteins: UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, PEBP (P31044), NSE (P07323), CK-BB (P07335),
Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0062] In another preferred embodiment, the kit comprises a
composition or panel of biomarkers comprises: .alpha. II spectrin,
SPDB-1, NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0063] Preferably, the biological sample is a fluid in
communication with the nervous system of the subject prior to being
isolated from the subject; for example, CSF or blood, and the agent
can be an antibody, aptamer, or other molecule that specifically
binds at least one or more of the neural proteins. The kit can also
include a detectable label such as one conjugated to the agent, or
one conjugated to a substance that specifically binds to the agent
(e.g., a secondary antibody).
[0064] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The invention is pointed out with particularity in the
appended claims. The above and further advantages of this invention
may be better understood by referring to the following description
taken in conjunction with the accompanying drawings, in which:
[0066] FIG. 1 is a schematic illustration showing the fate of brain
injury biomarkers. The pathway of genesis of biomarkers from the
brain to the eventual release of such biomarkers into biofluids,
such as CSF, blood, urine, saliva, sweat etc. provide an
opportunity for biomarker detection with low invasiveness.
[0067] FIG. 2 is a schematic illustration showing sources of brain
injury biomarkers from different cell types (neurons, astro-glia
cells, Microglia cells, oligodendrocyte or Schwann cell) and from
different subcellular structural structure of a neuron (dendrites,
axons, cell body, presynaptic terminal and postsynaptic
density)
[0068] FIG. 3A is a Western Blot showing the detection and
accumulation of Novel brain-specific marker #1: UCH-L1 neural
protein in CSF of rodents after experimental traumatic brain injury
in rats.
[0069] FIG. 3B is a graph showing the elevation of Novel
brain-specific marker #1: Ubiquitin C-terminal hydrolase L1
(UCH-L1) in rat CSF 48 h after experimental brain injury:
craniotomy and controlled cortical impact (CCI)-induced brain
injury when compared to CSF from naive control rats.
[0070] FIG. 4A is a Western Blot showing the detection and
accumulation of Novel brain-specific marker #2: neuronal
microtubule binding protein (p24) in CSF of rodents after
experimental traumatic brain injury in rats.
[0071] FIG. 4B is a graph showing the elevation of Novel
brain-specific marker #2: neuronal microtubule binding protein
(p24) in rat CSF 48 h after experimental brain injury: craniotomy
and controlled cortical impact (CCI)-induced brain injury when
compared to CSF from naive control rats.
[0072] FIG. 5A is a Western Blot showing the detection and
accumulation of Novel brain-specific marker #3: Neuronal protein
.alpha.-synuclein in CSF of rodents after experimental traumatic
brain injury in rats.
[0073] FIG. 5B is a graph showing the elevation of Novel
brain-specific marker #3: Neuronal protein .alpha.-synuclein in rat
CSF 48 h after experimental brain injury: craniotomy and controlled
cortical impact (CCI)-induced brain injury when compared to CSF
from naive control rats.
[0074] FIG. 6A is a Western Blot showing the detection and
accumulation of Neuronal biomarker #1 UCH-L1 levels are elevated in
human CSF 24 h after TBI.
[0075] FIG. 6B is a graph showing the elevation of Neuronal
biomarker #1 UCH-L1 levels are elevated in human CSF 24 h after
traumatic brain injury, when compared to CSF from neurological
controls with no apparent brain injury.
[0076] FIG. 7A is a Western Blot showing the detection and
accumulation of Novel brain-specific marker #2: neuronal
microtubule binding protein (p24) in human CSF after traumatic
brain injury
[0077] FIG. 7B is a graph showing the elevation of Neuronal
biomarker Novel brain-specific marker #2: neuronal microtubule
binding protein (p24) in human CSF 24 h after traumatic brain
injury when compared to CSF from neurological controls with no
apparent brain injury.
[0078] FIG. 8A are the results from a quantitative SW ELISA for
synaptophysin showing the detection of Novel brain-specific marker
#4: synaptophysin in rat CSF after traumatic brain injury when
compared to CSF from neurological controls with no apparent brain
injury.
[0079] FIG. 8B is a graph showing the elevation of Neuronal
biomarker Novel brain-specific marker #2: neuronal microtubule
binding protein (p24) in human CSF 24 h after traumatic brain
injury when compared to CSF from neurological controls with no
apparent brain injury.
[0080] FIG. 9A is a graph showing the elevation of Novel
brain-specific marker #1: Ubiquitin C-terminal hydrolase L1
(UCH-L1) as measure by quantitative sandwich ELISA with samples
from human CSF and serum from patients with severe traumatic brain
injury
[0081] FIG. 9B is a graph showing the temporal changes measured by
quantitative sandwich ELISA in levels of UCH-L1 measured in serum
for a patient with severe TBI. Serum samples were taken at the time
the patient was admitted to the hospital (0 d), and at 12 hours (1
d), 48 hours (2 d), 72 hours (3 d), and 120 hours (5 d) after the
time of injury.
[0082] FIG. 10A is a Western Blot showing detection and
accumulation of neurensin (p24) in cerebral spinal fluid (CSF) in
human patients with brain injury showing p24 accumulation and
spectrin breakdown product (SPDP) 150 kDa and 145 kDa measured at
12, 30, 42, 48, 66, 78 and 84 hours after injury compared to
controls N5 and N6.
[0083] FIG. 10B is a graph showing densiometric quantification of
CSF p24 levels in CSF in human brain injured patients at 12, 30,
42, 48, 66, 78 and 84 hours after injury compared to a control
N.
[0084] FIG. 11A is a Western Blot showing neurensin (p24) biomarker
immunoblotting detection in human serum using a centrifuging
filtration/concentration technique in molecular weight range of
30-50 kDa fraction at 24 hours after injury; 1 shows human TBI CSF
(7.5 ul); 2 is human TBI serum (200 ul with MW kDa cutoff); 3 is
human TBI serum (175 .mu.l plus human 25 .mu.l CSF); 4 is serum
(10-30 kDa cut off); 5 is serum plus CSF (10-30 kDa cut off).
[0085] FIG. 11B is a graph showing densiometric quantification of
serum p24 levels. The same method applied to normal control serum
samples showed no detection of p24 levels (level=0; data not
shown). Serum was pooled from 2 human patients.
[0086] FIG. 12A is a graph showing alpha-synuclein biomarker
elevation in human TBI patient CSF detected by sandwich ELISA.
Alpha-synuclein levels in control non-brain injured CSF were
compared to TBI patient CSF samples collected at different post
injury time (T=enrollment) or 12, 24, 48, 72, 96, 120 and 168 hours
after injury.
[0087] FIG. 12B is a graph showing alpha-synuclein levels in normal
control (non-brain injured) serum compared to TBI patient serum
samples collected at different post-injury times (T=E (enrollment)
or 24, 72 and 96 hr after injury and showed significant elevation
compared to control serum from uninjured patients.
DETAILED DESCRIPTION
[0088] The present invention identifies biomarkers that are
diagnostic of nerve cell injury and/or neuronal disorders.
Detection of different biomarkers of the invention are also
diagnostic of the degree of severity of nerve injury, the cell(s)
involved in the injury, and the subcellular localization of the
injury. In particular, the invention employs a step of correlating
the presence or amount of one or more neural protein(s) with the
severity and/or type of nerve cell injury. The amount of a neural
protein, fragment or derivative thereof directly relates to
severity of nerve tissue injury as a more severe injury damages a
greater number of nerve cells which in turn causes a larger amount
of neural protein(s) to accumulate in the biological sample (e.g.,
CSF).
[0089] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
that will be used hereinafter.
[0090] "Marker" in the context of the present invention refers to a
polypeptide (of a particular apparent molecular weight) which is
differentially present in a sample taken from patients having
neural injury and/or neuronal disorders as compared to a comparable
sample taken from control subjects (e.g., a person with a negative
diagnosis, normal or healthy subject).
[0091] "Complementary" in the context of the present invention
refers to detection of at least two biomarkers, which when detected
together provides increased sensitivity and specificity as compared
to detection of one biomarker alone.
[0092] The phrase "differentially present" refers to differences in
the quantity and/or the frequency of a marker present in a sample
taken from patients having for example, neural injury as compared
to a control subject. For example, a marker can be a polypeptide
which is present at an elevated level or at a decreased level in
samples of patients with neural injury compared to samples of
control subjects. Alternatively, a marker can be a polypeptide
which is detected at a higher frequency or at a lower frequency in
samples of patients compared to samples of control subjects. A
marker can be differentially present in terms of quantity,
frequency or both.
[0093] A polypeptide is differentially present between the two
samples if the amount of the polypeptide in one sample is
statistically significantly different from the amount of the
polypeptide in the other sample. For example, a polypeptide is
differentially present between the two samples if it is present at
least about 120%, at least about 130%, at least about 150%, at
least about 180%, at least about 200%, at least about 300%, at
least about 500%, at least about 700%, at least about 900%, or at
least about 1000% greater than it is present in the other sample,
or if it is detectable in one sample and not detectable in the
other.
[0094] Alternatively or additionally, a polypeptide is
differentially present between the two sets of samples if the
frequency of detecting the polypeptide in samples of patients'
suffering from neural injury and/or neuronal disorders, is
statistically significantly higher or lower than in the control
samples. For example, a polypeptide is differentially present
between the two sets of samples if it is detected at least about
120%, at least about 130%, at least about 150%, at least about
180%, at least about 200%, at least about 300%, at least about
500%, at least about 700%, at least about 900%, or at least about
1000% more frequently or less frequently observed in one set of
samples than the other set of samples.
[0095] "Diagnostic" means identifying the presence or nature of a
pathologic condition. 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.
[0096] A "test amount" of a marker refers to an amount of a marker
present in a sample being tested. A test amount can be either in
absolute amount (e.g., .mu.g/ml) or a relative amount (e.g.,
relative intensity of signals).
[0097] A "diagnostic amount" of a marker refers to an amount of a
marker in a subject's sample that is consistent with a diagnosis of
neural injury and/or neuronal disorder. A diagnostic amount can be
either in absolute amount (e.g., .mu.g/ml) or a relative amount
(e.g., relative intensity of signals).
[0098] A "control amount" of a marker can be any amount or a range
of amount which is to be compared against a test amount of a
marker. For example, a control amount of a marker can be the amount
of a marker in a person without neural injury and/or neuronal
disorder. A control amount can be either in absolute amount (e.g.,
.mu.g/ml) or a relative amount (e.g., relative intensity of
signals).
[0099] "Probe" refers to a device that is removably insertable into
a gas phase ion spectrometer and comprises a substrate having a
surface for presenting a marker for detection. A probe can comprise
a single substrate or a plurality of substrates.
[0100] "Substrate" or "probe substrate" refers to a solid phase
onto which an adsorbent can be provided (e.g., by attachment,
deposition, etc.).
[0101] "Adsorbent" refers to any material capable of adsorbing a
marker. The term "adsorbent" is used herein to refer both to a
single material ("monoplex adsorbent") (e.g., a compound or
functional group) to which the marker is exposed, and to a
plurality of different materials ("multiplex adsorbent") to which
the marker is exposed. The adsorbent materials in a multiplex
adsorbent are referred to as "adsorbent species." For example, an
addressable location on a probe substrate can comprise a multiplex
adsorbent characterized by many different adsorbent species (e.g.,
anion exchange materials, metal chelators, or antibodies), having
different binding characteristics. Substrate material itself can
also contribute to adsorbing a marker and may be considered part of
an "adsorbent."
[0102] "Adsorption" or "retention" refers to the detectable binding
between an absorbent and a marker either before or after washing
with an eluant (selectivity threshold modifier) or a washing
solution.
[0103] "Eluant" or "washing solution" refers to an agent that can
be used to mediate adsorption of a marker to an adsorbent. Eluants
and washing solutions are also referred to as "selectivity
threshold modifiers." Eluants and washing solutions can be used to
wash and remove unbound materials from the probe substrate
surface.
[0104] "Resolve," "resolution," or "resolution of marker" refers to
the detection of at least one marker in a sample. Resolution
includes the detection of a plurality of markers in a sample by
separation and subsequent differential detection. Resolution does
not require the complete separation of one or more markers from all
other biomolecules in a mixture. Rather, any separation that allows
the distinction between at least one marker and other biomolecules
suffices.
[0105] "Gas phase ion spectrometer" refers to an apparatus that
measures a parameter which can be translated into mass-to-charge
ratios of ions formed when a sample is volatilized and ionized.
Generally ions of interest bear a single charge, and mass-to-charge
ratios are often simply referred to as mass. Gas phase ion
spectrometers include, for example, mass spectrometers, ion
mobility spectrometers, and total ion current measuring
devices.
[0106] "Mass spectrometer" refers to a gas phase ion spectrometer
that includes an inlet system, an ionization source, an ion optic
assembly, a mass analyzer, and a detector.
[0107] "Laser desorption mass spectrometer" refers to a mass
spectrometer which uses laser as means to desorb, volatilize, and
ionize an analyte.
[0108] "Detect" refers to identifying the presence, absence or
amount of the object to be detected.
[0109] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. Polypeptides can be modified, e.g., by the
addition of carbohydrate residues to form glycoproteins. The terms
"polypeptide," "peptide" and "protein" include glycoproteins, as
well as non-glycoproteins.
[0110] "Detectable moiety" or a "label" refers to a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means. For example, useful labels
include .sup.32P, .sup.35S, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA),
biotin-streptavidin, dioxigenin, haptens and proteins for which
antisera or monoclonal antibodies are available, or nucleic acid
molecules with a sequence complementary to a target. The detectable
moiety often generates a measurable signal, such as a radioactive,
chromogenic, or fluorescent signal, that can be used to quantify
the amount of bound detectable moiety in a sample. Quantitation of
the signal is achieved by, e.g., scintillation counting,
densitometry, or flow cytometry.
[0111] "Antibody" refers to a polypeptide ligand substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof, which specifically binds and recognizes an
epitope (e.g., an antigen). The recognized immunoglobulin genes
include the kappa and lambda light chain constant region genes, the
alpha, gamma, delta, epsilon and mu heavy chain constant region
genes, and the myriad immunoglobulin variable region genes.
Antibodies exist, e.g., as intact immunoglobulins or as a number of
well characterized fragments produced by digestion with various
peptidases. This includes, e.g., Fab' and F(ab)'.sub.2 fragments.
The term "antibody," as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA methodologies.
It also includes polyclonal antibodies, monoclonal antibodies,
chimeric antibodies, humanized antibodies, or single chain
antibodies. "Fc" portion of an antibody refers to that portion of
an immunoglobulin heavy chain that comprises one or more heavy
chain constant region domains, CH.sub.1, CH.sub.2 and CH.sub.3, but
does not include the heavy chain variable region.
[0112] "Immunoassay" is an assay that uses an antibody to
specifically bind an antigen (e.g., a marker). The immunoassay is
characterized by the use of specific binding properties of a
particular antibody to isolate, target, and/or quantify the
antigen.
[0113] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind to a particular protein at least two times the background and
do not substantially bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, polyclonal
antibodies raised to marker NF-200 from specific species such as
rat, mouse, or human can be selected to obtain only those
polyclonal antibodies that are specifically immunoreactive with
marker NF-200 and not with other proteins, except for polymorphic
variants and alleles of marker NF-200. This selection may be
achieved by subtracting out antibodies that cross-react with marker
NF-200 molecules from other species. A variety of immunoassay
formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies
specifically immunoreactive with a protein (see, e.g., Harlow &
Lane, Antibodies, A Laboratory Manual (1988), for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity). Typically a specific or selective
reaction will be at least twice background signal or noise and more
typically more than 10 to 100 times background.
[0114] "Energy absorbing molecule" or "EAM" refers to a molecule
that absorbs energy from an ionization source in a mass
spectrometer thereby aiding desorption of analyte, such as a
marker, from a probe surface. Depending on the size and nature of
the analyte, the energy absorbing molecule can be optionally used.
Energy absorbing molecules used in MALDI are frequently referred to
as "matrix." Cinnamic acid derivatives, sinapinic acid ("SPA"),
cyano hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid are
frequently used as energy absorbing molecules in laser desorption
of bioorganic molecules.
[0115] "Sample" is used herein in its broadest sense. A sample
comprising polynucleotides, polypeptides, peptides, antibodies and
the like may comprise a bodily fluid; a soluble fraction of a cell
preparation, or media in which cells were grown; a chromosome, an
organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound
to a substrate; a cell; a tissue; a tissue print; a fingerprint,
skin or hair; and the like.
[0116] "Substantially purified" refers to nucleic acid molecules or
proteins that are removed from their natural environment and are
isolated or separated, and are at least about 60% free, preferably
about 75% free, and most preferably about 90% free, from other
components with which they are naturally associated.
[0117] "Substrate" refers to any rigid or semi-rigid support to
which nucleic acid molecules or proteins are bound and includes
membranes, filters, chips, slides, wafers, fibers, magnetic or
nonmagnetic beads, gels, capillaries or other tubing, plates,
polymers, and microparticles with a variety of surface forms
including wells, trenches, pins, channels and pores.
[0118] As used herein, the term "injury or neural injury" is
intended to include a damage which directly or indirectly affects
the normal functioning of the CNS. For example, the injury can be
damage to retinal ganglion cells; a traumatic brain injury; a
stroke related injury; a cerebral aneurism related injury; a spinal
cord injury, including monoplegia, diplegia, paraplegia, hemiplegia
and quadriplegia; a neuroproliferative disorder or neuropathic pain
syndrome. Examples of CNS injuries or disease include TBI, stroke,
concussion (including post-concussion syndrome), cerebral ischemia,
neurodegenerative diseases of the brain such as Parkinson's
disease, Dementia Pugilistica, Huntington's disease and Alzheimer's
disease, Creutzfeldt-Jakob disease, brain injuries secondary to
seizures which are induced by radiation, exposure to ionizing or
iron plasma, nerve agents, cyanide, toxic concentrations of oxygen,
neurotoxicity due to CNS malaria or treatment with anti-malaria
agents, trypanosomes, malarial pathogens, and other CNS
traumas.
[0119] As used herein, the term "stroke" is art recognized and is
intended to include sudden diminution or loss of consciousness,
sensation, and voluntary motion caused by rapture or obstruction
(e.g. by a blood clot) of an artery of the brain.
[0120] As used herein, the term "Traumatic Brain Injury" is art
recognized and is intended to include the condition in which, a
traumatic blow to the head causes damage to the brain, often
without penetrating the skull. Usually, the initial trauma can
result in expanding hematoma, subarachnoid hemorrhage, cerebral
edema, raised intracranial pressure (ICP), and cerebral hypoxia,
which can, in turn, lead to severe secondary events due to low
cerebral blood flow (CBF).
[0121] "Neural cells" as defined herein, are cells that reside in
the brain, central and peripheral nerve systems, including, but not
limited to, nerve cells, glial cell, oligodendrocyte, microglia
cells or neural stem cells.
[0122] "Neuronal specific or neuronally enriched proteins" are
defined herein, as proteins that are present in neural cells and
not in non-neuronal cells, such as, for example, cardiomyocytes,
myocytes, in skeletal muscles, hepatocytes, kidney cells and cells
in testis. Non-limiting examples of neural proteins are shown in
Table 1 below.
[0123] "Neural (neuronal) defects, disorders or diseases" as used
herein refers to any neurological disorder, including but not
limited to neurodegenerative disorders (Parkinson's; Alzheimer's)
or autoimmune disorders (multiple sclerosis) of the central nervous
system; memory loss; long term and short term memory disorders;
learning disorders; autism, depression, benign forgetfulness,
childhood learning disorders, close head injury, and attention
deficit disorder; autoimmune disorders of the brain, neuronal
reaction to viral infection; brain damage; depression; psychiatric
disorders such as bi-polarism, schizophrenia and the like;
narcolepsy/sleep disorders(including circadian rhythm disorders,
insomnia and narcolepsy); severance of nerves or nerve damage;
severance of the cerebrospinal nerve cord (CNS) and any damage to
brain or nerve cells; neurological deficits associated with AIDS;
tics (e.g. Giles de la Tourette's syndrome); Huntington's chorea,
schizophrenia, traumatic brain injury, tinnitus, neuralgia,
especially trigeminal neuralgia, neuropathic pain, inappropriate
neuronal activity resulting in neurodysthesias in diseases such as
diabetes, MS and motor neurone disease, ataxias, muscular rigidity
(spasticity) and temporomandibular joint dysfunction; Reward
Deficiency Syndrome (RDS) behaviors in a subject.
[0124] As used herein, "RDS" behaviors are those behaviors that
manifests as one or more behavioral disorders related to an
individual's feeling of well-being with anxiety, anger or a craving
for a substance. RDS behaviors include, alcoholism, SUD, smoking,
BMI or obesity, pathological gambling, carbohydrate bingeing, axis
11 diagnosis, SAB, ADD/ADHD, CD, TS, family history of SUD, and
Obesity. All these behaviors, and others described herein as
associated with RDS behaviors or genes involved in the neurological
pathways related to RDS, are included as RDS behaviors as part of
this invention. Additionally, many of the clinical terms used
herein for many specific disorders that are RDS disorders are found
in the Quick Reference to the Diagnostic Criteria From DSM-IV.TM.,
The American Psychiatric Association, Washington, D.C., 1994.
[0125] Affective disorders, including major depression, and the
bipolar, manic-depressive illness, are characterized by changes in
mood as the primary clinical manifestation. Major depression is the
most common of the significant mental illnesses, and it must be
distinguished clinically from periods of normal grief, sadness and
disappointment, and the related dysphoria or demoralization
frequently associated with medical illness. Depression is
characterized by feelings of intense sadness, and despair, mental
slowing and loss of concentration, pessimistic worry, agitation,
and self-deprecation. Physical changes can also occur, including
insomnia, anorexia, and weight loss, decreased energy and libido,
and disruption of hormonal circadian rhythms.
[0126] Mania, as well as depression, is characterized by changes in
mood as the primary symptom. Either of these two extremes of mood
may be accompanied by psychosis with disordered thought and
delusional perceptions. Psychosis may have, as a secondary symptom,
a change in mood, and it is this overlap with depression that
causes much confusion in diagnosis. Severe mood changes without
psychosis frequently occur in depression and are often accompanied
by anxiety.
[0127] Parkinson's disease, independent of a specific etiology, is
a chronic, progressive central nervous system disorder which
usually appears insidiously in the latter decades of life. The
disease produces a slowly increasing disability in purposeful
movement. It is characterized by four major clinical features of
tremor, bradykinesia, rigidity and a disturbance of posture. Often
patients have an accompanying dementia. In idiopathic Parkinsonism,
there is usually a loss of cells in the substantia nigra, locus
ceruleus, and other pigmented neurons of the brain, and a decrease
of dopamine content in nerve axon terminals of cells projecting
from the substantia nigra. The understanding that Parkinsonism is a
syndrome of dopamine deficiency and the discovery of levodopa as an
important drug for the treatment of the disease were the logical
culmination of a series of related basic and clinical observations,
which serves as the rationale for drug treatment.
[0128] As used herein, the term "schizophrenia" refers to a
psychiatric disorder that includes at least two of the following:
delusions, hallucinations, disorganized speech, grossly
disorganized or catatonic behavior, or negative symptoms. (APA,
1994, Diagnostic and Statistical Manual of Mental Disorders (Fourth
Edition), Washington, D.C.).
[0129] The term "Alzheimer's Disease" refers to a progressive
mental deterioration manifested by memory loss, confusion and
disorientation beginning in late middle life and typically
resulting in death in five to ten years. Pathologically,
Alzheimer's Disease can be characterized by thickening,
conglutination, and distortion of the intracellular neurofibrils,
neurofibrillary tangles and senile plaques composed of granular or
filamentous argentophilic masses with an amyloid core. Methods for
diagnosing Alzheimer's Disease are known in the art. For example,
the National Institute of Neurological and Communicative Disorders
and Stroke-Alzheimer's Disease and the Alzheimer's Disease and
Related Disorders Association (NINCDS-ADRDA) criteria can be used
to diagnose Alzheimer's Disease (McKhann et al., 1984, Neurology
34:939-944). The patient's cognitive function can be assessed by
the Alzheimer's Disease Assessment Scale-cognitive subscale
(ADAS-cog; Rosen et al., 1984, Am. J. Psychiatry
141:1356-1364).
[0130] As used herein, the term "autism" refers to a state of
mental introversion characterized by morbid self-absorption, social
failure, language delay, and stereotyped behavior.
[0131] As used herein, the term "depression" refers to a clinical
syndrome that includes a persistent sad mood or loss of interest in
activities, which lasts for at least two weeks in the absence of
treatment.
[0132] The term "benign forgetfulness," as used herein, refers to a
mild tendency to be unable to retrieve or recall information that
was once registered, learned, and stored in memory (e.g., an
inability to remember where one placed one's keys or parked one's
car). Benign forgetfulness typically affects individuals after 40
years of age and can be recognized by standard assessment
instruments such as the Wechsler Memory Scale (Russell, 1975, J.
Consult Clin. Psychol. 43:800-809).
[0133] As used herein, the term "childhood learning disorders"
refers to an impaired ability to learn, as experienced by certain
children.
[0134] The term "close head injury," as used herein, refers to a
clinical condition after head injury or trauma which condition can
be characterized by cognitive and memory impairment. Such a
condition can be diagnosed as "amnestic disorder due to a general
medical condition" according to DSM-IV.
[0135] The term "attention deficit disorder," as used herein,
refers to a disorder that is most commonly exhibited by children
and which can be characterized by increased motor activity and a
decreased attention span. Attention-deficit disorder ("ADD") is a
common behavioral learning disorder in children which adversely
affects school performance and family relationships. Symptoms and
signs include hyperactivity (e.g., ADDH and AD/HD, DSM-IV),
impulsivity, emotional lability, motor incoordination and some
perceptual difficulties. Treatment has included psychostimulants,
which while effective are controversial, and may cause troubling
side effects such as dysphoria, headache and growth retardation.
Other drugs, including the tricyclic antidepressants, appear to
improve attention, but may be less effective than the
psychostimulants.
[0136] As used herein, "subcellular localization" refers to defined
subcellular structures within a single nerve cell. These
subcellularly defined structures are matched with unique neural
proteins derived from, for example, dendritic, axonal, myelin
sheath, presynaptic terminal and postsynaptic locations as
illustrated in FIG. 2. By monitoring the release of proteins unique
to each of these regions, one can therefore monitor and define
subcellular damage after brain injury. Furthermore, mature neurons
are differentiated into dedicated subtype fusing a primary neural
transmitter such as cholinergic (nicotinic and mucarinic),
glutamatergic, gabaergic, serotonergic, dopaminergic. Each of this
neuronal subtype express unique neural proteins such as those
dedicated for the synthesis, metabolism and transporter and
receptor of each unique neurotransmitter system (Table 1).
[0137] As used herein, a "pharmaceutically acceptable" component is
one that is suitable for use with humans and/or animals without
undue adverse side effects (such as toxicity, irritation, and
allergic response) commensurate with a reasonable benefit/risk
ratio.
[0138] The terms "patient" or "individual" are used interchangeably
herein, and is meant a mammalian subject to be treated, with human
patients being preferred. In some cases, the methods of the
invention find use 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.
[0139] As used herein, "ameliorated" or "treatment" refers to a
symptom which is approaches a normalized value, e.g., is less than
50% different from a normalized value, preferably is less than
about 25% different from a normalized value, more preferably, is
less than 10% different from a normalized value, and still more
preferably, is not significantly different from a normalized value
as determined using routine statistical tests. For example,
amelioration or treatment of depression includes, for example,
relief from the symptoms of depression which include, but are not
limited to changes in mood, feelings of intense sadness and
despair, mental slowing, loss of concentration, pessimistic worry,
agitation, and self-deprecation. Physical changes may also be
relieved, including insomnia, anorexia and weight loss, decreased
energy and libido, and the return of normal hormonal circadian
rhythms. Another example, when using the terms "treating
Parkinson's disease" or "ameliorating" as used herein means relief
from the symptoms of Parkinson's disease which include, but are not
limited to tremor, bradykinesia, rigidity, and a disturbance of
posture.
Protein Biomarkers
[0140] In a preferred embodiment, detection of one or more neural
biomarkers is diagnostic of neural damage and/or neuronal disease.
Examples of neural biomarkers, include but are not limited to:
neural proteins, such as for example, axonal proteins-NF-200
(NF-H), NF-160 (NF-M), NF-68 (NF-L); amyloid precursor protein;
dendritic proteins-alpha-tubulin (P02551), beta-tubulin (PO 4691),
MAP-2A/B, MAP-2C, Tau, Dynamin-1 (P21575), Dynactin (Q13561), P24;
somal proteins-UCH-L1 (Q00981), PEBP (P31044), NSE (P07323), Thy
1.1, Prion, Huntington; presynaptic proteins-synapsin-1,
synapsin-2, alpha-synuclein (p37377), beta-synuclein (Q63754),
GAP43, synaptophysin, synaptotagmin (P21707), syntaxin;
post-synaptic proteins-PSD95, PSD93, NMDA-receptor (including all
subtypes); demyelination biomarkers-myelin basic protein (MBP),
myelin proteolipid protein; glial proteins-GFAP (P47819), protein
disulfide isomerase (PDI-P04785); neurotransmitter
biomarkers-cholinergic biomarkers: acetylcholine esterase, choline
acetyltransferase; dopaminergic biomarkers-tyrosine hydroxylase
(TH), phospho-TH, DARPP32; noradrenergic biomarkers-dopamine
beta-hydroxylase (DbH); serotonergic biomarkers-tryptophan
hydroxylase (TrH); glutamatergic biomarkers-glutaminase, glutamine
synthetase; GABAergic biomarkers-GABA transaminase
(4-aminobutyrate-2-ketoglutarate transaminase [GABAT]), glutamic
acid decarboxylase (GAD25, 44, 65, 67); neurotransmitter
receptors-beta-adrenoreceptor subtypes, (e.g. beta (2)),
alpha-adrenoreceptor subtypes, (e.g. (alpha (2c)), GABA receptors
(e.g. GABA(B)), metabotropic glutamate receptor (e.g. mGluR3), NMDA
receptor subunits (e.g. NR1A2B), Glutamate receptor subunits (e.g.
GluR4), 5-HT serotonin receptors (e.g. 5-HT(3)), dopamine receptors
(e.g. D4), muscarinic Ach receptors (e.g. M1), nicotinic
acetylcholine receptor (e.g. alpha-7); neurotransmitter
transporters-norepinephrine transporter (NET), dopamine transporter
(DAT), serotonin transporter (SERT), vesicular transporter proteins
(VMAT1 and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), glutamate transporter (e.g. GLT1),
vesicular acetylcholine transporter, choline transporter (e.g.
CHT1); other protein biomarkers include, but not limited to
vimentin (P31000), CK-BB (P07335), 14-3-3-epsilon (P42655), MMP2,
MMP9.
[0141] In another preferred embodiment, a composition or panel of
biomarkers comprises: Axonal Proteins: .alpha. II spectrin (and
SPDB)-1, NF-68 (NF-L)-2, Tau-3, .alpha. II, III spectrin, NF-200
(NF-H), NF-160 (NF-M), Amyloid precursor protein, .alpha.
internexin; Dendritic Proteins: beta III-tubulin-1, p24
microtubule-associated protein-2, alpha-Tubulin (P02551),
beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4, Dynamin-1
(P21575), Phocein, Dynactin (Q13561), Vimentin (P31000), Dynamin,
Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB (P07335),
Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0142] In another preferred embodiment, the panel of biomarkers
comprise at least one biomarker from each neural cell type. The
composition of biomarkers is diagnostic of neural injury, damage
and/or neural disorders. The composition comprises: .alpha. II
spectrin, SPDB-1, NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0143] Without wishing to be bound by theory, upon injury,
structural and functional integrity of the cell membrane and blood
brain barrier are compromised. Brain-specific and brain-enriched
proteins are released into the extracellular space and subsequently
into the CSF and blood. This is shown in a schematic illustration
in FIG. 1.
[0144] In a preferred embodiment, detection of at least one neural
protein in CSF, blood, or other biological fluids, is diagnostic of
the severity of brain injury and/or the monitoring of the
progression of therapy. Preferably, the neural proteins are
detected during the early stages of injury. An increase in the
amount of neural proteins, fragments or derivatives thereof, in a
patient suffering from a neural injury, neuronal disorder as
compared to a normal healthy individual, will be diagnostic of a
neural injury and/or neuronal disorder.
[0145] In another preferred embodiment, detection of at least one
neural protein in CSF, blood, or other biological fluids, is
diagnostic of the severity of injury following a variety of CNS
insults, such as for example, stroke, spinal cord injury, or
neurotoxicity caused by alcohol or substance abuse (e.g. ecstacy,
methamphetamine, etc.)
[0146] In a preferred embodiment, biomarkers of brain injury,
neural injury and/or neural disorders comprises proteins from the
neural system (CNS and PNS). The CNS comprises many brain-specific
and brain-enriched proteins that are preferable biomarkers in the
diagnosis of brain injury, neural injury, neural disorders and the
like. Non-limiting examples are shown in Table 1 and FIG. 2. For
example, the neural specific biomarkers can include Axonal
Proteins: .alpha. II spectrin (and SPDB)-1, NF-68 (NF-L)-2, Tau-3,
.alpha. II, III spectrin, NF-200 (NF-H), NF-160 (NF-M), Amyloid
precursor protein, .alpha. internexin; Dendritic Proteins: beta
III-tubulin-1, p24 microtubule-associated protein-2, alpha-Tubulin
(P02551), beta-Tubulin (P04691), MAP-2A/B-3, MAP-2C-3, Stathmin-4,
Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000),
Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)--P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2. Furthermore, proteins such as
GFAP and protein disulfide isomerase (PDI) are only synthesized in
glial cells of the CNS, a feature that is used to further detect
and diagnose the extent of damage to the CNS.
[0147] In another preferred embodiment, the invention provides for
the quantitative detection of damage to the CNS, PNS and/or brain
injury at a subcellular level. Depending on the type and severity
of injury, neurons can undergo damage in specific cellular regions.
For example, detection of certain biomarkers, such as for example,
axonal proteins, fragments and derivatives thereof include, but not
limited to: NF-200 (NF-H), NF-160 (NF-M), NF-68 (NF-L), and the
like, differentiates between axonal versus dendritic damage.
Non-limiting examples of dendritic proteins, peptides, fragments
and derivatives thereof, include, but not limited to: alpha-tubulin
(P02551), beta-tubulin (PO 4691), MAP-2A/B, MAP-2C, Tau, Dynamin-1
(P21575), Dynactin (Q13561), p24 (neural-specific MAP).
Furthermore, detection of different biomarkers not only
differentiate between, for example, axonal or dendritic damage, but
allow for the assessment of synaptic pathology, specific injury to
elements of the pre-synaptic terminal and post-synaptic density.
See table 1 for examples of biomarkers from each cellular,
sub-cellular and anatomical locations, detection of which detects
the location of injury.
[0148] In a preferred embodiment, biomarkers indicative of neural
injury in different anatomical in vivo locations include but not
limited to: Hippocampus: Stathmin, Hippocalcin, SCG10; Cerebellum:
Purkinje cell protein-2 (Pcp2), Calbindin D9K, Calbindin D28K
(NP_114190), Cerebellar CaBP, spot 35; Cerebrocortex: Cortexin-1
(P60606), H-2Z1 gene product; Thalamus: CD15
(3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin. For
example, to determine injury in a certain anatomical location,
detection of Stathmin and/or Hippocalcin and/or SCG10 is diagnostic
of injury in the Hippocampus. Detection of Purkinje cell protein-2
(Pcp2) and/or Calbindin D9K and/or Calbindin D28K (NP_114190)
and/or Cerebellar CaBP, spot 35 is diagnostic of injury in the
cerebellum. Detection of a combination of biomarkers, such as
Stathmin and/or Hippocalcin and/or SCG10 Purkinje cell protein-2
(Pcp2) and/or Calbindin D9K and/or Calbindin D28K (NP_114190)
and/or Cerebellar CaBP, spot 35 is diagnostic of injury in the
Hippocampus and cerebellum. Therefore, detection of one or more or
combinations of biomarkers is diagnostic of the location of neural
injury.
[0149] In another preferred embodiment, the amount of marker
detected, for example, in .mu.g/ml is diagnostic of the extent of
damage or injury. Quantitation of each biomarker is described in
the specification and in the Examples to follow. Assays include
immunoassays (such as ELISA's), spectrophotometry, HPLC, SELDI,
biochips and the like. Therefore, if for example, 10 .mu.g/ml of
stathmin and 0.001 .mu.g/ml of CaBP is diagnostic that the main
injury is to the Hippocampus with some injury to the cerebellum.
Detection of biomarkers from subcellular locations is diagnostic of
which cells are injured. For example, detection of axonal
biomarkers vs. dendritic biomarkers vs. microglial biomarkers is
diagnostic of the type of cells injured. As discussed, infra, the
quantitation of each as compared to a normal individual is
diagnostic of the extent of injury.
[0150] In another preferred embodiment, detection of certain
biomarkers are diagnostic of the specific cell type affected
following injury since neurons and glia possess distinct proteins.
For example, detection of glial proteins, peptides, fragments and
derivatives thereof is diagnostic of glial cell damage. Examples of
glial proteins, include, but not limited to: GFAP (P47819), Protein
disulfide isomerase (PDI)--P04785, Neurocalcin delta, S100beta.
[0151] The ability to detect and monitor levels of these proteins
after CNS injury provides enhanced diagnostic capability by
allowing clinicians (1) to determine the level of injury severity
in patients with various CNS injuries, (2) to monitor patients for
signs of secondary CNS injuries that may elicit these cellular
changes and (3) to monitor the effects of therapy by examination of
these proteins in CSF or blood. Unlike other organ-based diseases
where rapid diagnostics for surrogate biomarkers prove invaluable
to the course of action taken to treat the disease, no such rapid,
definitive diagnostic tests exist for traumatic or ischemic brain
injury that might provide physicians with quantifiable
neurochemical markers to help determine the seriousness of the
injury, the anatomical and cellular pathology of the injury, and
the implementation of appropriate medical management and
treatment.
[0152] In an illustrative example, not meant to limit or construe
the invention in any way, identification of which brain-specific
and brain-enriched proteins are elevated in CSF following traumatic
brain injury (TBI) is diagnostic, for example, of brain injury, the
degree of brain injury, type of cellular damage and degree of
cellular damage. Furthermore, detection of certain brain-specific
and brain-enriched proteins, fragments and derivatives thereof, is
diagnostic of the type and degree of cellular damage. For example,
increased levels of a variety of brain-specific and brain-enriched
proteins in the CSF 48 hours following injury, were detected.
Specifically, elevated levels of the somal protein ubiquitin
C-terminal hydrolase L1 (UCH-L1) the dendritic protein p24, and
.alpha.-synuclein, a pre-synaptic protein were detected following
injury.
[0153] In comparison to currently existing products, the invention
provides several superior advantages and benefits. First, the
identification of neuronal biomarkers provide more rapid and less
expensive diagnosis of injury severity than existing diagnostic
devices such as computed tomography (CT) and magnetic resonance
imaging (MRI). The invention also allows quantitative detection and
high content assessment of damage to the CNS at a subcellular level
(i.e. axonal versus dendritic). The invention also allows
identification of the specific cell type affected (for example,
neurons versus glia). In addition, levels of these brain-specific
and brain-enriched proteins provides more accurate information
regarding the level of injury severity than what is on the
market.
[0154] In another preferred embodiment, nerve cell damage in a
subject is analyzed by (a) providing a biological sample isolated
from a subject suspected of having a damaged nerve cell; (b)
detecting in the sample the presence or amount of at least one
marker selected from one or more neural proteins; and (c)
correlating the presence or amount of the marker with the presence
or type of nerve cell damage in the subject. Preferably, neural
cells, such as those cells that reside in the central and
peripheral nerve systems, including nerve cells, glial cell,
oligodendrocyte, microglia cells or neural stem cells) in in vitro
culture or in situ in an animal subjects express higher levels of
neural proteins ("neuronal specific or neuronally enriched"
proteins; examples are outlined in Table 1) as compared to
non-neuronal cells, such as cardiomyocytes, myocytes in skeletal
muscles, hepatocytes, kidney cells and cells in testis. Preferably,
the samples comprise neural cells, for example, a biopsy of a
central nervous system or peripheral nervous system tissue are
suitable biological samples for use in the invention. In addition,
after injury to the nervous system (such as brain injury), the
neural cell membrane is compromised, leading to the efflux of these
neural proteins first into the extracellular fluid or space and to
the cerebrospinal fluid and eventually in the circulating blood (as
assisted by the compromised blood brain barrier) and other
biofluids (e.g. urine, sweat, s saliva, etc.). Thus, other suitable
biological samples include, but not limited to such cells or fluid
secreted from these cells. Obtaining biological fluids such as
cerebrospinal fluid, blood, plasma, serum, saliva and urine, from a
subject is typically much less invasive and traumatizing than
obtaining a solid tissue biopsy sample. Thus, samples, which are
biological fluids, are preferred for use in the invention. CSF, in
particular, is preferred for detecting nerve damage in a subject as
it is in immediate contact with the nervous system and is readily
obtainable.
[0155] In a preferred embodiment, detection of nerve cell damage
comprises detection of one or more biomarkers comprising: Axonal
Proteins: .alpha. II spectrin (and SPDB)-1, NF-68 (NF-L)-2, Tau-3,
.alpha. II, III spectrin, NF-200 (NF-H), NF-160 (NF-M), Amyloid
precursor protein, .alpha. internexin; Dendritic Proteins: beta
III-tubulin-1, p24 microtubule-associated protein-2, alpha-Tubulin
(P02551), beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4,
Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000),
Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43, B as
soon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1, 2,
Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477); Post-Synaptic
Proteins: PSD95-1, NMDA-receptor (and all subtypes)-2, PSD93,
AMPA-kainate receptor (all subtypes), mGluR (all subtypes),
Calmodulin dependent protein kinase II (CAMPK)-alpha, beta, gamma,
CaMPK-IV, SNAP-25, a-/b-SNAP; Myelin-Oligodendrocyte: Myelin basic
protein (MBP) and fragments, Myelin proteolipid protein (PLP),
Myelin Oligodendrocyte specific protein (MOSP), Myelin
Oligodendrocyte glycoprotein (MOG), myelin associated protein
(MAG), Oligodendrocyte NS-1 protein; Glial Protein Biomarkers: GFAP
(P47819), Protein disulfide isomerase (PDI)-P04785, Neurocalcin
delta, S100beta; Microglia protein Biomarkers: Iba1, OX-42, OX-8,
OX-6, ED-1, PTPase (CD45), CD40, CD68, CD11b, Fractalkine (CX3CL1)
and Fractalkine receptor (CX3CR1), 5-d-4 antigen; Schwann cell
markers: Schwann cell myelin protein; Glia Scar: Tenascin;
Hippocampus: Stathmin, Hippocalcin, SCG10; Cerebellum: Purkinje
cell protein-2 (Pcp2), Calbindin D9K, Calbindin D28K (NP_114190),
Cerebellar CaBP, spot 35; Cerebrocortex: Cortexin-1 (P60606), H-2Z1
gene product; Thalamus: CD15 (3-fucosyl-N-acetyl-lactosamine)
epitope; Hypothalamus: Orexin receptors (OX-1R and OX-2R)-appetite,
Orexins (hypothalamus-specific peptides); Corpus callosum: MBP,
MOG, PLP, MAG; Spinal Cord: Schwann cell myelin protein; Striatum:
Striatin, Rhes (Ras homolog enriched in striatum); Peripheral
ganglia: Gadd45a; Peripherial nerve fiber(sensory+motor):
Peripherin, Peripheral myelin protein 22 (AAH91499); Other
Neuron-specific proteins: PH8 (S Serotonergic Dopaminergic, PEP-19,
Neurocalcin (NC), a neuron-specific EF-hand Ca.sup.2+-binding
protein, Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin,
Visinin; Neurotransmitter Receptors: NMDA receptor subunits (e.g.
NR1A2B), Glutamate receptor subunits (AMPA, Kainate receptors (e.g.
GluR1, GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0156] In another preferred embodiment, detection of neural damage
comprises detection of one or more biomarkers comprising at least
one biomarker from each neural cell type. The composition of
biomarkers is diagnostic of neural injury, damage and/or neural
disorders. The composition or panel of biomarkers comprises:
.alpha. II spectrin, SPDB-1, NF-68, NF-L-2, Tau-3,
.beta.III-tubulin-1, p24 microtubule-associated protein-2, UCH-L1
(Q00981)-1, Glycogen phosphorylase-BB-2, NeuN-1, Synaptophysin-1,
synaptotagmin (P21707), Synaptojanin-1 (Q62910), Synaptojanin-2,
PSD95-1, NMDA-receptor-2 and subtypes, myelin basic protein (MBP)
and fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1,
Schwann cell myelin protein, tenascin, stathmin, Purkinje cell
protein-2 (Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R,
OX-2R), Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0157] A biological sample can be obtained from a subject by
conventional techniques. For example, CSF can be obtained by lumbar
puncture. Blood can be obtained by venipuncture, while plasma and
serum can be obtained by fractionating whole blood according to
known methods. Surgical techniques for obtaining solid tissue
samples are well known in the art. For example, methods for
obtaining a nervous system tissue sample are described in standard
neurosurgery texts such as Atlas of Neurosurgery: Basic Approaches
to Cranial and Vascular Procedures, by F. Meyer, Churchill
Livingstone, 1999; Stereotactic and Image Directed Surgery of Brain
Tumors, 1st ed., by David G. T. Thomas, W B Saunders Co., 1993; and
Cranial Microsurgery: Approaches and Techniques, by L. N. Sekhar
and E. De Oliveira, 1st ed., Thieme Medical Publishing, 1999.
Methods for obtaining and analyzing brain tissue are also described
in Belay et al., Arch. Neurol. 58: 1673-1678 (2001); and Seijo et
al., J. Clin. Microbiol. 38: 3892-3895 (2000).
[0158] Any animal that expresses the neural proteins, such as for
example, those listed in Table 1, can be used as a subject from
which a biological sample is obtained. Preferably, the subject is a
mammal, such as for example, a human, dog, cat, horse, cow, pig,
sheep, goat, primate, rat, mouse and other vertebrates such as
fish, birds and reptiles. More preferably, the subject is a human.
Particularly preferred are subjects suspected of having or at risk
for developing traumatic or non-traumatic nervous system injuries,
such as victims of brain injury caused by traumatic insults (e.g.
gunshots wounds, automobile accidents, sports accidents, shaken
baby syndrome), ischemic events (e.g. stroke, cerebral hemorrhage,
cardiac arrest), spinal cord injury, neurodegenerative disorders
(such as Alzheimer's, Huntington's, and Parkinson's diseases;
Prion-related disease; other forms of dementia, and spinal cord
degeneration), epilepsy, substance abuse (e.g., from amphetamines,
methamphetamine/Speed, Ecstasy/MDMA, or ethanol and cocaine), and
peripheral nervous system pathologies such as diabetic neuropathy,
chemotherapy-induced neuropathy and neuropathic pain, peripheral
nerve damage or atrophy (ALS), multiple sclerosis (MS).
TABLE-US-00001 Subcellular neuronal markers Axonal Proteins .alpha.
II spectrin (and SPDB) -1 NF-68 (NF-L) - 2 Tau - 3 .beta. II, III
spectrin NF-200 (NF-H) NF-160 (NF-M) Amyloid precursor protein
.alpha. internexin Dendritic Proteins beta III-tubulin - 1 p24
microtubule-associated protein - 2 alpha-Tubulin (P02551)
beta-Tubulin (P04691) MAP-2A/B - 3 MAP-2C -3 Stathmin - 4 Dynamin-1
(P21575) Phocein Dynactin (Q13561) Vimentin (P31000) Dynamin
Profilin Cofilin 1, 2 Somal Proteins UCH-L1 (Q00981) - 1 Gyocogen
phosphorylase-BB - 2 PEBP (P31044) NSE (P07323) CK-BB (P07335) Thy
1.1 Prion protein Huntingtin 14-3-3 proteins (e.g. 14-3-3-epsolon
(P42655)) SM22-.alpha. Calgranulin AB alpha-Synuclein (P37377)
beta-Synuclein (Q63754) HNP 22 Neural nuclear proteins NeuN - 1
S/G(2) nuclear autoantigen (SG2NA) Huntingtin Presynaptic Proteins
Synaptophysin - 1 Synaptotagmin (P21707) Synaptojanin-1 (Q62910)
Synaptojanin-2 Synapsin1 (Synapsin-Ia) Synapsin2 (Q63537) Synapsin3
GAP43 Bassoon(NP_003449) Piccolo (aczonin) (NP_149015) Syntaxin
CRMP1, 2 Amphiphysin -1 (NP_001626) Amphiphysin -2 (NP_647477)
Post-Synaptic Proteins PSD95 - 1 NMDA-receptor (and all subtypes)
-2 PSD93 AMPA-kainate receptor (all subtypes) mGluR (all subtypes)
Calmodulin dependent protein kinase II (CAMPK)-alpha, beta, gamma
CaMPK-IV SNAP-25 a-/b-SNAP Nervous Cell subtype Biomarkers
Myelin-Oligodendrocyte Myelin basic protein (MBP) and fragments
Myelin proteolipid protein (PLP) Myelin Oligodendrocyte specific
protein (MOSP) Myelin Oligodendrocyte glycoprotein (MOG) myelin
associated protein (MAG) Oligodendrocyte NS-1 protein Glial Protein
Biomarkers GFAP (P47819) Protein disulfide isomerase (PDI) - P04785
Neurocalcin delta S100beta Microglia protein Biomarkers Iba1 OX-42
OX-8 OX-6 ED-1 PTPase (CD45) CD40; CD68 CD11b Fractalkine (CX3CL1)
and Fractalkine receptor (CX3CR1) 5-d-4 antigen Schwann cell
markers Schwann cell myelin protein Glia Scar Tenascin Anatomical
brain biomarkers (CNS + PNS) Hippocampus Stathmin, Hippocalcin
SCG10 Cerebellum Purkinje cell protein-2 (Pcp2) Calbindin D9K,
Calbindin D28K (NP_114190) Cerebellar CaBP, spot 35 Cerebrocortex
Cortexin-1. P60606 H-2Z1 gene product Thalamus CD15
(3-fucosyl-N-acetyl-lactosamine) epitope Hypothalamus Orexin
receptors (OX-1R and OX-2R)- appetite Orexins
(hypothalamus-specific peptides) Corpus callosum MBP, MOG, PLP MAG
Spinal Cord Schwann cell myelin protein Striatum Striatin Rhes (Ras
homolog enriched in striatum) Peripheral ganglia Gadd45a
Peripherial nerve fiber(sensory + motor) Peripherin Peripheral
myelin protein 22 (AAH91499) Other Neuron-specific proteins PH8 (S
Serotonergic Dopaminergic PEP-19, a neuron-specific protein
Neurocalcin (NC), a neuron-specific EF-hand Ca2+-binding protein
Encephalopsin Striatin SG2NA Zinedin, Recoverin Visinin Neuron
Subtypes based on Neurotransmitter receptors and transporters
Neurotransmitter Receptors NMDA receptor subunits (e.g. NR1A2B)
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4) beta-adrenoceptor subtypes (e.g. beta(2))
Alpha-adrenoceptors subtypes (e.g. alpha(2c)) GABA receptors (e.g.
GABA(B)) Metabotropic glutamate receptor (e.g. mGluR3) 5-HT
serotonin receptors (e.g. 5-HT(3)) Dopamine receptors (e.g. D4)
Muscarinic Ach receptors (e.g. M1) Nicotinic Acetylcholine Receptor
(e.g. alpha-7) Neurotransmitter Transporters Norepinephrine
Transporter (NET) Dopamine transporter (DAT) Serotonin transporter
(SERT) Vesicular transporter proteins (VMAT1 and VMAT2) GABA
transporter vesicular inhibitory amino acid transporter
(VIAAT/VGAT) Glutamate Transporter (e.g. GLT1) Vesicular
acetylcholine transporter Vesicular Glutamate Transporter 1
[VGLUT1; BNPI] and VGLUT2 Choline transporter, (e.g. CHT1) Neuron
Subtypes based on Neurotransmitter system Cholinergic Biomarkers
Acetylcholine Esterase Choline acetyltransferase [ChAT]
Dopaminergic Biomarkers Tyrosine Hydroxylase (TH) Phospho-TH
DARPP32 Noradrenergic Biomarkers Dopamine beta-hydroxylase (DbH)
Adrenergic Biomarkers Phenylethanolamine N-methyltransferase (PNMT)
Serotonergic Biomarkers Tryptophan Hydroxylase (TrH) Glutamatergic
Biomarkers Glutaminase Glutamine synthetase GABAergic Biomarkers
GABA transaminase [GABAT]) GABA-B-R2
[0159] As described above, the invention provides the step of
correlating the presence or amount of one or more neural protein(s)
with the severity and/or type of nerve cell injury. The amount of a
neural proteins, peptides, fragments, derivatives or the modified
forms, thereof, directly relates to severity of nerve tissue injury
as more severe injury damages a greater number of nerve cells which
in turn causes a larger amount of neural protein(s) to accumulate
in the biological sample (e.g., CSF). Whether a nerve cell injury
triggers an apoptotic, oncotic (necrotic) or type 2 (autophagic)
cell death, can be determined by examining the unique proteins
released into the biofluid in response to different cell death
phenotype. The unique proteins are detected from the many cell
types that comprise the nervous system. For example, astroglia,
oligodendrocytes, microglia cells, Schwann cells, fibroblast,
neuroblast, neural stem cells and mature neurons. Furthermore,
mature neurons are differentiated into dedicated subtype fusing a
primary neural transmitter such as cholinergic (nicotinic and
mucarinic), glutamatergic, gabaergic, serotonergic, dopaminergic.
Each of this neuronal subtype express unique neural proteins such
as those dedicated for the synthesis, metabolism and transporter
and receptor of each unique neurotransmitter system (Table 1).
Lastly, within a single nerve cell, there are subcellularly defined
structures matched with unique neural proteins (dendritic, axonal,
myelin sheath, presynaptic terminal and postsynaptic density). By
monitoring the release of proteins unique to each of these regions,
subcellular damage can be monitored and defined after brain injury
(FIG. 2).
[0160] The biomarkers of the invention can be detected in a sample
by any means. Methods for detecting the biomarkers are described in
detail in the materials and methods and Examples which follow. For
example, immunoassays, include but are not limited to competitive
and non-competitive assay systems using techniques such as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, fluorescent immunoassays and the like. Such
assays are routine and well known in the art (see, e.g., Ausubel et
al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John
Wiley & Sons, Inc., New York, which is incorporated by
reference herein in its entirety). Exemplary immunoassays are
described briefly below (but are not intended by way of
limitation).
[0161] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding an antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody to immunoprecipitate
a particular antigen can be assessed by, e.g., western blot
analysis. One of skill in the art would be knowledgeable as to the
parameters that can be modified to increase the binding of the
antibody to an antigen and decrease the background (e.g.,
pre-clearing the cell lysate with sepharose beads). For further
discussion regarding immunoprecipitation protocols see, e.g.,
Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,
Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
[0162] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
.sup.32P or .sup.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0163] ELISAs comprise preparing antigen (i.e. neural biomarker),
coating the well of a 96 well microtiter plate with the antigen,
adding the antibody of interest conjugated to a detectable compound
such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline phosphatase) to the well and incubating for a period of
time, and detecting the presence of the antigen. In ELISAs the
antibody of interest does not have to be conjugated to a detectable
compound; instead, a second antibody (which recognizes the antibody
of interest) conjugated to a detectable compound may be added to
the well. Further, instead of coating the well with the antigen,
the antibody may be coated to the well. In this case, a second
antibody conjugated to a detectable compound may be added following
the addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York at 11.2.1.
Identification of New Markers and Quantitation of Markers
[0164] In a preferred embodiment, a biological sample is obtained
from a patient with neural injury. Biological samples comprising
biomarkers from other patients and control subjects (i.e. normal
healthy individuals of similar age, sex, physical condition) are
used as comparisons. Biological samples are extracted as discussed
above. Preferably, the sample is prepared prior to detection of
biomarkers. Typically, preparation involves fractionation of the
sample and collection of fractions determined to contain the
biomarkers. Methods of pre-fractionation include, for example, size
exclusion chromatography, ion exchange chromatography, heparin
chromatography, affinity chromatography, sequential extraction, gel
electrophoresis and liquid chromatography. The analytes also may be
modified prior to detection. These methods are useful to simplify
the sample for further analysis. For example, it can be useful to
remove high abundance proteins, such as albumin, from blood before
analysis.
[0165] In one embodiment, a sample can be pre-fractionated
according to size of proteins in a sample using size exclusion
chromatography. For a biological sample wherein the amount of
sample available is small, preferably a size selection spin column
is used. In general, the first fraction that is eluted from the
column ("fraction 1") has the highest percentage of high molecular
weight proteins; fraction 2 has a lower percentage of high
molecular weight proteins; fraction 3 has even a lower percentage
of high molecular weight proteins; fraction 4 has the lowest amount
of large proteins; and so on. Each fraction can then be analyzed by
immunoassays, gas phase ion spectrometry, and the like, for the
detection of markers.
[0166] In another embodiment, a sample can be pre-fractionated by
anion exchange chromatography. Anion exchange chromatography allows
pre-fractionation of the proteins in a sample roughly according to
their charge characteristics. For example, a Q anion-exchange resin
can be used (e.g., Q HyperD F, Biosepra), and a sample can be
sequentially eluted with eluants having different pH's. Anion
exchange chromatography allows separation of biomarkers in a sample
that are more negatively charged from other types of biomarkers.
Proteins that are eluted with an eluant having a high pH is likely
to be weakly negatively charged, and a fraction that is eluted with
an eluant having a low pH is likely to be strongly negatively
charged. Thus, in addition to reducing complexity of a sample,
anion exchange chromatography separates proteins according to their
binding characteristics.
[0167] In yet another embodiment, a sample can be pre-fractionated
by heparin chromatography. Heparin chromatography allows
pre-fractionation of the markers in a sample also on the basis of
affinity interaction with heparin and charge characteristics.
Heparin, a sulfated mucopolysaccharide, will bind markers with
positively charged moieties and a sample can be sequentially eluted
with eluants having different pH's or salt concentrations. Markers
eluted with an eluant having a low pH are more likely to be weakly
positively charged. Markers eluted with an eluant having a high pH
are more likely to be strongly positively charged. Thus, heparin
chromatography also reduces the complexity of a sample and
separates markers according to their binding characteristics.
[0168] In yet another embodiment, a sample can be pre-fractionated
by isolating proteins that have a specific characteristic, e.g. are
glycosylated. For example, a CSF sample can be fractionated by
passing the sample over a lectin chromatography column (which has a
high affinity for sugars). Glycosylated proteins will bind to the
lectin column and non-glycosylated proteins will pass through the
flow through. Glycosylated proteins are then eluted from the lectin
column with an eluant containing a sugar, e.g.,
N-acetyl-glucosamine and are available for further analysis.
[0169] Thus there are many ways to reduce the complexity of a
sample based on the binding properties of the proteins in the
sample, or the characteristics of the proteins in the sample.
[0170] In yet another embodiment, a sample can be fractionated
using a sequential extraction protocol. In sequential extraction, a
sample is exposed to a series of adsorbents to extract different
types of biomarkers from a sample. For example, a sample is applied
to a first adsorbent to extract certain proteins, and an eluant
containing non-adsorbent proteins (i.e., proteins that did not bind
to the first adsorbent) is collected. Then, the fraction is exposed
to a second adsorbent. This further extracts various proteins from
the fraction. This second fraction is then exposed to a third
adsorbent, and so on.
[0171] Any suitable materials and methods can be used to perform
sequential extraction of a sample. For example, a series of spin
columns comprising different adsorbents can be used. In another
example, a multi-well comprising different adsorbents at its bottom
can be used. In another example, sequential extraction can be
performed on a probe adapted for use in a gas phase ion
spectrometer, wherein the probe surface comprises adsorbents for
binding biomarkers. In this embodiment, the sample is applied to a
first adsorbent on the probe, which is subsequently washed with an
eluant. Markers that do not bind to the first adsorbent are removed
with an eluant. The markers that are in the fraction can be applied
to a second adsorbent on the probe, and so forth. The advantage of
performing sequential extraction on a gas phase ion spectrometer
probe is that markers that bind to various adsorbents at every
stage of the sequential extraction protocol can be analyzed
directly using a gas phase ion spectrometer.
[0172] In yet another embodiment, biomarkers in a sample can be
separated by high-resolution electrophoresis, e.g., one or
two-dimensional gel electrophoresis. A fraction containing a marker
can be isolated and further analyzed by gas phase ion spectrometry.
Preferably, two-dimensional gel electrophoresis is used to generate
two-dimensional array of spots of biomarkers, including one or more
markers. See, e.g., Jungblut and Thiede, Mass Spectr. Rev.
16:145-162 (1997).
[0173] The two-dimensional gel electrophoresis can be performed
using methods known in the art. See, e.g., Deutscher ed., Methods
In Enzymology vol. 182. Typically, biomarkers in a sample are
separated by, e.g., isoelectric focusing, during which biomarkers
in a sample are separated in a pH gradient until they reach a spot
where their net charge is zero (i.e., isoelectric point). This
first separation step results in one-dimensional array of
biomarkers. The biomarkers in one dimensional array is further
separated using a technique generally distinct from that used in
the first separation step. For example, in the second dimension,
biomarkers separated by isoelectric focusing are further separated
using a polyacrylamide gel, such as polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate
(SDS-PAGE). SDS-PAGE gel allows further separation based on
molecular mass of biomarkers. Typically, two-dimensional gel
electrophoresis can separate chemically different biomarkers in the
molecular mass range from 1000-200,000 Da within complex
mixtures.
[0174] Biomarkers in the two-dimensional array can be detected
using any suitable methods known in the art. For example,
biomarkers in a gel can be labeled or stained (e.g., Coomassie Blue
or silver staining). If gel electrophoresis generates spots that
correspond to the molecular weight of one or more markers of the
invention, the spot can be further analyzed by densitometric
analysis or gas phase ion spectrometry. For example, spots can be
excised from the gel and analyzed by gas phase ion spectrometry.
Alternatively, the gel containing biomarkers can be transferred to
an inert membrane by applying an electric field. Then a spot on the
membrane that approximately corresponds to the molecular weight of
a marker can be analyzed by gas phase ion spectrometry. In gas
phase ion spectrometry, the spots can be analyzed using any
suitable techniques, such as MALDI or SELDI.
[0175] Prior to gas phase ion spectrometry analysis, it may be
desirable to cleave biomarkers in the spot into smaller fragments
using cleaving reagents, such as proteases (e.g., trypsin). The
digestion of biomarkers into small fragments provides a mass
fingerprint of the biomarkers in the spot, which can be used to
determine the identity of markers if desired.
[0176] In yet another embodiment, high performance liquid
chromatography (HPLC) can be used to separate a mixture of
biomarkers in a sample based on their different physical
properties, such as polarity, charge and size. HPLC instruments
typically consist of a reservoir of mobile phase, a pump, an
injector, a separation column, and a detector. Biomarkers in a
sample are separated by injecting an aliquot of the sample onto the
column. Different biomarkers in the mixture pass through the column
at different rates due to differences in their partitioning
behavior between the mobile liquid phase and the stationary phase.
A fraction that corresponds to the molecular weight and/or physical
properties of one or more markers can be collected. The fraction
can then be analyzed by gas phase ion spectrometry to detect
markers.
[0177] Optionally, a marker can be modified before analysis to
improve its resolution or to determine its identity. For example,
the markers may be subject to proteolytic digestion before
analysis. Any protease can be used. Proteases, such as trypsin,
that are likely to cleave the markers into a discrete number of
fragments are particularly useful. The fragments that result from
digestion function as a fingerprint for the markers, thereby
enabling their detection indirectly. This is particularly useful
where there are markers with similar molecular masses that might be
confused for the marker in question. Also, proteolytic
fragmentation is useful for high molecular weight markers because
smaller markers are more easily resolved by mass spectrometry. In
another example, biomarkers can be modified to improve detection
resolution. For instance, neuraminidase can be used to remove
terminal sialic acid residues from glycoproteins to improve binding
to an anionic adsorbent and to improve detection resolution. In
another example, the markers can be modified by the attachment of a
tag of particular molecular weight that specifically bind to
molecular markers, further distinguishing them. Optionally, after
detecting such modified markers, the identity of the markers can be
further determined by matching the physical and chemical
characteristics of the modified markers in a protein database
(e.g., SwissProt).
[0178] After preparation, biomarkers in a sample are typically
captured on a substrate for detection. Traditional substrates
include antibody-coated 96-well plates or nitrocellulose membranes
that are subsequently probed for the presence of proteins.
Preferably, the biomarkers are identified using immunoassays as
described above. However, preferred methods also include the use of
biochips. Preferably the biochips are protein biochips for capture
and detection of proteins. Many protein biochips are described in
the art. These include, for example, protein biochips produced by
Packard BioScience Company (Meriden Conn.), Zyomyx (Hayward,
Calif.) and Phylos (Lexington, Mass.). In general, protein biochips
comprise a substrate having a surface. A capture reagent or
adsorbent is attached to the surface of the substrate. Frequently,
the surface comprises a plurality of addressable locations, each of
which location has the capture reagent bound there. The capture
reagent can be a biological molecule, such as a polypeptide or a
nucleic acid, which captures other biomarkers in a specific manner.
Alternatively, the capture reagent can be a chromatographic
material, such as an anion exchange material or a hydrophilic
material. Examples of such protein biochips are described in the
following patents or patent applications: U.S. Pat. No. 6,225,047
(Hutchens and Yip, "Use of retentate chromatography to generate
difference maps," May 1, 2001), International publication WO
99/51773 (Kuimelis and Wagner, "Addressable protein arrays," Oct.
14, 1999), International publication WO 00/04389 (Wagner et al.,
"Arrays of protein-capture agents and methods of use thereof," Jul.
27, 2000), International publication WO 00/56934 (Englert et al.,
"Continuous porous matrix arrays," Sep. 28, 2000).
[0179] In general, a sample containing the biomarkers is placed on
the active surface of a biochip for a sufficient time to allow
binding. Then, unbound molecules are washed from the surface using
a suitable eluant. In general, the more stringent the eluant, the
more tightly the proteins must be bound to be retained after the
wash. The retained protein biomarkers now can be detected by
appropriate means.
[0180] Analytes captured on the surface of a protein biochip can be
detected by any method known in the art. This includes, for
example, mass spectrometry, fluorescence, surface plasmon
resonance, ellipsometry and atomic force microscopy. Mass
spectrometry, and particularly SELDI mass spectrometry, is a
particularly useful method for detection of the biomarkers of this
invention.
[0181] Preferably, a laser desorption time-of-flight mass
spectrometer is used in embodiments of the invention. In laser
desorption mass spectrometry, a substrate or a probe comprising
markers is introduced into an inlet system. The markers are
desorbed and ionized into the gas phase by laser from the
ionization source. The ions generated are collected by an ion optic
assembly, and then in a time-of-flight mass analyzer, ions are
accelerated through a short high voltage field and let drift into a
high vacuum chamber. At the far end of the high vacuum chamber, the
accelerated ions strike a sensitive detector surface at a different
time. Since the time-of-flight is a function of the mass of the
ions, the elapsed time between ion formation and ion detector
impact can be used to identify the presence or absence of markers
of specific mass to charge ratio.
[0182] Matrix-assisted laser desorption/ionization mass
spectrometry, or MALDI-MS, is a method of mass spectrometry that
involves the use of an energy absorbing molecule, frequently called
a matrix, for desorbing proteins intact from a probe surface. MALDI
is described, for example, in U.S. Pat. No. 5,118,937 (Hillenkamp
et al.) and U.S. Pat. No. 5,045,694 (Beavis and Chait). In MALDI-MS
the sample is typically mixed with a matrix material and placed on
the surface of an inert probe. Exemplary energy absorbing molecules
include cinnamic acid derivatives, sinapinic acid ("SPA"), cyano
hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid. Other
suitable energy absorbing molecules are known to those skilled in
this art. The matrix dries, forming crystals that encapsulate the
analyte molecules. Then the analyte molecules are detected by laser
desorption/ionization mass spectrometry. MALDI-MS is useful for
detecting the biomarkers of this invention if the complexity of a
sample has been substantially reduced using the preparation methods
described above.
[0183] Surface-enhanced laser desorption/ionization mass
spectrometry, or SELDI-MS represents an improvement over MALDI for
the fractionation and detection of biomolecules, such as proteins,
in complex mixtures. SELDI is a method of mass spectrometry in
which biomolecules, such as proteins, are captured on the surface
of a protein biochip using capture reagents that are bound there.
Typically, non-bound molecules are washed from the probe surface
before interrogation. SELDI is described, for example, in: U.S.
Pat. No. 5,719,060 ("Method and Apparatus for Desorption and
Ionization of Analytes," Hutchens and Yip, Feb. 17, 1998) U.S. Pat.
No. 6,225,047 ("Use of Retentate Chromatography to Generate
Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et
al., "Time-of-flight mass spectrometry," in Encyclopedia of
Analytical Chemistry, R. A. Meyers, ed., pp 11915-11918 John Wiley
& Sons Chichesher, 2000.
[0184] Markers on the substrate surface can be desorbed and ionized
using gas phase ion spectrometry. Any suitable gas phase ion
spectrometers can be used as long as it allows markers on the
substrate to be resolved. Preferably, gas phase ion spectrometers
allow quantitation of markers.
[0185] In one embodiment, a gas phase ion spectrometer is a mass
spectrometer. In a typical mass spectrometer, a substrate or a
probe comprising markers on its surface is introduced into an inlet
system of the mass spectrometer. The markers are then desorbed by a
desorption source such as a laser, fast atom bombardment, high
energy plasma, electrospray ionization, thermospray ionization,
liquid secondary ion MS, field desorption, etc. The generated
desorbed, volatilized species consist of preformed ions or neutrals
which are ionized as a direct consequence of the desorption event.
Generated ions are collected by an ion optic assembly, and then a
mass analyzer disperses and analyzes the passing ions. The ions
exiting the mass analyzer are detected by a detector. The detector
then translates information of the detected ions into
mass-to-charge ratios. Detection of the presence of markers or
other substances will typically involve detection of signal
intensity. This, in turn, can reflect the quantity and character of
markers bound to the substrate. Any of the components of a mass
spectrometer (e.g., a desorption source, a mass analyzer, a
detector, etc.) can be combined with other suitable components
described herein or others known in the art in embodiments of the
invention.
[0186] In another embodiment, an immunoassay can be used to detect
and analyze markers in a sample. This method comprises: (a)
providing an antibody that specifically binds to a marker; (b)
contacting a sample with the antibody; and (c) detecting the
presence of a complex of the antibody bound to the marker in the
sample.
[0187] To prepare an antibody that specifically binds to a marker,
purified markers or their nucleic acid sequences can be used.
Nucleic acid and amino acid sequences for markers can be obtained
by further characterization of these markers. For example, each
marker can be peptide mapped with a number of enzymes (e.g.,
trypsin, V8 protease, etc.). The molecular weights of digestion
fragments from each marker can be used to search the databases,
such as SwissProt database, for sequences that will match the
molecular weights of digestion fragments generated by various
enzymes. Using this method, the nucleic acid and amino acid
sequences of other markers can be identified if these markers are
known proteins in the databases.
[0188] Alternatively, the proteins can be sequenced using protein
ladder sequencing. Protein ladders can be generated by, for
example, fragmenting the molecules and subjecting fragments to
enzymatic digestion or other methods that sequentially remove a
single amino acid from the end of the fragment. Methods of
preparing protein ladders are described, for example, in
International Publication WO 93/24834 (Chait et al.) and U.S. Pat.
No. 5,792,664 (Chait et al.). The ladder is then analyzed by mass
spectrometry. The difference in the masses of the ladder fragments
identify the amino acid removed from the end of the molecule.
[0189] If the markers are not known proteins in the databases,
nucleic acid and amino acid sequences can be determined with
knowledge of even a portion of the amino acid sequence of the
marker. For example, degenerate probes can be made based on the
N-terminal amino acid sequence of the marker. These probes can then
be used to screen a genomic or cDNA library created from a sample
from which a marker was initially detected. The positive clones can
be identified, amplified, and their recombinant DNA sequences can
be subcloned using techniques which are well known. See, e.g.,
Current Protocols for Molecular Biology (Ausubel et al., Green
Publishing Assoc. and Wiley-Interscience 1989) and Molecular
Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring
Harbor Laboratory, N Y 2001).
[0190] Using the purified markers or their nucleic acid sequences,
antibodies that specifically bind to a marker can be prepared using
any suitable methods known in the art. See, e.g., Coligan, Current
Protocols in Immunology (1991); Harlow & Lane, Antibodies: A
Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles
and Practice (2 d ed. 1986); and Kohler & Milstein, Nature
256:495-497 (1975). Such techniques include, but are not limited
to, antibody preparation by selection of antibodies from libraries
of recombinant antibodies in phage or similar vectors, as well as
preparation of polyclonal and monoclonal antibodies by immunizing
rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281
(1989); Ward et al., Nature 341:544-546 (1989)).
[0191] After the antibody is provided, a marker can be detected
and/or quantified using any of suitable immunological binding
assays known in the art (see, e.g., U.S. Pat. Nos. 4,366,241;
4,376,110; 4,517,288; and 4,837,168). Useful assays include, for
example, an enzyme immune assay (EIA) such as enzyme-linked
immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western
blot assay, or a slot blot assay. These methods are also described
in, e.g., Methods in Cell Biology: Antibodies in Cell Biology,
volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites
& Ten, eds., 7th ed. 1991); and Harlow & Lane, supra. The
detection and quantitation of biomarkers is described in detail in
the Examples which follow.
[0192] Generally, a sample obtained from a subject can be contacted
with the antibody that specifically binds the marker. Optionally,
the antibody can be fixed to a solid support to facilitate washing
and subsequent isolation of the complex, prior to contacting the
antibody with a sample. Examples of solid supports include glass or
plastic in the form of, e.g., a microtiter plate, a stick, a bead,
or a microbead. Antibodies can also be attached to a probe
substrate or ProteinChip.RTM. array described above. The sample is
preferably a biological fluid sample taken from a subject. Examples
of biological fluid samples include cerebrospinal fluid, blood,
serum, plasma, neuronal cells, tissues, urine, tears, saliva etc.
In a preferred embodiment, the biological fluid comprises
cerebrospinal fluid. The sample can be diluted with a suitable
eluant before contacting the sample to the antibody.
[0193] After incubating the sample with antibodies, the mixture is
washed and the antibody-marker complex formed can be detected. This
can be accomplished by incubating the washed mixture with a
detection reagent. This detection reagent may be, e.g., a second
antibody which is labeled with a detectable label. Exemplary
detectable labels include magnetic beads (e.g., DYNABEADS.TM.),
fluorescent dyes, radiolabels, enzymes (e.g., horse radish
peroxide, alkaline phosphatase and others commonly used in an
ELISA), and colorimetric labels such as colloidal gold or colored
glass or plastic beads. Alternatively, the marker in the sample can
be detected using an indirect assay, wherein, for example, a
second, labeled antibody is used to detect bound marker-specific
antibody, and/or in a competition or inhibition assay wherein, for
example, a monoclonal antibody which binds to a distinct epitope of
the marker is incubated simultaneously with the mixture.
[0194] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, marker, volume of solution,
concentrations and the like. Usually the assays will be carried out
at ambient temperature, although they can be conducted over a range
of temperatures, such as 10.degree. C. to 40.degree. C.
[0195] Immunoassays can be used to determine presence or absence of
a marker in a sample as well as the quantity of a marker in a
sample. First, a test amount of a marker in a sample can be
detected using the immunoassay methods described above. If a marker
is present in the sample, it will form an antibody-marker complex
with an antibody that specifically binds the marker under suitable
incubation conditions described above. The amount of an
antibody-marker complex can be determined by comparing to a
standard. A standard can be, e.g., a known compound or another
protein known to be present in a sample. As noted above, the test
amount of marker need not be measured in absolute units, as long as
the unit of measurement can be compared to a control.
[0196] The methods for detecting these markers in a sample have
many applications. For example, one or more markers can be measured
to aid in the diagnosis of spinal injury, brain injury, the degree
of injury, neural injury due to neuronal disorders, alcohol and
drug abuse, fetal injury due to alcohol and/or drug abuse by
pregnant mothers, etc. In another example, the methods for
detection of the markers can be used to monitor responses in a
subject to treatment. In another example, the methods for detecting
markers can be used to assay for and to identify compounds that
modulate expression of these markers in vivo or in vitro.
[0197] Data generated by desorption and detection of markers can be
analyzed using any suitable means. In one embodiment, data is
analyzed with the use of a programmable digital computer. The
computer program generally contains a readable medium that stores
codes. Certain code can be devoted to memory that includes the
location of each feature on a probe, the identity of the adsorbent
at that feature and the elution conditions used to wash the
adsorbent. The computer also contains code that receives as input,
data on the strength of the signal at various molecular masses
received from a particular addressable location on the probe. This
data can indicate the number of markers detected, including the
strength of the signal generated by each marker.
[0198] Data analysis can include the steps of determining signal
strength (e.g., height of peaks) of a marker detected and removing
"outliers" (data deviating from a predetermined statistical
distribution). The observed peaks can be normalized, a process
whereby the height of each peak relative to some reference is
calculated. For example, a reference can be background noise
generated by instrument and chemicals (e.g., energy absorbing
molecule) which is set as zero in the scale. Then the signal
strength detected for each marker or other biomolecules can be
displayed in the form of relative intensities in the scale desired
(e.g., 100). Alternatively, a standard (e.g., a CSF protein) may be
admitted with the sample so that a peak from the standard can be
used as a reference to calculate relative intensities of the
signals observed for each marker or other markers detected.
[0199] The computer can transform the resulting data into various
formats for displaying. In one format, referred to as "spectrum
view or retentate map," a standard spectral view can be displayed,
wherein the view depicts the quantity of marker reaching the
detector at each particular molecular weight. In another format,
referred to as "peak map," only the peak height and mass
information are retained from the spectrum view, yielding a cleaner
image and enabling markers with nearly identical molecular weights
to be more easily seen. In yet another format, referred to as "gel
view," each mass from the peak view can be converted into a
grayscale image based on the height of each peak, resulting in an
appearance similar to bands on electrophoretic gels. In yet another
format, referred to as "3-D overlays," several spectra can be
overlaid to study subtle changes in relative peak heights. In yet
another format, referred to as "difference map view," two or more
spectra can be compared, conveniently highlighting unique markers
and markers which are up- or down-regulated between samples. Marker
profiles (spectra) from any two samples may be compared visually.
In yet another format, Spotfire Scatter Plot can be used, wherein
markers that are detected are plotted as a dot in a plot, wherein
one axis of the plot represents the apparent molecular mass of the
markers detected and another axis represents the signal intensity
of markers detected. For each sample, markers that are detected and
the amount of markers present in the sample can be saved in a
computer readable medium. This data can then be compared to a
control (e.g., a profile or quantity of markers detected in
control, e.g., normal, healthy subjects in whom neural injury is
undetectable).
Diagnosis of Neural Injury
[0200] In another aspect, the invention provides methods for aiding
a human neural injury and/or neural disorder diagnosis using one or
more markers. For example, proteins identified in Table 1,
peptides, fragments or derivatives thereof. These markers can be
used singularly or in combination with other markers in any set,
for example, axonal and dendritic. The markers are differentially
present in samples of a human patient, for example a TBI patient,
and a normal subject in whom neural injury is undetectable. For
example, some of the markers are expressed at an elevated level
and/or are present at a higher frequency in human patients with
neural injury and/or neuronal disorders than in normal subjects.
Therefore, detection of one or more of these markers in a person
would provide useful information regarding the probability that the
person may have neural injury and/or neuronal disorder.
[0201] Nervous system diseases, neuronal disorders, and/or
conditions, which can be treated, prevented, and/or diagnosed with
the compositions of the invention (e.g., polypeptides,
polynucleotides, and/or agonists or antagonists), include, but are
not limited to, nervous system injuries, and diseases, disorders,
and/or conditions which result in either a disconnection of axons,
a diminution or degeneration of neurons, or demyelination. Nervous
system lesions which may be treated, prevented, and/or diagnosed in
a patient (including human and non-human mammalian patients)
according to the invention, include but are not limited to, the
following lesions of either the central (including spinal cord,
brain) or peripheral nervous systems: (1) ischemic lesions, in
which a lack of oxygen in a portion of the nervous system results
in neuronal injury or death, including cerebral infarction or
ischemia, or spinal cord infarction or ischemia; (2) traumatic
lesions, including lesions caused by physical injury or associated
with surgery, for example, lesions which sever a portion of the
nervous system, or compression injuries; (3) malignant lesions, in
which a portion of the nervous system is destroyed or injured by
malignant tissue which is either a nervous system associated
malignancy or a malignancy derived from non-nervous system tissue;
(4) infectious lesions, in which a portion of the nervous system is
destroyed or injured as a result of infection, for example, by an
abscess or associated with infection by human immunodeficiency
virus, herpes zoster, or herpes simplex virus or with Lyme disease,
tuberculosis, syphilis; (5) degenerative lesions, in which a
portion of the nervous system is destroyed or injured as a result
of a degenerative process including but not limited to degeneration
associated with Parkinson's disease, Alzheimer's disease,
Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6)
lesions associated with nutritional diseases, disorders, and/or
conditions, in which a portion of the nervous system is destroyed
or injured by a nutritional disorder or disorder of metabolism
including but not limited to, vitamin B12 deficiency, folic acid
deficiency, Wernicke disease, tobacco-alcohol amblyopia,
Marchiafava-Bignami disease (primary degeneration of the corpus
callosum), and alcoholic cerebellar degeneration; (7) neurological
lesions associated with systemic diseases including, but not
limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic
lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused
by toxic substances including alcohol, lead, or particular
neurotoxins; and (9) demyelinated lesions in which a portion of the
nervous system is destroyed or injured by a demyelinating disease
including, but not limited to, multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy
or various etiologies, progressive multifocal leukoencephalopathy,
and central pontine myelinolysis.
[0202] Accordingly, embodiments of the invention include methods
for aiding human neural injury and/or neuronal disorders, wherein
the method comprises: (a) detecting at least one marker in a
sample, wherein the marker is selected from any one of the markers
listed in Table 1, peptides, fragments and derivatives thereof; and
(b) correlating the detection of the marker or markers with a
probable diagnosis of human neural injury and/or neuronal disorder.
The correlation may take into account the amount of the marker or
markers in the sample compared to a control amount of the marker or
markers (up or down regulation of the marker or markers) (e.g., in
normal subjects in whom human neural injury is undetectable). The
correlation may take into account the presence or absence of the
markers in a test sample and the frequency of detection of the same
markers in a control. The correlation may take into account both of
such factors to facilitate determination of whether a subject has
neural injury, the degree of severity of the neural injury, and
subcellular location of the injury, or not.
[0203] In a preferred embodiment, the method of diagnosing and
detecting neural injury and/or neural disorders comprises detecting
one or more biomarkers: Axonal Proteins: .alpha. II spectrin (and
SPDB)-1, NF-68 (NF-L)-2, Tau-3, .alpha. II, III spectrin, NF-200
(NF-H), NF-160 (NF-M), Amyloid precursor protein, .alpha.
internexin; Dendritic Proteins: beta III-tubulin-1, p24
microtubule-associated protein-2, alpha-Tubulin (P02551),
beta-Tubulin (P04691), MAP-2A/B-3, MAP-2C-3, Stathmin-4, Dynamin-1
(P21575), Phocein, Dynactin (Q13561), Vimentin (P31000), Dynamin,
Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB (P07335),
Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0204] In another preferred embodiment, the method of diagnosing
and detecting neural injury and/or neural disorders comprises
detecting at least one biomarker from each neural cell type. The
composition of biomarkers is diagnostic of neural injury, damage
and/or neural disorders. The composition comprises: .alpha. II
spectrin, SPDB-1, NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Thal, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0205] Any suitable samples can be obtained from a subject to
detect markers. Preferably, a sample is a cerebrospinal fluid
sample from the subject. If desired, the sample can be prepared as
described above to enhance detectability of the markers. For
example, to increase the detectability of markers, a blood serum
sample from the subject can be preferably fractionated by, e.g.,
Cibacron blue agarose chromatography and single stranded DNA
affinity chromatography, anion exchange chromatography and the
like. Sample preparations, such as pre-fractionation protocols, is
optional and may not be necessary to enhance detectability of
markers depending on the methods of detection used. For example,
sample preparation may be unnecessary if antibodies that
specifically bind markers are used to detect the presence of
markers in a sample.
[0206] Any suitable method can be used to detect a marker or
markers in a sample. For example, an immunoassay or gas phase ion
spectrometry can be used as described above. Using these methods,
one or more markers can be detected. Preferably, a sample is tested
for the presence of a plurality of markers. Detecting the presence
of a plurality of markers, rather than a single marker alone, would
provide more information for the diagnostician. Specifically, the
detection of a plurality of markers in a sample would increase the
percentage of true positive and true negative diagnoses and would
decrease the percentage of false positive or false negative
diagnoses.
[0207] The detection of the marker or markers is then correlated
with a probable diagnosis of neural injury and/or neuronal
disorders. In some embodiments, the detection of the mere presence
or absence of a marker, without quantifying the amount of marker,
is useful and can be correlated with a probable diagnosis of neural
injury and/or neuronal disorders. For example, neural proteins,
fragments or derivatives thereof, such as for example, axonal
proteins--NF-200 (NF-H), NF-160 (NF-M), NF-68 (NF-L); can be more
frequently detected in patients with neuronal injury than in normal
subjects.
[0208] In other embodiments, the detection of markers can involve
quantifying the markers to correlate the detection of markers with
a probable diagnosis of neural injury, degree of severity of neural
injury, diagnosis of neural disorders and the like. Thus, if the
amount of the markers detected in a subject being tested is higher
compared to a control amount, then the subject being tested has a
higher probability of having such injuries and/or neural
disorders.
[0209] Similarly, in another embodiment, the detection of markers
can further involve quantifying the markers to correlate the
detection of markers with a probable diagnosis of neural injury,
degree of severity of neural injury, diagnosis of neural disorders
and the like, wherein the markers are present in lower quantities
in CSF or blood serum samples from patients than in blood serum
samples of normal subjects. Thus, if the amount of the markers
detected in a subject being tested is lower compared to a control
amount, then the subject being tested has a higher probability of
having neural injury and/or neural disorder.
[0210] When the markers are quantified, it can be compared to a
control. A control can be, e.g., the average or median amount of
marker present in comparable samples of normal subjects in whom
neural injury and/or neural disorders, is undetectable. The control
amount is measured under the same or substantially similar
experimental conditions as in measuring the test amount. For
example, if a test sample is obtained from a subject's
cerebrospinal fluid and/or blood serum sample and a marker is
detected using a particular probe, then a control amount of the
marker is preferably determined from a serum sample of a patient
using the same probe. It is preferred that the control amount of
marker is determined based upon a significant number of samples
from normal subjects who do not have neural injury and/or neuronal
disorders so that it reflects variations of the marker amounts in
that population.
[0211] Data generated by mass spectrometry can then be analyzed by
a computer software. The software can comprise code that converts
signal from the mass spectrometer into computer readable form. The
software also can include code that applies an algorithm to the
analysis of the signal to determine whether the signal represents a
"peak" in the signal corresponding to a marker of this invention,
or other useful markers. The software also can include code that
executes an algorithm that compares signal from a test sample to a
typical signal characteristic of "normal" and human neural injury
and determines the closeness of fit between the two signals. The
software also can include code indicating which the test sample is
closest to, thereby providing a probable diagnosis.
Production of Antibodies to Detect Neural Biomarkers
[0212] Neural biomarkers obtained from samples in patients
suffering from varying neural injuries, degrees of severity of
injury, neuronal disorders and the like, can be prepared as
described above. Furthermore, neural biomarkers can be subjected to
enzymatic digestion to obtain fragments or peptides of the
biomarkers for the production of antibodies to different antigenic
epitopes that can be present in a peptide versus the whole protein.
Antigenic epitopes are useful, for example, to raise antibodies,
including monoclonal antibodies, that specifically bind the
epitope. Antigenic epitopes can be used as the target molecules in
immunoassays. (See, for instance, Wilson et al., Cell 37:767-778
(1984); Sutcliffe et al., Science 219:660-666 (1983)).
[0213] In a preferred embodiment, antibodies are directed to
epitopes (specifically bind) of biomarkers Axonal Proteins: .alpha.
II spectrin (and SPDB)-1, NF-68 (NF-L)-2, Tau-3, .alpha. II, III
spectrin, NF-200 (NF-H), NF-160 (NF-M), Amyloid precursor protein,
.alpha. internexin; Dendritic Proteins: beta III-tubulin-1, p24
microtubule-associated protein-2, alpha-Tubulin (P02551),
beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4, Dynamin-1
(P21575), Phocein, Dynactin (Q13561), Vimentin (P31000), Dynamin,
Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB (P07335),
Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Iba1, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0214] In another preferred embodiment, the antibodies of the
invention bind to at least one biomarker from each neural cell
type. The composition of biomarkers is diagnostic of neural injury,
damage and/or neural disorders. The composition comprises: .alpha.
II spectrin, SPDB-1, NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0215] Neural biomarker epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Neural polypeptides comprising
one or more immunogenic epitopes may be presented for eliciting an
antibody response together with a carrier protein, such as an
albumin, to an animal system (such as rabbit or mouse), or, if the
polypeptide is of sufficient length (at least about 25 amino
acids), the polypeptide may be presented without a carrier.
However, immunogenic epitopes comprising as few as 3 to 10 amino
acids have been shown to be sufficient to raise antibodies capable
of binding to, at the very least, linear epitopes in a denatured
polypeptide (e.g., in Western blotting).
[0216] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0217] Nucleic acids neural biomarker epitopes can also be
recombined with a gene of interest as an epitope tag (e.g., the
hemagglutinin ("HA") tag or flag tag) to aid in detection and
purification of the expressed polypeptide. For example, a system
described by Janknecht et al. allows for the ready purification of
non-denatured fusion proteins expressed in human cell lines
(Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976).
In this system, the gene of interest is subcloned into a vaccinia
recombination plasmid such that the open reading frame of the gene
is translationally fused to an amino-terminal tag consisting of six
histidine residues. The tag serves as a matrix binding domain for
the fusion protein. Extracts from cells infected with the
recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose column and histidine-tagged proteins can be
selectively eluted with imidazole-containing buffers.
[0218] The antibodies of the present invention may be generated by
any suitable method known in the art. The antibodies of the present
invention can comprise polyclonal antibodies. Methods of preparing
polyclonal antibodies are known to the skilled artisan (Harlow, et
al., Antibodies: A Laboratory Manual, (Cold spring Harbor
Laboratory Press, 2.sup.nd ed. (1988), which is hereby incorporated
herein by reference in its entirety). For example, a polypeptide of
the invention can be administered to various host animals
including, but not limited to, rabbits, mice, rats, etc. to induce
the production of sera containing polyclonal antibodies specific
for the antigen. The administration of the polypeptides of the
present invention may entail one or more injections of an
immunizing agent and, if desired, an adjuvant. Various adjuvants
may be used to increase the immunological response, depending on
the host species, and include but are not limited to, Freund's
(complete and incomplete), mineral gels such as aluminum hydroxide,
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Such
adjuvants are also well known in the art. For the purposes of the
invention, "immunizing agent" may be defined as a polypeptide of
the invention, including fragments, variants, and/or derivatives
thereof, in addition to fusions with heterologous polypeptides and
other forms of the polypeptides as may be described herein.
[0219] Typically, the immunizing agent and/or adjuvant will be
injected in the mammal by multiple subcutaneous or intraperitoneal
injections, though they may also be given intramuscularly, and/or
through IV. The immunizing agent may include polypeptides of the
present invention or a fusion protein or variants thereof.
Depending upon the nature of the polypeptides (i.e., percent
hydrophobicity, percent hydrophilicity, stability, net charge,
isoelectric point etc.), it may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized. Such conjugation includes either chemical
conjugation by derivatizing active chemical functional groups to
both the polypeptide of the present invention and the immunogenic
protein such that a covalent bond is formed, or through
fusion-protein based methodology, or other methods known to the
skilled artisan. Examples of such immunogenic proteins include, but
are not limited to keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Various adjuvants may
be used to increase the immunological response, depending on the
host species, including but not limited to Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum. Additional
examples of adjuvants which may be employed includes the MPL-TDM
adjuvant (monophosphoryl lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one
skilled in the art without undue experimentation.
[0220] The antibodies of the present invention can also comprise
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma methods, such as those described by Kohler and Milstein,
Nature, 256:495 (1975) and U.S. Pat. No. 4,376,110, by Harlow, et
al., Antibodies: A Laboratory Manual, (Cold spring Harbor
Laboratory Press, 2.sup.nd ed. (1988), by Hammerling, et al.,
Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, N.Y.,
(1981)), or other methods known to the artisan. Other examples of
methods which may be employed for producing monoclonal antibodies
includes, but are not limited to, the human B-cell hybridoma
technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,
1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the
EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies
And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies
may be of any immunoglobulin class including IgG, IgM, IgE, IgA,
IgD and any subclass thereof. The hybridoma producing the mAb of
this invention may be cultivated in vitro or in vivo. Production of
high titers of mAbs in vivo makes this the presently preferred
method of production.
[0221] In a hybridoma method, a mouse, a humanized mouse, a mouse
with a human immune system, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro.
[0222] The immunizing agent will typically include neural
polypeptides, fragments or a fusion protein thereof. Generally,
either peripheral blood lymphocytes ("PBLs") are used if cells of
human origin are desired, or spleen cells or lymph node cells are
used if non-human mammalian sources are desired. The lymphocytes
are then fused with an immortalized cell line using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, (1986), pp. 59-103). Immortalized cell lines are usually
transformed mammalian cells, particularly myeloma cells of rodent,
bovine and human origin. Usually, rat or mouse myeloma cell lines
are employed. The hybridoma cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent
the growth of HGPRT-deficient cells.
[0223] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. As inferred
throughout the specification, human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, Marcel Dekker, Inc., New York, (1987)
pp. 51-63).
[0224] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the neural polypeptides of the present invention.
Preferably, the binding specificity of monoclonal antibodies
produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoadsorbant assay
(ELISA). Such techniques are known in the art and within the skill
of the artisan. The binding affinity of the monoclonal antibody
can, for example, be determined by the Scatchard analysis of Munson
and Pollart, Anal. Biochem., 107:220 (1980).
[0225] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, supra). Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640. Alternatively, the hybridoma cells may be grown in
vivo as ascites in a mammal.
[0226] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-sepharose, hydroxyapatite chromatography, gel
exclusion chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
[0227] The skilled artisan would acknowledge that a variety of
methods exist in the art for the production of monoclonal
antibodies and thus, the invention is not limited to their sole
production in hybridomas. For example, the monoclonal antibodies
may be made by recombinant DNA methods, such as those described in
U.S. Pat. No. 4,816,567. In this context, the term "monoclonal
antibody" refers to an antibody derived from a single eukaryotic,
phage, or prokaryotic clone. The DNA encoding the monoclonal
antibodies of the invention can be readily isolated and sequenced
using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding
the heavy and light chains of murine antibodies, or such chains
from human, humanized, or other sources). The hybridoma cells of
the invention serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transformed into host cells such as Simian COS cells, Chinese
hamster ovary (CHO) cells, or myeloma cells that do not otherwise
produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells.
[0228] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with a biomarker
polypeptide or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well-known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by limited dilution. The hybridoma clones are then assayed
by methods known in the art for cells that secrete antibodies
capable of binding a polypeptide of the invention. Ascites fluid,
which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0229] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention. The antibodies detecting neural biomarkers, peptides and
derivatives thereof, can be used in immunoassays and other methods
to identify new neural biomarkers and for use in the diagnosis of
neural injury, degree of severity of injury and/or neurological
disorders.
[0230] Other methods can also be used for the large scale
production of neural biomarker specific antibodies. For example,
antibodies can also be generated using various phage display
methods known in the art. In phage display methods, functional
antibody domains are displayed on the surface of phage particles
which carry the polynucleotide sequences encoding them. In a
particular embodiment, such phage can be utilized to display
antigen binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab,
Fv or disulfide stabilized Fv antibody domains recombinantly fused
to either the phage gene III or gene VIII protein. Examples of
phage display methods that can be used to make the antibodies of
the present invention include those disclosed in Brinkman et al.,
J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.
Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et
al., Advances in Immunology 57:191-280 (1994); PCT application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0231] The antibodies of the present invention have various
utilities. For example, such antibodies may be used in diagnostic
assays to detect the presence or quantification of the polypeptides
of the invention in a sample. Such a diagnostic assay can comprise
at least two steps. The first, subjecting a sample with the
antibody, wherein the sample is a tissue (e.g., human, animal,
etc.), biological fluid (e.g., blood, urine, sputum, semen,
amniotic fluid, saliva, etc.), biological extract (e.g., tissue or
cellular homogenate, etc.), a protein microchip (e.g., See Arenkov
P, et al., Anal Biochem., 278(2):123-131 (2000)), or a
chromatography column, etc. And a second step involving the
quantification of antibody bound to the substrate. Alternatively,
the method may additionally involve a first step of attaching the
antibody, either covalently, electrostatically, or reversibly, to a
solid support, and a second step of subjecting the bound antibody
to the sample, as defined above and elsewhere herein.
[0232] Various diagnostic assay techniques are known in the art,
such as competitive binding assays, direct or indirect sandwich
assays and immunoprecipitation assays conducted in either
heterogeneous or homogenous phases (Zola, Monoclonal Antibodies: A
Manual of Techniques, CRC Press, Inc., (1987), pp147-158). The
antibodies used in the diagnostic assays can be labeled with a
detectable moiety. The detectable moiety should be capable of
producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety may be a radioisotope, such as
.sup.2H, .sup.14C, .sup.32P, or .sup.125I, a florescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase, green fluorescent protein, or
horseradish peroxidase. Any method known in the art for conjugating
the antibody to the detectable moiety may be employed, including
those methods described by Hunter et al., Nature, 144:945 (1962);
David et al., Biochem., 13:1014 (1974); Pain et al., J. Immunol.
Methods, 40:219(1981); and Nygren, J. Histochem. and Cytochem.,
30:407 (1982).
Kits
[0233] In yet another aspect, the invention provides kits for
aiding a diagnosis of neural injury, degree of severity of injury,
subcellular localization and/or neuronal disorders, wherein the
kits can be used to detect the markers of the present invention.
For example, the kits can be used to detect any one or more of the
markers described herein, which markers are differentially present
in samples of a patient and normal subjects. The kits of the
invention have many applications. For example, the kits can be used
to differentiate if a subject has axonal injury versus, for
example, dendritic, or has a negative diagnosis, thus aiding
neuronal injury diagnosis. In another example, the kits can be used
to identify compounds that modulate expression of one or more of
the markers in in vitro or in vivo animal models to determine the
effects of treatment.
[0234] In one embodiment, a kit comprises (a) an antibody that
specifically binds to a marker; and (b) a detection reagent. Such
kits can be prepared from the materials described above, and the
previous discussion regarding the materials (e.g., antibodies,
detection reagents, immobilized supports, etc.) is fully applicable
to this section and will not be repeated. Optionally, the kit may
further comprise pre-fractionation spin columns. In some
embodiments, the kit may further comprise instructions for suitable
operation parameters in the form of a label or a separate
insert.
[0235] In another embodiment, the kit comprises (a) a panel or
composition of biomarkers (b) a detecting agent. The panel or
composition of biomarkers included in a kit include at least one
biomarker and/or a plurality of biomarkers in order to diagnose in
vivo location of neural injury. These biomarkers include: Axonal
Proteins: .alpha. II spectrin (and SPDB)-1, NF-68 (NF-L)-2, Tau-3,
.alpha. II, III spectrin, NF-200 (NF-H), NF-160 (NF-M), Amyloid
precursor protein, .alpha. internexin; Dendritic Proteins: beta
III-tubulin-1, p24 microtubule-associated protein-2, alpha-Tubulin
(P02551), beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4,
Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000),
Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43, B as
soon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1, 2,
Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477); Post-Synaptic
Proteins: PSD95-1, NMDA-receptor (and all subtypes)-2, PSD93,
AMPA-kainate receptor (all subtypes), mGluR (all subtypes),
Calmodulin dependent protein kinase II (CAMPK)-alpha, beta, gamma,
CaMPK-IV, SNAP-25, a-/b-SNAP; Myelin-Oligodendrocyte: Myelin basic
protein (MBP) and fragments, Myelin proteolipid protein (PLP),
Myelin Oligodendrocyte specific protein (MOSP), Myelin
Oligodendrocyte glycoprotein (MOG), myelin associated protein
(MAG), Oligodendrocyte NS-1 protein; Glial Protein Biomarkers: GFAP
(P47819), Protein disulfide isomerase (PDI)-P04785, Neurocalcin
delta, S100beta; Microglia protein Biomarkers: Iba1, OX-42, OX-8,
OX-6, ED-1, PTPase (CD45), CD40, CD68, CD11b, Fractalkine (CX3CL1)
and Fractalkine receptor (CX3CR1), 5-d-4 antigen; Schwann cell
markers: Schwann cell myelin protein; Glia Scar: Tenascin;
Hippocampus: Stathmin, Hippocalcin, SCG10; Cerebellum: Purkinje
cell protein-2 (Pcp2), Calbindin D9K, Calbindin D28K (NP_114190),
Cerebellar CaBP, spot 35; Cerebrocortex: Cortexin-1 (P60606), H-2Z1
gene product; Thalamus: CD15 (3-fucosyl-N-acetyl-lactosamine)
epitope; Hypothalamus: Orexin receptors (OX-1R and OX-2R)-appetite,
Orexins (hypothalamus-specific peptides); Corpus callosum: MBP,
MOG, PLP, MAG; Spinal Cord: Schwann cell myelin protein; Striatum:
Striatin, Rhes (Ras homolog enriched in striatum); Peripheral
ganglia: Gadd45a; Peripherial nerve fiber(sensory+motor):
Peripherin, Peripheral myelin protein 22 (AAH91499); Other
Neuron-specific proteins: PH8 (S Serotonergic Dopaminergic, PEP-19,
Neurocalcin (NC), a neuron-specific EF-hand Ca.sup.2+-binding
protein, Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin,
Visinin; Neurotransmitter Receptors: NMDA receptor subunits (e.g.
NR1A2B), Glutamate receptor subunits (AMPA, Kainate receptors (e.g.
GluR1, GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0236] In another preferred embodiment, the panel of biomarkers in
a kit at least one biomarker from each neural cell type. The
composition of biomarkers is diagnostic of neural injury, damage
and/or neural disorders. The composition comprises: .alpha. II
spectrin, SPDB-1, NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0237] In another preferred embodiment, the antibodies in a kit are
specific for a panel of biomarkers and one or more antibodies can
be used. Antibodies are specific for biomarkers: Axonal Proteins:
.alpha. II spectrin (and SPDB)-1, NF-68 (NF-L)-2, Tau-3, .alpha.
II, III spectrin, NF-200 (NF-H), NF-160 (NF-M), Amyloid precursor
protein, .alpha. internexin; Dendritic Proteins: beta
III-tubulin-1, p24 microtubule-associated protein-2, alpha-Tubulin
(P02551), beta-Tubulin (P04691), MAP-2A/B--3, MAP-2C-3, Stathmin-4,
Dynamin-1 (P21575), Phocein, Dynactin (Q13561), Vimentin (P31000),
Dynamin, Profilin, Cofilin 1,2; Somal Proteins: UCH-L1 (Q00981)-1,
Glycogen phosphorylase-BB--2, PEBP (P31044), NSE (P07323), CK-BB
(P07335), Thy 1.1, Prion protein, Huntingtin, 14-3-3 proteins (e.g.
14-3-3-epsolon (P42655)), SM22-.alpha., Calgranulin AB,
alpha-Synuclein (P37377), beta-Synuclein (Q63754), HNP 22; Neural
nuclear proteins: NeuN-1, S/G(2) nuclear autoantigen (SG2NA),
Huntingtin; Presynaptic Proteins: Synaptophysin-1, Synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, Synapsin1
(Synapsin-Ia), Synapsin2 (Q63537), Synapsin3, GAP43,
Bassoon(NP_003449), Piccolo (aczonin) (NP_149015), Syntaxin, CRMP1,
2, Amphiphysin-1 (NP_001626), Amphiphysin-2 (NP_647477);
Post-Synaptic Proteins: PSD95-1, NMDA-receptor (and all
subtypes)-2, PSD93, AMPA-kainate receptor (all subtypes), mGluR
(all subtypes), Calmodulin dependent protein kinase II
(CAMPK)-alpha, beta, gamma, CaMPK-IV, SNAP-25, a-/b-SNAP;
Myelin-Oligodendrocyte: Myelin basic protein (MBP) and fragments,
Myelin proteolipid protein (PLP), Myelin Oligodendrocyte specific
protein (MOSP), Myelin Oligodendrocyte glycoprotein (MOG), myelin
associated protein (MAG), Oligodendrocyte NS-1 protein; Glial
Protein Biomarkers: GFAP (P47819), Protein disulfide isomerase
(PDI)-P04785, Neurocalcin delta, S100beta; Microglia protein
Biomarkers: Thal, OX-42, OX-8, OX-6, ED-1, PTPase (CD45), CD40,
CD68, CD11b, Fractalkine (CX3CL1) and Fractalkine receptor
(CX3CR1), 5-d-4 antigen; Schwann cell markers: Schwann cell myelin
protein; Glia Scar: Tenascin; Hippocampus: Stathmin, Hippocalcin,
SCG10; Cerebellum: Purkinje cell protein-2 (Pcp2), Calbindin D9K,
Calbindin D28K (NP_114190), Cerebellar CaBP, spot 35;
Cerebrocortex: Cortexin-1 (P60606), H-2Z1 gene product; Thalamus:
CD15 (3-fucosyl-N-acetyl-lactosamine) epitope; Hypothalamus: Orexin
receptors (OX-1R and OX-2R)-appetite, Orexins
(hypothalamus-specific peptides); Corpus callosum: MBP, MOG, PLP,
MAG; Spinal Cord: Schwann cell myelin protein; Striatum: Striatin,
Rhes (Ras homolog enriched in striatum); Peripheral ganglia:
Gadd45a; Peripherial nerve fiber(sensory+motor): Peripherin,
Peripheral myelin protein 22 (AAH91499); Other Neuron-specific
proteins: PH8 (S Serotonergic Dopaminergic, PEP-19, Neurocalcin
(NC), a neuron-specific EF-hand Ca.sup.2+-binding protein,
Encephalopsin, Striatin, SG2NA, Zinedin, Recoverin, Visinin;
Neurotransmitter Receptors: NMDA receptor subunits (e.g. NR1A2B),
Glutamate receptor subunits (AMPA, Kainate receptors (e.g. GluR1,
GluR4), beta-adrenoceptor subtypes (e.g. beta(2)),
Alpha-adrenoceptors subtypes (e.g. alpha(2c)), GABA receptors (e.g.
GABA(B)), Metabotropic glutamate receptor (e.g. mGluR3), 5-HT
serotonin receptors (e.g. 5-HT(3)), Dopamine receptors (e.g. D4),
Muscarinic Ach receptors (e.g. M1), Nicotinic Acetylcholine
Receptor (e.g. alpha-7); Neurotransmitter Transporters:
Norepinephrine Transporter (NET), Dopamine transporter (DAT),
Serotonin transporter (SERT), Vesicular transporter proteins (VMAT1
and VMAT2), GABA transporter vesicular inhibitory amino acid
transporter (VIAAT/VGAT), Glutamate Transporter (e.g. GLT1),
Vesicular acetylcholine transporter, Vesicular Glutamate
Transporter 1, [VGLUT1; BNPI] and VGLUT2, Choline transporter,
(e.g. CHT1); Cholinergic Biomarkers: Acetylcholine Esterase,
Choline acetyltransferase [ChAT]; Dopaminergic Biomarkers: Tyrosine
Hydroxylase (TH), Phospho-TH, DARPP32; Noradrenergic Biomarkers:
Dopamine beta-hydroxylase (DbH); Adrenergic Biomarkers:
Phenylethanolamine N-methyltransferase (PNMT); Serotonergic
Biomarkers: Tryptophan Hydroxylase (TrH); Glutamatergic Biomarkers:
Glutaminase, Glutamine synthetase; GABAergic Biomarkers: GABA
transaminase [GABAT]), GABA-B-R2.
[0238] In another preferred embodiment, the antibodies are specific
for at least one biomarker from each neural cell type. The
composition of biomarkers is diagnostic of neural injury, damage
and/or neural disorders. The antibodies bind to: .alpha. II
spectrin, SPDB-1, NF-68, NF-L-2, Tau-3, .beta.III-tubulin-1, p24
microtubule-associated protein-2, UCH-L1 (Q00981)-1, Glycogen
phosphorylase-BB-2, NeuN-1, Synaptophysin-1, synaptotagmin
(P21707), Synaptojanin-1 (Q62910), Synaptojanin-2, PSD95-1,
NMDA-receptor-2 and subtypes, myelin basic protein (MBP) and
fragments, GFAP (P47819), Iba1, OX-42, OX-8, OX-6, ED-1, Schwann
cell myelin protein, tenascin, stathmin, Purkinje cell protein-2
(Pcp2), Cortexin-1 (P60606), Orexin receptors (OX-1R, OX-2R),
Striatin, Gadd45a, Peripherin, peripheral myelin protein 22
(AAH91499), and Neurocalcin (NC).
[0239] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0240] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St.
Louis, Mo.).
[0241] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0242] Optionally, the kit may further comprise a standard or
control information so that the test sample can be compared with
the control information standard to determine if the test amount of
a marker detected in a sample is a diagnostic amount consistent
with a diagnosis of neural injury, degree of severity of the
injury, subcellular localization, neuronal disorder and/or effect
of treatment on the patient.
[0243] In another embodiment, a kit comprises: (a) a substrate
comprising an adsorbent thereon, wherein the adsorbent is suitable
for binding a marker, and (b) instructions to detect the marker or
markers by contacting a sample with the adsorbent and detecting the
marker or markers retained by the adsorbent. In some embodiments,
the kit may comprise an eluant (as an alternative or in combination
with instructions) or instructions for making an eluant, wherein
the combination of the adsorbent and the eluant allows detection of
the markers using gas phase ion spectrometry. Such kits can be
prepared from the materials described above, and the previous
discussion of these materials (e.g., probe substrates, adsorbents,
washing solutions, etc.) is fully applicable to this section and
will not be repeated.
[0244] In another embodiment, the kit may comprise a first
substrate comprising an adsorbent thereon (e.g., a particle
functionalized with an adsorbent) and a second substrate onto which
the first substrate can be positioned to form a probe which is
removably insertable into a gas phase ion spectrometer. In other
embodiments, the kit may comprise a single substrate which is in
the form of a removably insertable probe with adsorbents on the
substrate. In yet another embodiment, the kit may further comprise
a pre-fractionation spin column (e.g., Cibacron blue agarose
column, anti-HSA agarose column, size exclusion column, Q-anion
exchange spin column, single stranded DNA column, lectin column,
etc.).
[0245] Optionally, the kit can further comprise instructions for
suitable operational parameters in the form of a label or a
separate insert. For example, the kit may have standard
instructions informing a consumer how to wash the probe after a
sample is contacted on the probe. In another example, the kit may
have instructions for pre-fractionating a sample to reduce
complexity of proteins in the sample. In another example, the kit
may have instructions for automating the fractionation or other
processes.
[0246] The following examples are offered by way of illustration,
not by way of limitation. While specific examples have been
provided, the above description is illustrative and not
restrictive. Any one or more of the features of the previously
described embodiments can be combined in any manner with one or
more features of any other embodiments in the present invention.
Furthermore, many variations of the invention will become apparent
to those skilled in the art upon review of the specification. The
scope of the invention should, therefore, be determined not with
reference to the above description, but instead should be
determined with reference to the appended claims along with their
full scope of equivalents.
[0247] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted. By their citation of
various references in this document, Applicants do not admit any
particular reference is "prior art" to their invention.
EXAMPLES
Materials and Methods
[0248] Abbreviations:
[0249] AEBSF, 4-(2-aminoethyl)-benzenesulfonylflouride; EDTA,
ethylenediaminetetraacetic acid; EGTA,
ethylenebis(oxyethylenenitrilo) tetra acetic acid; DMEM, Dulbecco's
modified Eagle's medium; BSA, bovine serum albumin; DPBS,
Dulbecco's phosphate buffered saline; DTT, dithiothreitol; FDA,
fluorescein diacetate; GFAP, glial fibrillary acid protein; HBSS,
Hanks' balanced salt solution; MAP-2, microtubule associated
protein-2; PI, propidium iodide; PMSF, phenylmethylsulfonyl
fluoride; SDS, sodium dedocyl sulfate; TEMED,
N,N,N',N'-tetramethyletheylenediamine; CalpInh-II, calpain
inhibitor II (N-acetyl-Leu-Leu-methioninal); Z-D-DCB, pan-caspase
inhibitor(carbobenzoxy-Asp-CH.sub.2--OC(O)-2-6-dichlorobenzene);
PBS, phosphate buffered saline; TLCK, Na-p-tosyl-L-Lysine chloro
methyl; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.
[0250] Surgical Procedures
[0251] Controlled cortical impact traumatic brain injury. A
cortical impact injury device was used to produce TBI in rodents.
Cortical impact TBI results in cortical deformation within the
vicinity of the impactor tip associated with contusion, and
neuronal and axonal damage that is constrained in the hemisphere
ipsilateral to the site of injury. Adult male (280-300 g)
Sprague-Dawley rats (Harlan; Indianapolis, Ind.) were initially
anesthetized with 4% isoflurane in a carrier gas of 1:1
O.sub.2/N.sub.2O (4 min.) followed by maintenance anesthesia of
2.5% isoflurane in the same carrier gas. Core body temperature was
monitored continuously by a rectal thermistor probe and maintained
at 37.+-.1.degree. C. by placing an adjustable temperature
controlled heating pad beneath the rats. Animals were mounted in a
stereotactic frame in a prone position and secured by ear and
incisor bars.
[0252] A midline cranial incision was made, the soft tissues were
reflected, and a unilateral (ipsilateral to site of impact)
craniotomy (7 mm diameter) was performed adjacent to the central
suture, midway between bregma and lambda. The dura mater was kept
intact over the cortex. Brain trauma in rats was produced by
impacting the right cortex (ipsilateral cortex) with a 5 mm
diameter aluminum impactor tip (housed in a pneumatic cylinder) at
a velocity of 3.5 m/s with a 2.0 mm compression and 150 ms dwell
time (compression duration). Velocity was controlled by adjusting
the pressure (compressed N.sub.2) supplied to the pneumatic
cylinder. Velocity and dwell time were measured by a linear
velocity displacment transducer (Lucas Shaevitz.TM. model 500 HR;
Detroit, Mich.) that produces an analogue signal that was recorded
by a storage-trace oscilloscope (BK Precision, model 2522B;
Placentia, Calif.). Sham-injured animals underwent identical
surgical procedures but did not receive an impact injury.
Appropriate pre- and post-injury management was maintained.
[0253] Preparation of Cortical Tissue And CSF
[0254] CSF and brain cortices were collected from animals at
various intervals after sham-injury or TBI. At the appropriate
time-points, TBI or sham-injured animals were anesthetized as
described above and secured in a stereotactic frame with the head
allowed to move freely along the longitudinal axis. The head was
flexed so that the external occipital protuberance in the neck was
prominent and a dorsal midline incision was made over the cervical
vertebrae and occiput. The atlanto-occipital membrane was exposed
by blunt dissection and a 25G needle attached to polyethylene
tubing was carefully lowered into the cisterna magna. Approximately
0.1 to 0.15 ml of CSF was collected from each rat. Following CSF
collection, animals were removed from the stereotactic frame and
immediately killed by decapitation.
[0255] Ipsilateral and contralateral (to the impact site) cortices
were then rapidly dissected, rinsed in ice cold PBS, and snap
frozen in liquid nitrogen. Cortices beneath the craniotomies were
excised to the level of the white matter and extended .about.4 mm
laterally and -7 mm rostrocaudally. CSF samples were centrifuged at
4000 g for 4 min. at 4.degree. C. to clear any contaminating
erythrocytes. Cleared CSF and frozen tissue samples were stored at
-80.degree. C. until ready for use. Cortices were homogenized in a
glass tube with a TEFLON dounce pestle in 15 volumes of an ice-cold
triple detergent lysis buffer (20 mM Hepes, 1 mM EDTA, 2 mM EGTA,
150 mM NaCl, 0.1% SDS, 1.0% IGEPAL 40, 0.5% deoxycholic acid, pH
7.5) containing a broad range protease inhibitor cocktail (Roche
Molecular Biochemicals, cat. #1-836-145).
[0256] Human CSF samples were obtained with informed consent from
human subjects suffering from TBI, and from control patients
without TBI, having hydrocephaly.
[0257] Sandwich ELISA.
[0258] Anti-Biomarker specific rabbit polyclonal antibody and
monoclonal antibodies are produced in the laboratory. To determine
reactivity and specificity of the antibodies a tissue panel is
probed by Western blot. An indirect ELISA is used with the
recombinant biomarker protein attached to the ELISA plate to
determine the optimal concentrations of the antibodies used in the
assay. This assay determines a robust concentration of
anti-biomarker to use in the assay. 96-well microplate wells are
coated with 50 ng/well and the rabbit and mouse anti-biomarker
antibodies are diluted serially starting with a 1:250 dilution down
to 1:10,000 to determine the optimum concentration to use for the
assay. A secondary anti-rabbit (or mouse)-horseradish peroxidase
(HRP) labeled detection antibody and Ultra-TMB are used as
detection substrate to evaluate the results.
[0259] Once the concentration of antibody for maximum signal are
determined, maximum detection limit of the indirect ELISA for each
antibody is determined. 96-well microplates are coated with a
concentration from 50 ng/well serially diluted to <1 pg/well.
For detection antibodies are diluted to the concentration
determined above. This provides a sensitivity range for the
Biomarker ELISA assays and determines which antibody to choose for
capture and detection antibody.
[0260] Optimization and enhancement of signal in the sandwich
ELISA: The detection antibody is directly labeled with HRP to avoid
any cross reactivity and to be able to enhance the signal with the
amplification system, which is very sensitive. This format is used
in detecting all the biomarkers. The wells of the 96-well plate are
coated with saturating concentrations of purified antibody
(.about.250 ng/well), the concentration of biomarker antigen ranges
from 50 ng to <1 pg/well and the detection antibody is at the
concentration determined above. Initially the complex is detected
with a HRP-labeled secondary antibody to confirm the SW ELISA
format, and the detection system is replaced by the HRP-labeled
detection antibody.
[0261] Standard curves of biomarkers and samples from control and
injured animals are used. This also determines parallelism between
the serum samples and the standard curve. Serum samples are spiked
with a serial dilution of each biomarker, similar to the standard
curve. Parallel results are equal to 80-100% recovery. If any high
concentrations of serum have interfering substances, the minimum
dilution required is determined to remove the interference. The
assay is used to evaluate biomarker levels in serum from injured
animals having injuries of different magnitudes followed over
time.
[0262] The ELISA has been developed and optimized as a standard
96-well format ELISA which is specific for the biomarkers and
sensitivity in the range measured in rat and human CSF and serum.
Antibodies that recognize the UCH-L1 protein with high specificity
and sensitivity were used as capture and detection antibodies. The
detection antibody is labeled with horseradish peroxidase (HRP) and
colorimetric development is achieved using Ultra-TMB.
[0263] Validation of UCH-L1 as a Biomarker for TBI
[0264] Using rat and human samples obtained from the University of
Florida (Gainesville, Fla. and Banyan Biomarkers, Alachua Fla.) has
confirmed that UCH-L1 is a reliable and sensitive biomarker for
TBI. Rat CSF and serum samples were obtained from animals that had
received an experimental brain injury using controlled cortical
impact. UCH-L1 levels in CSF and serum were significantly higher in
brain injured animals than they were in uninjured or sham-injured
controls. Likewise, high levels of UCH-L1 can be measured in serum
from human patients with brain injuries but are below the level of
assay detection in normal healthy people.
[0265] Gel Electrophoresis and Immunoblot Analyses of CSF
[0266] Protein concentrations of CSF were determined by
bicinchoninic acid microprotein assays (Pierce Inc., Rockford,
Ill.) with albumin standards. Protein balanced samples were
prepared for sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) in twofold loading buffer containing
0.25 M Tris (pH 6.8), 0.2 M DTT, 8% SDS, 0.02% bromophenol blue,
and 20% glycerol in distilled H.sub.2O. Samples were heated for 2
min. at 90.degree. C. and centrifuged for 1 min. at 10,000 rpm in a
microcentrifuge at ambient temperature. Twenty to forty micrograms
of protein per lane was routinely resolved by SDS-PAGE on 6.5%
Tris/glycine gels for 1 hour at 200V. Following electrophoresis,
separated proteins were laterally transferred to polyvinylidene
fluoride (PVDF) membranes in a transfer buffer containing 400 mM
glycine and 0.025 M Tris (pH 8.9) with 5% methanol at a constant
voltage of 125 V for 2 hour at 4.degree. C. Blots were blocked for
1 hour at ambient temperature in 5% nonfat milk in TBST (25 mM
TrisHCl pH 7.4, 150 mM NaCl, 0.05% Tween-20, 0.02% sodium
azide).
[0267] Immunoblots containing brain or CSF protein were probed with
an anti-neural protein specific primary antibodies (e.g.
anti-UCH-L1, anti-alpha-synuclein and anti-p24). Following an
overnight incubation at 4.degree. C. with the primary antibodies in
5% nonfat milk in TBST, blots were incubated for 1 hour at ambient
temperature in 5% nonfat milk that contained an alkaline
phosphatase or horseradish peroxidase-conjugated goat anti-mouse
IgG (1:10,000 dilution) or goat-anti-rabbit IgG (1:3000). Alkaline
phosphatase-based colorimetric development (BCIP-NBT substrate) or
enhanced chemiluminescence (ECL, Amersham) reagents were used to
visualize immunolabeling on Kodak Biomax ML chemiluminescent
film.
[0268] Assessing Neural Protein Release
[0269] SDS-Polyacrylamide (SDS-PAGE) gel electrophoresis and
immunoblotting. At the end of an experiment, cells were harvested
from 5 identical culture wells and collected in 15 ml centrifuge
tubes and centrifuged at 3000 g for 5 min. The medium was removed
and the pellet cells were rinsed with 1.times.DPBS. Cells were
lysed in ice cold homogenization buffer [20 mM PIPES (pH 7.6), 1 mM
EDTA, 2 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 50 .mu.g/mL Leupeptin, and
10 .mu.g/mL each of AEBSF, aprotinin, pepstatin, TLCK and TPCK for
30 min., and sheared through a 1.0 mL syringe with a 25 gauge
needle 15 times. Protein content in the samples was assayed by the
Micro BCA method (Pierce, Rockford, Ill., USA).
[0270] For protein electrophoresis, equal amounts of total protein
(30 .mu.g) were prepared in two fold loading buffer containing 0.25
M Tris (pH6.8), 0.2 M DTT, 8% SDS, 0.02% bromophenol blue, and 20%
glycerol, and heated at 95.degree. C. for 10 min. Samples were
resolved in a vertical electrophoresis chamber using a 4% stacking
gel over a 7% acrylamide resolving gel for 1 hour at 200V. For
immunoblotting, separated proteins were laterally transferred to
nitrocellulose membranes (0.45 .mu.M) using a transfer buffer
consisting of 0.192 M glycine and 0.025 M Tris (pH 8.3) with 10%
methanol at a constant voltage (100 V) for 1 hour at 4.degree. C.
Blots were blocked overnight in 5% non-fat milk in 20 mM Tris, 0.15
M NaCl, and 0.005% Tween-20 at 4.degree. C. Coomassie blue and
Panceau red (Sigma, St. Louis, Mo.) were used to stain gels and
nitrocellulose membranes (respectively) to confirm that equal
amounts of protein were loaded in each lane.
[0271] Immunoblots were probed as described below with a primary
antibody (e.g. anti-UCH-L1 monoclonal antibody raised in mouse
(Chemicon), anti-alpha-synuclein monoclonal antibody raised in
mouse (Chemicon), anti-p24 monoclonal antibody raised in mouse
(Becton Dickson Bioscience). Following incubation with the primary
antibody (1:2000) for 2 hours at room temperature, the blots were
incubated in peroxidase-conjugated sheep anti-mouse IgG for 1 hour
(1:10,000). Enhanced chemiluminescence reagents (ECL, Amersham)
were used to visualize the immunolabeling on Hyperfilm (Hyperfilm
ECL, Amersham).
[0272] Statistical Analyses.
[0273] Quantitative evaluation of protein levels detected by
immunoblotting was performed by computer-assisted densitometric
scanning (ImageJ-NIH). Data were acquired as integrated
densitometric values and transformed to percentages of the
densitometric levels obtained on scans from sham-injured animals
visualized on the same blot. Data was evaluated by least squares
linear regression followed by ANOVA. All values are given as
mean.+-.SEM. Differences were considered significant if
p<0.05.
[0274] Example 1: Detection of Neural proteins UCH-L1, p24, and
alpha-synuclein in CSF of Rodents Following TBI.
[0275] TBI was induced in rodents as described above. Following TBI
or sham operation or naive rats, samples of CSF were collected and
analyzed for presence of three novel neural protein biomarkers
(e.g. UCH-L1 (FIGS. 3A and 3B), p24 (FIGS. 4A and 4B) and
alpha-synuclein (FIGS. 5A and 5B). Results, shown in FIGS. 3A, 3B,
4A, 4B, 5A, and 5B, demonstrated independent or concurrent
accumulation of UCH-L1 (see FIGS. 3A and 3B), p24 (see FIGS. 4A and
4B) and alpha-synuclein (see FIGS. 5A and 5B), in the CSF of
rodents after TBI. Significantly less of these neural proteins were
observed in sham-injured and naive controls. Each lane in the blots
represents a different animal. The sensitivity of this assay
permits detection of inter-animal differences, which is valuable
for prediction of outcome. The results of this study demonstrated
that after TBI, neural proteins accumulated in the CSF in
sufficient levels to be easily detectable on Western blots or by
other immunoassays such as ELISA.
[0276] Example 2: Detection of Neural proteins UCH-L1 and p24 in
CSF of human TBI.
[0277] Accumulation of novel neural markers (UCH-L1 and p24) was
analyzed in samples of human CSF taken at 24 hr after TBI. From
five patients who experienced severe TBI and five neurological
controls (normal pressure hydrocephalus. As in the rodent models of
TBI, the neural proteins examined (UCH-L1 and p24) were prominent
in CSF samples TBI. Levels of these neural proteins were much
higher in the TBI patients than in the control patients (e.g.
UCH-L1 (FIGS. 6A and 6B), p24 (FIGS. 7A and 7B). These data
demonstrated that after TBI, neural proteins accumulated in human
CSF in sufficient levels to be easily detectable on Western blots
or by other immunoassays such as ELISA.
[0278] Example 3 shows that standard immunodetection method can be
used to detect and quantify P24/Neurorensin biomarker elevation in
Human TBI patient CSF versus control CSF. FIG. 10A shows
immunoblotting detection of P24 and SBDP biomarker in human TBI
patient CSF (12, 30, 42, 48, 78 and 84 h after injury) versus
controls (N) and FIG. 10B shows densitometric quantification of CSF
P24 levels are demonstrated.
[0279] Example 4 shows that standard immunodetection method can
also be used to detect and quantify P24/Neurorensin biomarker
detection in Human TBI patient Serum. FIG. 11A shows immunoblotting
detection of P24 in human TBI serum using sequential centrifuging
filtration/concentration units (1.5 mL) and technique in molecular
weight rang of 30-50 kDa fraction, and FIG. 11B shows densitometric
quantification of serum P24 levels is demonstrated. Same method
applied to normal control serum samples show no detection of P24
(level=zero; data not shown).
[0280] Example 5 shows that standard immunodetection method (ELISA)
can also be used to detect and quantify alpha-synuclein biomarker
elevation in human TBI patient CSF (FIG. 12A) and serum (FIG. 12B).
Sandwich ELISA based detection of alpha-synuclein was used. In FIG.
12A, alpha-synuclein levels in control non-brain injured CSF were
compared to TBI patient CSF samples collected at different
post-injured time (T=E (enrollment) or 12, 24, 48, 72, 96, 120 and
168 h after injury. In FIG. 12B, alpha-synuclein levels in normal
control (non-brain injured) serum were compared to TBI patient
serum samples collected at different post-injured time (T=E
(enrollment) or 24, 72, 96 h after injury).
OTHER EMBODIMENTS
[0281] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *