U.S. patent application number 13/058748 was filed with the patent office on 2011-06-16 for biomarker detection process and assay of neurological condition.
This patent application is currently assigned to Banyan Blomakers, Inc.. Invention is credited to Ronald L. Hayes, Uwe R. Mueller, Kevin Ka-wang Wang, Zhiqun Zhang.
Application Number | 20110143375 13/058748 |
Document ID | / |
Family ID | 41669607 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143375 |
Kind Code |
A1 |
Wang; Kevin Ka-wang ; et
al. |
June 16, 2011 |
BIOMARKER DETECTION PROCESS AND ASSAY OF NEUROLOGICAL CONDITION
Abstract
The subject invention provides a robust, quantitative, and
reproducible process and assay for diagnosis of a neurological
condition in a subject. The invention provides measurement of two
or more biomarkers in a biological fluid such as CSF or serum
resulting in a synergistic mechanism for determining the extent of
neurological damage in a subject with an abnormal neurological
condition and for discerning subtypes thereof or tissue types
subjected to damage.
Inventors: |
Wang; Kevin Ka-wang;
(Gainesville, FL) ; Hayes; Ronald L.; (Alachua,
FL) ; Mueller; Uwe R.; (Alachua, FL) ; Zhang;
Zhiqun; (Alachua, FL) |
Assignee: |
Banyan Blomakers, Inc.
Alachua
FL
|
Family ID: |
41669607 |
Appl. No.: |
13/058748 |
Filed: |
August 11, 2009 |
PCT Filed: |
August 11, 2009 |
PCT NO: |
PCT/US09/53376 |
371 Date: |
February 11, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61218727 |
Jun 19, 2009 |
|
|
|
61097622 |
Sep 17, 2008 |
|
|
|
61188554 |
Aug 11, 2008 |
|
|
|
Current U.S.
Class: |
435/7.21 ;
422/69; 435/287.1; 435/287.2; 435/7.4; 435/7.8; 436/501;
436/86 |
Current CPC
Class: |
G01N 2800/28 20130101;
G01N 2800/2871 20130101; G01N 2800/60 20130101; G01N 33/6896
20130101 |
Class at
Publication: |
435/7.21 ;
436/86; 436/501; 435/7.4; 435/287.1; 422/69; 435/287.2;
435/7.8 |
International
Class: |
G01N 33/53 20060101
G01N033/53; G01N 33/573 20060101 G01N033/573; C12M 1/34 20060101
C12M001/34; B01J 19/00 20060101 B01J019/00 |
Goverment Interests
GOVERNMENTAL SUPPORT
[0002] Portions of this work were supported by grants
N14-06-1-1029, W81XWH-8-1-0376 and W81XWH-07-01-0701 from the
United States Department of Defense.
Claims
1. A process for determining the neurological condition of a
subject or cells from the subject comprising: measuring a sample
obtained from the subject or cells from the subject at a first time
for a quantity of a first biomarker selected from the group of
GFAP, UCH-L1, NSE, MAP2, S100b, or a SBDP, and a quantity of at
least one additional neuroactive biomarker; and comparing the
quantity of said first biomarker and the quantity of said at least
one additional neuroactive biomarker to normal levels of said first
biomarker and said at least one additional neuroactive biomarker to
determine the neurological condition of the subject.
2. The process of claim 1 wherein the sample is cerebrospinal fluid
or blood serum.
3. The process of claim 1 wherein the sample is a culture of the
cells exposed to a drug candidate or an environmental
contaminant.
4. The process of claim 1 wherein said at least one additional
neuroactive biomarker is GFAP, UCH-L1, NSE, SBDP150, SBDP145,
SBDP120, S100b, MAP2, MAP1, MAP3, MAP4, MAPS, MBP, Tau,
Neurofilament protein (NF), Cannabinoid Receptor CB, CAM, Synaptic
protein, CRMP, iNOS, NeuN, CNPase, Neuroserpin, alpha-internexin,
LC3, Neurofascin, EAAT, Nestin, Cortin-1, or BIII-Tubulin.
5. The process of claim 1 wherein is at least one additional
neuroactive biomarker is one of GFAP, UCH-L1, NSE, SBDP150,
SBDP150i, SBDP145, SBDP120, or MAP2.
6. The process of claim 1 further comprising measuring a second
quantity of said first biomarker and a second quantity of said at
least one additional neuroactive biomarker at a second time to
yield a kinetic profile for said first biomarker and said at least
one additional neuroactive biomarker.
7. The process of claim 1 further comprising comparing the quantity
of said first biomarker and the quantity of said at least one
additional neuroactive biomarker between normal levels in the
subject to other individuals of the same gender as the subject.
8. The process of claim 1 wherein said at least one additional
neuroactive biomarker is GFAP.
9. The process of claim 7 wherein said first biomarker is UCH-L1
and determined if the subject or cells from the subject has been
exposed to some degree of traumatic brain injury ranging from mild
to severe.
10. The process of claim 9 further comprising predicting mortality
based on the quantity of UCH-L1 and the quantity of GFAP.
11. The process of claim 9 wherein mild traumatic brain injury and
moderate traumatic brain injury have detection cutoffs for UCH-L1
and GFAP in serum of 0.39 ng/ml and 1.4 ng/ml, respectively.
12. The process of claim 1 wherein the at least one additional
neuroactive biomarker is S100b.
13. The process of claim 1 wherein the at least one additional
neuroactive biomarker is a SBDP of SBDP150, SBDP150i, SBDP145, or
SBDP120.
14. The process of claim 1 wherein the at least one additional
neuroactive biomarker is NSE.
15. The process of claim 1 wherein the at least one additional
neuroactive biomarker is a MAP of MAP2, MAP1, MAP3, MAP4, or
MAPS.
16. The process of any of claims 1 to 15 wherein the first
biomarker is UCH-L1.
17. An assay for determining the neurological condition of a
subject or cells from the subject comprising: a substrate for
holding a sample isolated from the subject or the cells; a first
biomarker specifically binding agent wherein a first biomarker is
one of GFAP, UCH-L1, NSE, MAP2, S100b, or a SBDP; an agent
specifically binding at least one additional neuroactive biomarker;
and printed instructions for reacting said first biomarker specific
agent with a first portion of the sample so as to detect an amount
of said first biomarker and reacting said at least one additional
neuroactive biomarker specific agent with a second portion of the
sample and said at least one additional neuroactive biomarker in
the sample so as to detect an amount of said at least one
additional neuroactive biomarker to determine the neurological
condition of the subject according to the process of claim 1.
18. The assay of claim 17 wherein the first biomarker specific
agent is one of anti-GFAP antibody, anti-UCH-L1 antibody, anti-NSE
antibody, anti-MAP2 antibody, or an anti-SBDP antibody.
19. The assay of claim 17 wherein the agent specifically binding at
least one additional neuroactive biomarker binds one of GFAP, NSE,
SBDP, SBDP150, SBDP150i, SBDP145, SBDP120, S100b, MAP2, MAP1, MAP3,
MAP4, MAPS, MBP, Tau, Neurofilament protein (NF), Cannabinoid
Receptor CB, CAM, Synaptic protein, CRMP, iNOS, NeuN, CNPase,
Neuroserpin, alpha-internexin, LC3, Neurofascin, EAAT, Nestin,
Cortin-1, or BIII-Tubulin.
20. The assay of claim 17 wherein the agent specifically binding at
least one additional neuroactive biomarker binds GFAP.
21. The assay of claim 17 wherein the agent specifically binding at
least one additional neuroactive biomarker binds S100b.
22. The assay of claim 17 wherein the agent specifically binding at
least one additional neuroactive biomarker binds a SBDP of SBDP150,
SBDP150i, SBDP145, or SBDP120.
23. The assay of any of claims 17 to 22 wherein the first biomarker
specific agent is anti-UCH-L1 antibody.
24. The assay of claim 17 wherein said first biomarker specifically
binding agent and said agent specifically binding at least one
additional neuroactive biomarker are both bound to the
substrate.
25. The assay of claim 24 wherein said first biomarker specifically
binding agent and said agent specifically binding at least one
additional neuroactive biomarker are both bound to the substrate
with spatial overlap.
26. The assay of claim 17 wherein the first portion and the second
portion of the sample and the second portion of the sample are the
same, and detection of the amount of said first biomarker and the
amount of said at least one additional neuroactive biomarker occurs
simultaneously.
27. The assay of claim 26 wherein detection of the amount of said
first biomarker and the amount of said at least one additional
neuroactive biomarker occurs with spatial overlap.
28. The assay of any of claims 17 or 24 to 26 further comprising a
separate first biomarker detection species for said first biomarker
and a separate discernable at least one additional neuroactive
biomarker detection species for said at least one additional
neuroactive biomarker.
29. The assay of claim 17 wherein the neurological condition is
stroke, ischemic stroke, hemorrhagic stroke, or subarachnoid
hemorrhage (SAH).
30. The assay of claim 17 wherein the neurological condition is
mild traumatic brain injury or moderate traumatic brain injury.
31. A process for determining if a subject has suffered mild
traumatic brain injury or moderate traumatic brain injury in an
event comprising: measuring a sample obtained from the subject or
cells from the subject at a first time after the event for a
quantity of GFAP; and comparing the quantity of said GFAP to normal
levels of GFAP in a control to determine if the subject has
suffered mild traumatic brain injury or moderate traumatic brain
injury in the event.
32. The process of claim 31 wherein the first time is within 48
hours of the event.
33. The process of claim 31 wherein the sample is blood serum.
34. The process of any of claims 31 to 33 wherein a mean GFAP value
for the subject having suffered suffered mild traumatic brain
injury or moderate traumatic brain injury in the event is
approximately 0.28 nanograms per milliliter of the sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/188,554 filed Aug. 11, 2008; U.S. Provisional
Application No. 61/097,622 filed Sep. 17, 2008; U.S. Provisional
Application No. 61/218,727 filed Jun. 19, 2009; and U.S.
Provisional Application No. 61/271,135 filed Jul. 18, 2009. The
contents of each provisional application is incorporated herein by
reference as if each were explicitly and fully expressed
herein.
FIELD OF THE INVENTION
[0003] The present invention in general relates to determination of
neurological condition of an individual and in particular to
measuring the quantity of a neuroprotective biomarker such as glial
fibrillary acidic protein (GFAP) in concert with another biomarker
of neurological condition.
BACKGROUND OF THE INVENTION
[0004] The field of clinical neurology remains frustrated by the
recognition that secondary injury to a central nervous system
tissue associated with physiologic response to the initial insult
could be lessened if only the initial insult could be rapidly
diagnosed or in the case of a progressive disorder before stress on
central nervous system tissues reached a preselected threshold.
Traumatic, ischemic, and neurotoxic chemical insult, along with
generic disorders, all present the prospect of brain damage. While
the diagnosis of severe forms of each of these causes of brain
damage is straightforward through clinical response testing,
computed tomography (CT), and magnetic resonance imaging (MRI), the
imaging diagnostics are limited by both the high cost of
spectroscopic imaging and long diagnostic time. The clinical
response testing of incapacitated individuals is of limited value
and often precludes a nuanced diagnosis. Additionally, owing to the
limitations of existing diagnostics, situations arise wherein a
subject experiences a stress to their neurological condition but
are often unaware that damage has occurred or fail seek treatment
as the subtle symptoms often quickly resolve. The lack of treatment
of these mild to moderate challenges to neurologic condition of a
subject can have a cumulative effect or otherwise result in a
severe brain damage event, either of which have a poor clinical
prognosis.
[0005] In order to overcome the limitations associated with
spectroscopic and clinical response diagnosis of neurological
condition, there is increasing attention on the use of biomarkers
as internal indicators of change to molecular or cellular level
health condition of a subject. As biomarker detection uses a sample
obtained from a subject, typically cerebrospinal fluid, blood, or
plasma, and detects the biomarkers in that sample, biomarker
detection holds the prospect of inexpensive, rapid, and objective
measurement of neurological condition. The attainment of rapid and
objective indicators of neurological condition allows one to
determine severity of a non-normal brain condition with a
previously unrealized degree of objectivity, predict outcome, guide
therapy of the condition, as well as monitor subject responsiveness
and recovery. Additionally, such information as obtained from
numerous subjects allows one to gain a degree of insight into the
mechanism of brain injury.
[0006] A number of biomarkers have been identified as being
associated with severe traumatic brain injury as is often seen in
vehicle collision and combat wounded subjects. These biomarkers
included spectrin breakdown products such as SBDP150, SBDP150i,
SBDP145 (calpain mediated acute neural necrosis), SBDP120 (caspase
mediated delayed neural apoptosis), UCH-L1 (neuronal cell body
damage marker), and MAP2 dendritic cell injury associated marker.
The nature of these biomarkers is detailed in U.S. Pat. Nos.
7,291,710 and 7,396,654, the contents of which are hereby
incorporated by reference.
[0007] Glial Fibrillary Acidic Protein (GFAP), a member of the
cytoskeletal protein family, is the principal 8-9 nanometer
intermediate filament of glial cells such as mature astrocytes of
the central nervous system (CNS). GFAP is a monomeric molecule with
a molecular mass between 40 and 53 kDa and an isoelectric point
between 5.7 and 5.8. GFAP is highly brain specific protein that is
not found outside the CNS. GFAP is released into the blood and CSF
soon after brain injury. In the CNS following injury, either as a
result of trauma, disease, genetic disorders, or chemical insult,
astrocytes become reactive in a way that is characterized by rapid
synthesis of GFAP termed astrogliosis or gliosis. However, GFAP
normally increases with age and there is a wide variation in the
concentration and metabolic turnover of GFAP in brain tissue.
[0008] Thus, there exists a need for a process and an assay for
providing improved measurement of neurological condition through
the quantification of a first biomarker such as GFAP in combination
with another biomarker associated with neurological condition.
SUMMARY OF THE INVENTION
[0009] A process for determining the neurological condition of a
subject or cells from the subject includes measuring a sample
obtained from the subject or cells from the subject at a first time
for a quantity of a first biomarker selected from the group of
GFAP, UCH-L1, NSE, MAP2, or SBDP. The sample is also measured for a
quantity of at least one additional neuroactive biomarker. Through
comparison of the quantity of the first biomarker and the quantity
of the at least one additional neuroactive biomarker to normal
levels for each biomarker, the neurological condition of the
subject is determined. When the subject have been exposed to an
event that could cause mild traumatic brain injury and moderate
traumatic brain injury, a process of measuring UCH-L1 and GFAP,
such injuries have detection cutoffs for UCH-L1 and GFAP in serum
of 0.39 nanograms per milliliter (ng/ml) and 1.4 ng/ml,
respectively.
[0010] An assay for determining the neurological condition of a
subject or neural cells from the subject is also provided. The
assay includes: (a) a substrate for holding a sample isolated from
a subject or the cells; (b) a first biomarker specifically binding
agent wherein a first biomarker is one of GFAP, UCH-L1, NSE, MAP2,
or SBDP; (c) a binding agent specific for another neuroactive
biomarker (including one of GFAP, UCH-L1, NSE, MAP2, or SBDP not
chosen as the first biomarker); and (d) printed instructions for
reacting the first biomarker specific agent with a first portion of
the sample so as to detect an amount of said first biomarker and
reacting said at least one additional neuroactive biomarker
specific agent with a second portion of the sample and the at least
one additional neuroactive biomarker in the sample so as to detect
an amount of said at least one additional neuroactive biomarker for
relation to the condition of the subject or cells derived the
subject.
[0011] A process for determining if a subject has suffered mild
traumatic brain injury or moderate traumatic brain injury in an
event is provided that includes measuring a sample obtained from
the subject or cells from the subject at a first time after the
event for a quantity of GFAP. By comparing the quantity of GFAP to
normal levels of GFAP in a control, one determines if the subject
has suffered mild traumatic brain injury or moderate traumatic
brain injury in the event.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 represents quantitative western blotting of UCH-L1 in
rat CSF following MCAO;
[0013] FIG. 2 represents UCH-L1 levels in CSF in sham and CCI
treated subjects;
[0014] FIG. 3 represents UCH-L1 levels in CSF following sham, mild
MCAO challenge, and severe MCAO challenge;
[0015] FIG. 4 represents UCH-L1 levels in serum following sham or
CCI at various timepoints;
[0016] FIG. 5 represents UCH-L1 levels in serum following sham,
mild MCAO challenge, and severe MCAO challenge;
[0017] FIG. 6 represents SBDP145 levels in CSF and serum following
sham, mild MCAO challenge, and severe MCAO challenge;
[0018] FIG. 7 represents SBDP120 levels in CSF and serum following
sham, mild MCAO challenge, and severe MCAO challenge;
[0019] FIG. 8 represents MAP2 elevation in CSF and serum following
sham, mild MCAO challenge, and severe MCAO challenge;
[0020] FIG. 9 are bar graphs of GFAP and other biomarkers for human
control and severe TBI subjects from CSF samples;
[0021] FIG. 10 are bar graphs of GFAP and other biomarkers for
human control and severe TBI subjects of FIG. 1 from serum
samples;
[0022] FIG. 11 are bar graphs of GFAP and other biomarkers for
human control and severe TBI subjects summarizing the data of FIGS.
9 and 10;
[0023] FIG. 12 are plots of arterial blood pressure (MABP),
intracranial pressure (ICP) and cerebral profusion pressure (CPP)
for a single human subject of traumatic brain injury as a function
of time;
[0024] FIG. 13 are plots of inventive biomarkers from CSF and serum
samples from the single human subject of traumatic brain injury of
FIG. 12 as a function of time;
[0025] FIG. 14 are plots of inventive biomarkers from CSF and serum
samples from another individual human subject of traumatic brain
injury as a function of time;
[0026] FIG. 15 are plots of UCH-L1 amounts being present in CSF and
serum post severe traumatic brain injury in a mouse subject;
[0027] FIG. 16 are bar graphs of GFAP concentration for controls,
as well as individuals in the mild/moderate traumatic brain injury
cohort as a function of CT scan results upon admission and 24 hours
thereafter;
[0028] FIG. 17 are bar graphs of parallel assays for UCH-L1
biomarker from the samples used for FIG. 16;
[0029] FIG. 18 are bar graphs showing the concentration of UCH-L1
and GFAP as well as a biomarker not selected for diagnosis of
neurological condition, S100 beta, provided as a function of injury
magnitude between control, mild, and moderate traumatic brain
injury;
[0030] FIG. 19 are bar graphs showing the concentration of the same
markers as depicted in FIG. 18 with respect to initial evidence
upon hospital admission as to lesions in tomography scans;
[0031] FIG. 20 represents biomarker levels in human subjects with
varying types of brain injury;
[0032] FIG. 21 are plots that represent ROC analysis of UCH-L1,
GFAP and SBDP145 in human CSF (severe TBI vs. Control A) First 24
hours post-injury;
[0033] FIG. 22 is a plot that represent ROC analysis of UCH-L1 and
GFAP in human CSF (mild TBI vs. normal Controls) a mean of 3h35'
with a range 15'-14h35 post-injury.
[0034] FIG. 23 are bar graphs of showing the elevation of brain
injury biomarkers (GFAP, UCH-L1 and MAP2) in plasma from stroke
patients.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] The present invention has utility in the diagnosis and
management of abnormal neurological condition. Through the
measurement of a biomarker such as GFAP from a subject in
combination with values obtained for an additional neuroactive
biomarker, a determination of subject neurological condition is
provided with greater specificity than previously attainable. The
present description is directed toward a first biomarker of GFAP
for illustrative purposes only and is not meant to be a limitation
on the practice or scope of the present invention. It is
appreciated that the invention encompasses several other first and
additional biomarkers illustratively including UCH-L1, NSE, MAP2,
and SBDP. The description is appreciated by one of ordinary skill
in the art as fully encompassing all inventive biomarkers as an
inventive first biomarker as described herein. Surprisingly, by
combining the detection of more than one biomarker, a synergistic
result is achieved. Illustratively, combining the detection of two
neuroactive biomarkers such as UCH-L1 and GFAP provides sensitive
detection that is unexpectedly able to discern the level and
severity of an abnormal neurological condition in a subject.
[0036] The present invention provides for the detection of a
neurological condition in a subject. A neurological condition may
be an abnormal neurological condition such as that caused by
genetic disorder, injury, or disease to nervous tissue. As such, it
is a further object of the present invention to provide a means for
detecting or diagnosing an abnormal neurological condition in a
subject.
[0037] The present invention also provides an assay for detecting
or diagnosing the neurological condition of a subject. As the
neurological condition may be the result of stress such as that
from exposure to environmental, therapeutic, or investigative
compounds, it is a further aspect of the present invention to
provide a process and assay for screening candidate drug or other
compounds or for detecting the effects of environmental
contaminants regardless of whether the subject itself or cells
derived there from are exposed to the drug candidate or other
possible stressors.
[0038] For purposes of the subject invention, brain injury is
divided into two levels, mild traumatic brain injury (MTBI), and
traumatic brain injury (TBI). An intermediate level of moderate TBI
is also referred to herein. The spectrum between MTBI and extending
through moderate TBI is also referred to synonymously mild to
moderate TBI or by the abbreviation MMTBI. TBI is defined as an
injury that correlates with a two-fold increase or greater of
two-fold decrease or greater in molecular marker levels or
activities. MTBI is defined and an injury that correlates with less
than a two-fold increase or two-fold decrease in molecular marker
levels or activities.
[0039] An inventive process preferably includes determining the
neurological condition of a subject by assaying a sample derived
from a subject at a first time for the presence of a first
biomarker. A biomarker is a cell, protein, nucleic acid, steroid,
fatty acid, metabolite, or other differentiator useful for
measurement of biological activity or response. Biomarkers operable
herein illustratively include: ubiquitin carboxyl-terminal
esterase, ubiquitin carboxy-terminal hydrolase, spectrin breakdown
product(s), a neuronally-localized intracellular protein, MAP-tau,
C-tau, MAP2, poly (ADP-ribose) polymerase (PARP), collapsin
response mediator protein, Annexin A11, Aldehyde dehydrogenase
family 7, Cofilin 1, Profilin 1, .alpha.-Enolase (non-neural
enolase), Enolase 1 protein, Glyceraldehyde-3-phosphate
dehydrogenase, Hexokinase 1, Aconitase 2, Acetyl-CoA synthetase 2,
Neuronal protein 22, Phosphoglycerate kinase 2, Phosphoglycerate
kinase 1, Hsc70-ps1, Glutamate dehydrogenase 1, Aldolase A,
Aldolase C, fructose-biphosphate, Dimethylarginine
dimethylaminohydrolase 1, Microtubule-associated protein 2,
Carbonic anhydrase, ADP-ribosylation factor 3, Transferrin, Liver
regeneration-related protein, Hemoglobin .alpha.-chain, Hemoglobin
.beta. chain, Liver regeneration-related protein, Fetuin .beta.,
3-Oxoacid-CoA transferase, Malate dehydrogenase 1, NAD (soluble),
Lactate dehydrogenase B, Malate dehydrogenase, Carboxylesterase E1
precursor, Serine protease inhibitor .alpha.1, Haptoglobin,
Ubiquitin carboxyl-terminal hydrolase L1, Serine protease inhibitor
2a, T-kininogen, .alpha.1 major acute phase protein, Albumin,
.alpha.1 major acute phase protein prepeptide, Murinoglobulin 1
homolog, Group-specific component protein, Guanosine diphosphate
dissociation inhibitor 1, Collapsin response mediator protein 2,
Murinoglobulin 1 homolog, Ferroxidase, Ceruloplasmin, Spectrin
.alpha.-chain, brain, C-reactive protein, Brain creatine kinase,
Proteasome subunit .alpha.-type 7, 14-3-3 protein, Synaptotagmin,
subtypes thereof, fragments thereof, breakdown products thereof, or
combinations thereof. Other potential biomarkers illustratively
include those identified by Kobeissy, F H, et al, Molecular &
Cellular Proteomics, 2006; 5:1887-1898, the contents of which are
incorporated herein by reference, or others known in the art.
[0040] A first biomarker is preferably a neuroactive biomarker.
Illustrative examples of neuroactive biomarkers include GFAP,
ubiquitin carboxyl-terminal esterase L1 (UCH-L1), Neuron specific
enolase (NSE), spectrin breakdown products (SBDP), preferably
SBDP150, SBDP150i SBDP145, SBDP120, S100 calcium binding protein B
(S100b), microtubule associated proteins (MAP), preferably MAP2,
MAP1, MAP3, MAP4, MAPS, myelin basic protein (MBP), Tau,
Neurofilament protein (NF), Cannabinoid Receptor (CB), CAM
proteins, Synaptic protein, collapsin response mediator proteins
(CRMP), inducible nitric oxide synthase (iNOS), Neuronal Nuclei
protein (NeuN), 2',3'-cyclic nucleotide-3'-phosphohydrolase
(CNPase), Neuroserpin, alpha-internexin, microtubule-associated
protein 1 light chain 3 (LC3), Neurofascin, the glutamate
transporters (EAAT), Nestin, Cortin-1, 2', and BIII-Tubulin.
[0041] The inventive process also includes assaying the sample for
at least one additional neuroactive biomarker. The one additional
neuroactive biomarker is preferably not the same biomarker as the
first biomarker. Any of the aforementioned inventive biomarkers are
operable as an additional neuroactive biomarker. Any number of
biomarkers can be detected such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more. Detection can be either simultaneous or sequential and may be
from the same biological sample or from multiple samples from the
same or different subjects. Preferably, detection of multiple
biomarkers is in the same assay chamber. The inventive process
further includes comparing the quantity of the first biomarker and
the quantity of the at least one additional neuroactive biomarker
to normal levels of each of the first biomarker and the one
additional neuroactive biomarker to determine the neurological
condition of the subject.
[0042] In a preferred embodiment a biomarker is GFAP. GFAP is
associated with glial cells such as astrocytes. Preferably, an
additional neuroactive biomarker is associated with the health of a
different type of cell associated with neural function. For
example, CNPase is found in the myelin of the central nervous
system, and NSE is found primarily in neurons. More preferably, the
other cell type is an axon, neuron, or dendrite.
[0043] In another preferred embodiment, especially for MBTI and
MMTBI, is UCH-L1 in combination with other biomarkers such as GFAP
and MAP2.
[0044] It is appreciated however, that multiple biomarkers may be
predictors of different modes or types of damage to the same cell
type. Through the use of an inventive assay inclusive of biomarkers
associated with glial cells as well as at least one other type of
neural cell, the type of neural cells being stressed or killed as
well as quantification of neurological condition results provides
rapid and robust diagnosis of traumatic brain injury type.
Measuring GFAP along with at least one additional neuroactive
biomarker and comparing the quantity of GFAP and the additional
biomarker to normal levels of the markers provides a determination
of subject neurological condition.
[0045] Preferably, specific biomarker levels that when measured in
concert with GFAP afford superior evaluation of subject
neurological condition include SBDP 150, SBDP150i, a combination of
SBDP145 (calpain mediated acute neural necrosis) and SBDP120
(caspase mediated delayed neural apoptosis), UCH-L1 (neuronal cell
body damage marker), and MAP2. This is noted to be of particular
value in measuring MMTBI and screening drug candidates or other
neural cell stressor compounds with cell cultures.
[0046] A sample is preferably a biological sample. Preferred
examples of biological samples are illustratively cells, tissues,
cerebral spinal fluid (CSF), artificial CSF, whole blood, serum,
plasma, cytosolic fluid, urine, feces, stomach fluids, digestive
fluids, saliva, nasal or other airway fluid, vaginal fluids, semen,
buffered saline, saline, water, or other biological fluid
recognized in the art. Most preferably, a biological sample is CSF
or blood serum. It is appreciated that two or more separate
biological samples are optionally assayed to elucidate the
neurological condition of the subject.
[0047] In addition to increased cell expression, biomarkers also
appear in biological fluids in communication with injured cells.
Obtaining biological fluids such as cerebrospinal fluid (CSF),
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 that 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. Serum is a preferred biological sample as it is easily
obtainable and presents much less risk of further injury or
side-effect to a donating subject.
[0048] To provide correlations between neurological condition and
measured quantities of GFAP and other neuroactive biomarkers,
samples of CSF or serum are collected from subjects with the
samples being subjected to measurement of GFAP as well as other
neuroactive biomarkers. The subjects vary in neurological
condition. Detected levels of GFAP and other neuroactive biomarkers
are optionally then correlated with CT scan results as well as GCS
scoring. Based on these results, an inventive assay is developed
and validated (Lee et al., Pharmacological Research 23:312-328,
2006). It is appreciated that GFAP and other neuroactive
biomarkers, in addition to being obtained from CSF and serum, are
also readily obtained from blood, plasma, saliva, urine, as well as
solid tissue biopsy. While CSF is a preferred sampling fluid owing
to direct contact with the nervous system, it is appreciated that
other biological fluids have advantages in being sampled for other
purposes and therefore allow for inventive determination of
neurological condition as part of a battery of tests performed on a
single sample such as blood, plasma, serum, saliva or urine.
[0049] A biological sample is obtained from a subject by
conventional techniques. For example, CSF is preferably obtained by
lumbar puncture. Blood is preferably obtained by venipuncture,
while plasma and serum are 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, WB 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).
[0050] After insult, nerve cells in in vitro culture or in situ in
a subject express altered levels or activities of one or more
proteins than do such cells not subjected to the insult. Thus,
samples that contain nerve cells, e.g., a biopsy of a central
nervous system or peripheral nervous system tissue are
illustratively suitable biological samples for use in the
invention. In addition to nerve cells, however, other cells express
illustratively .alpha.II-spectrin including, for example,
cardiomyocytes, myocytes in skeletal muscles, hepatocytes, kidney
cells and cells in testis. A biological sample including such cells
or fluid secreted from these cells might also be used in an
adaptation of the inventive methods to determine and/or
characterize an injury to such non-nerve cells.
[0051] A subject illustratively includes a dog, a cat, a horse, a
cow, a pig, a sheep, a goat, a chicken, non-human primate, a human,
a rat, and a mouse. Subjects who most benefit from the present
invention are those suspected of having or at risk for developing
abnormal neurological conditions, such as victims of brain injury
caused by traumatic insults (e.g., gunshot wounds, automobile
accidents, sports accidents, shaken baby syndrome), ischemic events
(e.g., stroke, cerebral hemorrhage, cardiac arrest),
neurodegenerative disorders (such as Alzheimer's, Huntington's, and
Parkinson's diseases; prion-related disease; other forms of
dementia), epilepsy, substance abuse (e.g., from amphetamines,
Ecstasy/MDMA, or ethanol), and peripheral nervous system
pathologies such as diabetic neuropathy, chemotherapy-induced
neuropathy and neuropathic pain.
[0052] Baseline levels of several biomarkers are those levels
obtained in the target biological sample in the species of desired
subject in the absence of a known neurological condition. These
levels need not be expressed in hard concentrations, but may
instead be known from parallel control experiments and expressed in
terms of fluorescent units, density units, and the like. Typically,
in the absence of a neurological condition SBDPs are present in
biological samples at a negligible amount. However, UCH-L1 is a
highly abundant protein in neurons. Determining the baseline levels
of UCH-L1 in neurons of particular species is well within the skill
of the art. Similarly, determining the concentration of baseline
levels of MAP2, GFAP, NSE, or other biomarker is well within the
skill of the art.
[0053] As used herein the term "diagnosing" means recognizing the
presence or absence of a neurological or other condition such as an
injury or disease. Diagnosing is optionally referred to as the
result of an assay wherein a particular ratio or level of a
biomarker is detected or is absent.
[0054] As used herein a "ratio" is either a positive ratio wherein
the level of the target is greater than the target in a second
sample or relative to a known or recognized baseline level of the
same target. A negative ratio describes the level of the target as
lower than the target in a second sample or relative to a known or
recognized baseline level of the same target. A neutral ratio
describes no observed change in target biomarker.
[0055] As used herein an injury is an alteration in cellular or
molecular integrity, activity, level, robustness, state, or other
alteration that is traceable to an event. Injury illustratively
includes a physical, mechanical, chemical, biological, functional,
infectious, or other modulator of cellular or molecular
characteristics. An event is illustratively, a physical trauma such
as an impact (percussive) or a biological abnormality such as a
stroke resulting from either blockade or leakage of a blood vessel.
An event is optionally an infection by an infectious agent. A
person of skill in the art recognizes numerous equivalent events
that are encompassed by the terms injury or event.
[0056] An injury is optionally a physical event such as a
percussive impact. An impact is the like of a percussive injury
such as resulting to a blow to the head that either leaves the
cranial structure intact or results in breach thereof.
Experimentally, several impact methods are used illustratively
including controlled cortical impact (CCI) at a 1.6 mm depression
depth, equivalent to severe TBI in human. This method is described
in detail by Cox, C D, et al., J Neurotrauma, 2008; 25(11):1355-65.
It is appreciated that other experimental methods producing impact
trauma are similarly operable.
[0057] TBI may also result from stroke. Ischemic stroke is
optionally modeled by middle cerebral artery occlusion (MCAO) in
rodents. UCH-L1 protein levels, for example, are increased
following mild MCAO which is further increased following severe
MCAO challenge. Mild MCAO challenge may result in an increase of
protein levels within two hours that is transient and returns to
control levels within 24 hours. In contrast, severe MCAO challenge
results in an increase in protein levels within two hours following
injury and may be much more persistent demonstrating statistically
significant levels out to 72 hours or more.
[0058] An exemplary process for detecting the presence or absence
of GFAP and one or more other neuroactive biomarkers in a
biological sample involves obtaining a biological sample from a
subject, such as a human, contacting the biological sample with a
compound or an agent capable of detecting of the marker being
analyzed, illustratively including an antibody or aptamer, and
analyzing binding of the compound or agent to the sample after
washing. Those samples having specifically bound compound or agent
express the marker being analyzed.
[0059] An inventive process can be used to detect GFAP and one or
more other neuroactive biomarkers in a biological sample in vitro,
as well as in vivo. The quantity of GFAP and one or more other
neuroactive biomarkers in a sample is compared with appropriate
controls such as a first sample known to express detectable levels
of the marker being analyzed (positive control) and a second sample
known to not express detectable levels of the marker being analyzed
(a negative control). For example, in vitro techniques for
detection of a marker illustratively include enzyme linked
immunosorbent assays (ELISAs), radioimmuno assay, radioassay,
western blot, Southern blot, northern blot, immunoprecipitation,
immunofluorescence, mass spectrometry, RT-PCR, PCR, liquid
chromatography, high performance liquid chromatography, enzyme
activity assay, cellular assay, positron emission tomography, mass
spectroscopy, combinations thereof, or other technique known in the
art. Furthermore, in vivo techniques for detection of a marker
include introducing a labeled agent that specifically binds the
marker into a biological sample or test subject. For example, the
agent can be labeled with a radioactive marker whose presence and
location in a biological sample or test subject can be detected by
standard imaging techniques. Optionally, the first biomarker
specifically binding agent and the agent specifically binding at
least one additional neuroactive biomarker are both bound to a
substrate. It is appreciated that a bound agent assay is readily
formed with the agents bound with spatial overlap, with detection
occurring through discernibly different detection for first
biomarker and each of at least one additional neuroactive
biomarkers. A color intensity based quantification of each of the
spatially overlapping bound biomarkers is representative of such
techniques.
[0060] Any suitable molecule that can specifically bind GFAP and
any suitable molecule that specifically binds one or more other
neuroactive biomarkers are operative in the invention to achieve a
synergistic assay. A preferred agent for detecting GFAP or the one
or more other neuroactive biomarkers is an antibody capable of
binding to the biomarker being analyzed. Preferably, an antibody is
conjugated with a detectable label. Such antibodies can be
polyclonal or monoclonal. An intact antibody, a fragment thereof
(e.g., Fab or F(ab').sub.2), or an engineered variant thereof
(e.g., sFv) can also be used. Such antibodies can be of any
immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof. Antibodies for numerous inventive biomarkers are
available from vendors known to one of skill in the art.
Illustratively, antibodies directed to inventive biomarkers are
available from Santa Cruz Biotechnology (Santa Cruz, Calif.).
Exemplary antibodies operative herein to detect a first biomarker
include anti-GFAP antibody, anti-UCH-L1 antibody, anti-NSE
antibody, anti-MAP2 antibody, or an anti-SBDP antibody. Other
biomarkers to be targeted as part of an inventive assay different
from the first biomarker include GFAP, NSE, SBDP, SBDP150, SBDP145,
SBDP120, S100b, MAP2, MAP1, MAP3, MAP4, MAPS, MBP, Tau,
Neurofilament protein (NF), Cannabinoid Receptor CB, CAM, Synaptic
protein, CRMP, iNOS, NeuN, CSPase, Neuroserpin, alpha-internexin,
LC3, Neurofascin, EAAT, Nestin, Cortin-1, or BIII-Tubulin
[0061] An antibody is optionally labeled. A person of ordinary
skill in the art recognizes numerous labels operable herein. Labels
and labeling kits are commercially available optionally from
Invitrogen Corp, Carlsbad, Calif. Labels illustratively include,
fluorescent labels, biotin, peroxidase, radionucleotides, or other
label known in the art. Alternatively, a detection species of
another antibody or other compound known to the art is used as form
detection of a biomarker bound by an antibody.
[0062] Antibody-based assays are preferred for analyzing a
biological sample for the presence of GFAP and one or more other
neuroactive biomarkers. Suitable western blotting methods are
described below in the examples section. For more rapid analysis
(as may be important in emergency medical situations),
immunosorbent assays (e.g., ELISA and RIA) and immunoprecipitation
assays may be used. As one example, the biological sample or a
portion thereof is immobilized on a substrate, such as a membrane
made of nitrocellulose or PVDF; or a rigid substrate made of
polystyrene or other plastic polymer such as a microtiter plate,
and the substrate is contacted with an antibody that specifically
binds GFAP, or one of the other neuroactive biomarkers under
conditions that allow binding of antibody to the biomarker being
analyzed. After washing, the presence of the antibody on the
substrate indicates that the sample contained the marker being
assessed. If the antibody is directly conjugated with a detectable
label, such as an enzyme, fluorophore, or radioisotope, the
presence of the label is optionally detected by examining the
substrate for the detectable label. Alternatively, a detectably
labeled secondary antibody that binds the marker-specific antibody
is added to the substrate. The presence of detectable label on the
substrate after washing indicates that the sample contained the
marker.
[0063] Numerous permutations of these basic immunoassays are also
operative in the invention. These include the biomarker-specific
antibody, as opposed to the sample being immobilized on a
substrate, and the substrate is contacted with GFAP or another
neuroactive biomarker conjugated with a detectable label under
conditions that cause binding of antibody to the labeled marker.
The substrate is then contacted with a sample under conditions that
allow binding of the marker being analyzed to the antibody. A
reduction in the amount of detectable label on the substrate after
washing indicates that the sample contained the marker.
[0064] Although antibodies are preferred for use in the invention
because of their extensive characterization, any other suitable
agent (e.g., a peptide, an aptamer, or a small organic molecule)
that specifically binds GFAP or another neuroactive biomarker is
optionally used in place of the antibody in the above described
immunoassays. For example, an aptamer that specifically binds all
spectrin and/or one or more of its SBDPs might be used. Aptamers
are nucleic acid-based molecules that bind specific ligands.
Methods for making aptamers with a particular binding specificity
are known as detailed in U.S. Pat. Nos. 5,475,096; 5,670,637;
5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985; 5,567,588;
5,683,867; 5,637,459; and 6,011,020.
[0065] A myriad of detectable labels that are operative in a
diagnostic assay for biomarker expression are known in the art.
Agents used in methods for detecting GFAP or another neuroactive
biomarker are conjugated to a detectable label, e.g., an enzyme
such as horseradish peroxidase. Agents labeled with horseradish
peroxidase can be detected by adding an appropriate substrate that
produces a color change in the presence of horseradish peroxidase.
Several other detectable labels that may be used are known. Common
examples of these include alkaline phosphatase, horseradish
peroxidase, fluorescent compounds, luminescent compounds, colloidal
gold, magnetic particles, biotin, radioisotopes, and other enzymes.
It is appreciated that a primary/secondary antibody system is
optionally used to detect one or more biomarkers. A primary
antibody that specifically recognizes one or more biomarkers is
exposed to a biological sample that may contain the biomarker of
interest. A secondary antibody with an appropriate label that
recognizes the species or isotype of the primary antibody is then
contacted with the sample such that specific detection of the one
or more biomarkers in the sample is achieved.
[0066] The present invention employs a step of correlating the
presence or amount of GFAP alone, or with one or more other
neuroactive biomarker in a biological sample with the severity
and/or type of nerve cell injury. GFAP measurement alone is shown
herein to be highly effective in detecting MMTBI. The amount of
GFAP and one or more other neuroactive biomarkers in the biological
sample are associated with a neurological condition such as
traumatic brain injury as detailed in the examples. The results of
an inventive assay to synergistically measure GFAP and one or more
other neuroactive biomarkers can help a physician or veterinarian
determine the type and severity of injury with implications as to
the types of cells that have been compromised. These results are in
agreement with CT scan and GCS results, yet are quantitative,
obtained more rapidly, and at far lower cost.
[0067] The present invention provides a step of comparing the
quantity of GFAP and the amount of at least one other neuroactive
biomarker to normal levels to determine the neurological condition
of the subject. It is appreciated that selection of additional
biomarkers allows one to identify the types of cells implicated in
an abnormal neurological condition as well as the nature of cell
death in the case of an axonal injury marker, namely an SBDP. The
practice of an inventive process provides a test which can help a
physician determine suitable therapeutics to administer for optimal
benefit of the subject. While the data provided in the examples
herein are provided with respect to a full spectrum of traumatic
brain injury, it is appreciated that these results are applicable
to ischemic events, neurodegenerative disorders, prion related
disease, epilepsy, chemical etiology and peripheral nervous system
pathologies. As is shown in the subsequently provided example data,
a gender difference is unexpectedly noted in abnormal subject
neurological condition.
[0068] An assay for analyzing cell damage in a subject or a cell
culture isolated therefrom is also provided. The assay includes:
(a) a substrate for holding a sample isolated from a subject
suspected of having a damaged nerve cell, the sample being a fluid
in communication with the nervous system of the subject prior to
being isolated from the subject; (b) a GFAP (or other biomarker)
specific binding agent; (c) a binding agent specific for another
neuroactive biomarker; and (d) printed instructions for performing
the assay illustratively for reacting: the specific binding agent
with the biological sample or a portion of the biological sample to
detect the presence or amount of biomarker, and the agent specific
for another neuroactive biomarker with the biological sample or a
portion of the biological sample to detect the presence or amount
of the at least one biomarker in the biological sample. The
inventive assay can be used to detect a neurological condition for
financial renumeration.
[0069] The assay optionally includes a detectable label such as one
conjugated to the agent, or one conjugated to a substance that
specifically binds to the agent, such as a secondary antibody.
[0070] An inventive process illustratively includes diagnosing a
neurological condition in a subject, treating a subject with a
neurological condition, or both. In a preferred embodiment an
inventive process illustratively includes obtaining a biological
sample from a subject. The biological sample is assayed by
mechanisms known in the art for detecting or identifying the
presence of one or more biomarkers present in the biological
sample. Based on the amount or presence of a target biomarker in a
biological sample, a ratio of one or more biomarkers is optionally
calculated. The ratio is optionally the level of one or more
biomarkers relative to the level of another biomarker in the same
or a parallel sample, or the ratio of the quantity of the biomarker
to a measured or previously established baseline level of the same
biomarker in a subject known to be free of a pathological
neurological condition. The ratio allows for the diagnosis of a
neurological condition in the subject. An inventive process also
optionally administers a therapeutic to the subject that will
either directly or indirectly alter the ratio of one or more
biomarkers.
[0071] An inventive process is also provided for diagnosing and
optionally treating a multiple-organ injury. Multiple organs
illustratively include subsets of neurological tissue such as
brain, spinal cord and the like, or specific regions of the brain
such as cortex, hippocampus and the like. Multiple injuries
illustratively include apoptotic cell death which is detectable by
the presence of caspase induced SBDPs, and oncotic cell death which
is detectable by the presence of calpain induced SBDPs. The
inventive process illustratively includes assaying for a plurality
of biomarkers in a biological sample obtained from a subject
wherein the biological was optionally in fluidic contact with an
organ suspected of having undergone injury or control organ when
the biological sample was obtained from the subject. The inventive
process determines a first subtype of organ injury based on a first
ratio of a plurality of biomarkers. The inventive process also
determines a second subtype of a second organ injury based on a
second ratio of the plurality of biomarkers in the biological
sample. The ratios are illustratively determined by processes
described herein or known in the art.
[0072] The subject invention illustratively includes a composition
for distinguishing the magnitude of a neurological condition in a
subject. An inventive composition is either an agent entity or a
mixture of multiple agents. In a preferred embodiment a composition
is a mixture. The mixture optionally contains a biological sample
derived from a subject. The subject is optionally suspected of
having a neurological condition. The biological sample in
communication with the nervous system of the subject prior to being
isolated from the subject. In inventive composition also contains
at least two primary agents, preferably antibodies, that
specifically and independently bind to at least two biomarkers that
may be present in the biological sample. In a preferred embodiment
the first primary agent is in antibody that specifically binds
GFAP. A second primary agent is preferably an antibody that
specifically binds a ubiquitin carboxyl-terminal hydrolase,
preferably UCH-L1, or a spectrin breakdown product.
[0073] The agents of the inventive composition are optionally
immobilized or otherwise in contact with a substrate. The inventive
teachings are also preferably labeled with at least one detectable
label. In a preferred embodiment the detectable label on each agent
is unique and independently detectable in either the same assay
chamber or alternate chambers. Optionally a secondary agent
specific for detecting or binding to the primary agent is labeled
with at least one detectable label. In the nonlimiting example the
primary agent is a rabbit derived antibody. A secondary agent is
optionally an antibody specific for a rabbit derived primary
antibody. Mechanisms of detecting antibody binding to an antigen
are well known in the art, and a person of ordinary skill in the
art readily envisions numerous methods and agents suitable for
detecting antigens or biomarkers in a biological sample.
[0074] The invention employs a step of correlating the presence or
amount of a biomarker in a biological sample with the severity
and/or type of nerve cell (or other biomarker-expressing cell)
injury. The amount of biomarker(s) in the biological sample
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 biomarker(s) to accumulate in the
biological sample (e.g., CSF; serum). Whether a nerve cell injury
triggers an apoptotic and/or necrotic type of cell death can also
be determined by examining the SBDPs present in the biological
sample. Necrotic cell death preferentially activates calpain,
whereas apoptotic cell death preferentially activates caspase-3.
Because calpain and caspase-3 SBDPs can be distinguished,
measurement of these markers indicates the type of cell damage in
the subject. For example, necrosis-induced calpain activation
results in the production of SBDP150 and SBDP145; apoptosis-induced
caspase-3 activation results in the production of SBDP150i and
SBDP120; and activation of both pathways results in the production
of all four markers. Also, the level of or kinetic extent of UCH-L1
present in a biological sample may optionally distinguish mild
injury from a more severe injury. In an illustrative example,
severe MCAO (2 h) produces increased UCH-L1 in both CSF and serum
relative to mild challenge (30 min) while both produce UCH-L1
levels in excess of uninjured subjects. Moreover, the persistence
or kinetic extent of the markers in a biological sample is
indicative of the severity of the injury with greater injury
indicating increases persistence of GFAP, UCH-L1, or SBDP in the
subject that is measured by an inventive process in biological
samples taken at several time points following injury.
[0075] The results of such a test can help a physician determine
whether the administration a particular therapeutic such as calpain
and/or caspase inhibitors or muscarinic cholinergic receptor
antagonists might be of benefit to a patient. This method may be
especially important in detecting age and gender difference in cell
death mechanism.
[0076] It is appreciated that other reagents such as assay grade
water, buffering agents, membranes, assay plates, secondary
antibodies, salts, and other ancillary reagents are available from
vendors known to those of skill in the art. Illustratively, assay
plates are available from Corning, Inc. (Corning, N.Y.) and
reagents are available from Sigma-Aldrich Co. (St. Louis, Mo.).
[0077] Methods involving conventional biological techniques are
described herein. Such techniques are generally known in the art
and are described in detail in methodology treatises such as
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Immunological methods (e.g.,
preparation of antigen-specific antibodies, immunoprecipitation,
and immunoblotting) are described, e.g., in Current Protocols in
Immunology, ed. Coligan et al., John Wiley & Sons, New York,
1991; and Methods of Immunological Analysis, ed. Masseyeff et al.,
John Wiley & Sons, New York, 1992.
[0078] Various aspects of the present invention are illustrated by
the following non-limiting examples. The examples are for
illustrative purposes and are not a limitation on any practice of
the present invention. It will be understood that variations and
modifications can be made without departing from the spirit and
scope of the invention. While the examples are generally directed
to mammalian tissue, specifically, analyses of mouse tissue, a
person having ordinary skill in the art recognizes that similar
techniques and other techniques known in the art readily translate
the examples to other mammals such as humans. Reagents illustrated
herein are commonly cross reactive between mammalian species or
alternative reagents with similar properties are commercially
available, and a person of ordinary skill in the art readily
understands where such reagents may be obtained. Variations within
the concepts of the invention are apparent to those skilled in the
art.
EXAMPLE 1
Materials for Biomarker Analyses
[0079] Illustrative reagents used in performing the subject
invention include Sodium bicarbonate (Sigma Cat #: C-3041),
blocking buffer (Startingblock T20-TBS) (Pierce Cat#: 37543), Tris
buffered saline with Tween 20 (TBST; Sigma Cat #: T-9039).
Phosphate buffered saline (PBS; Sigma Cat #: P-3813); Tween 20
(Sigma Cat #: P5927); Ultra TMB ELISA (Pierce Cat #: 34028); and
Nunc maxisorp ELISA plates (Fisher). Monoclonal and polyclonal GFAP
and UCH-L1 antibodies are made in-house or are obtained from Santa
Cruz Biotechnology, Santa Cruz, Calif. Antibodies directed to
.alpha.-II spectrin and breakdown products as well as to MAP2 are
available from Santa Cruz Biotechnology, Santa Cruz, Calif. Labels
for antibodies of numerous subtypes are available from Invitrogen,
Corp., Carlsbad, Calif. Protein concentrations in biological
samples are determined using bicinchoninic acid microprotein assays
(Pierce Inc., Rockford, Ill., USA) with albumin standards. All
other necessary reagents and materials are known to those of skill
in the art and are readily ascertainable.
EXAMPLE 2
Biomarker Assay Development
[0080] Anti-biomarker specific rabbit polyclonal antibody and
monoclonal antibodies are produced in the laboratory. To determine
reactivity specificity of the antibodies to detect a target
biomarker a known quantity of isolated or partially isolated
biomarker is analyzed or 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 optimal concentration of
the antibodies used in the assay. Microplate wells are coated with
rabbit polyclonal anti-human biomarker antibody. After determining
the concentration of rabbit anti-human biomarker antibody for a
maximum signal, the lower detection limit of the indirect ELISA for
each antibody is determined. An appropriate diluted sample is
incubated with a rabbit polyclonal antihuman biomarker antibody for
2 hours and then washed. Biotin labeled monoclonal anti-human
biomarker antibody is then added and incubated with captured
biomarker. After thorough wash, streptavidin horseradish peroxidase
conjugate is added. After 1 hour incubation and the last washing
step, the remaining conjugate is allowed to react with substrate of
hydrogen peroxide tetramethyl benzadine. The reaction is stopped by
addition of the acidic solution and absorbance of the resulting
yellow reaction product is measured at 450 nanometers. The
absorbance is proportional to the concentration of the biomarker. A
standard curve is constructed by plotting absorbance values as a
function of biomarker concentration using calibrator samples and
concentrations of unknown samples are determined using the standard
curve.
EXAMPLE 3
In Vivo Model of TBI Injury Model
[0081] A controlled cortical impact (CCI) device is used to model
TBI on rats as previously described (Pike et al, 1998). Adult male
(280-300 g) Sprague-Dawley rats (Harlan: Indianapolis, Ind.) are
anesthetized with 4% isoflurane in a carrier gas of 1:1
O.sub.2/N.sub.2O (4 min.) and maintained in 2.5% isoflurane in the
same carrier gas. Core body temperature is 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 are mounted in a stereotactic frame in a prone
position and secured by ear and incisor bars. Following a midline
cranial incision and reflection of the soft tissues, a unilateral
(ipsilateral to site of impact) craniotomy (7 mm diameter) is
performed adjacent to the central suture, midway between bregma and
lambda. The dura mater is kept intact over the cortex. Brain trauma
is produced by impacting the right (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 1.6 mm compression and 150 ms dwell
time. Sham-injured control animals are subjected to identical
surgical procedures but do not receive the impact injury.
Appropriate pre- and post-injury management is preformed to insure
compliance with guidelines set forth by the University of Florida
Institutional Animal Care and Use Committee and the National
Institutes of Health guidelines detailed in the Guide for the Care
and Use of Laboratory Animals. In addition, research is conducted
in compliance with the Animal Welfare Act and other federal
statutes and regulations relating to animals and experiments
involving animals and adhered to principles stated in the "Guide
for the Care and Use of Laboratory Animals, NRC Publication, 1996
edition."
EXAMPLE 4
Middle Cerebral Artery Occlusion (MCAO) Injury Model
[0082] Rats are incubated under isoflurane anesthesia (5%
isoflurane via induction chamber followed by 2% isoflurane via nose
cone), the right common carotid artery (CCA) of the rat is exposed
at the external and internal carotid artery (ECA and ICA)
bifurcation level with a midline neck incision. The ICA is followed
rostrally to the pterygopalatine branch and the ECA is ligated and
cut at its lingual and maxillary branches. A 3-0 nylon suture is
then introduced into the ICA via an incision on the ECA stump (the
suture's path was visually monitored through the vessel wall) and
advanced through the carotid canal approximately 20 mm from the
carotid bifurcation until it becomes lodged in the narrowing of the
anterior cerebral artery blocking the origin of the middle cerebral
artery. The skin incision is then closed and the endovascular
suture left in place for 30 minutes or 2 hours. Afterwards the rat
is briefly reanesthetized and the suture filament is retracted to
allow reperfusion. For sham MCAO surgeries, the same procedure is
followed, but the filament is advanced only 10 mm beyond the
internal-external carotid bifurcation and is left in place until
the rat is sacrificed. During all surgical procedures, animals are
maintained at 37.+-.1.degree. C. by a homeothermic heating blanket
(Harvard Apparatus, Holliston, Mass., U.S.A.). It is important to
note that at the conclusion of each experiment, if the rat brains
show pathologic evidence of subarachnoid hemorrhage upon necropsy
they are excluded from the study. Appropriate pre- and post-injury
management is preformed to insure compliance with all animal care
and use guidelines.
EXAMPLE 5
Tissue and Sample Preparation
[0083] At the appropriate time points (2, 6, 24 hours and 2, 3, 5
days) after injury, animals are anesthetized and immediately
sacrificed by decapitation. Brains are quickly removed, rinsed with
ice cold PBS and halved. The right hemisphere (cerebrocortex around
the impact area and hippocampus) is rapidly dissected, rinsed in
ice cold PBS, snap-frozen in liquid nitrogen, and stored at
-80.degree. C. until used. For immunohistochemistry, brains are
quick frozen in dry ice slurry, sectioned via cryostat (20 .mu.m)
onto SUPERFROST PLUS GOLD.RTM. (Fisher Scientific) slides, and then
stored at -80.degree. C. until used. For the left hemisphere, the
same tissue as the right side is collected. For Western blot
analysis, the brain samples are pulverized with a small mortar and
pestle set over dry ice to a fine powder. The pulverized brain
tissue powder is then lysed for 90 min at 4.degree. C. in a buffer
of 50 mM Tris (pH 7.4), 5 mM EDTA, 1% (v/v) Triton X-100, 1 mM DTT,
1.times. protease inhibitor cocktail (Roche Biochemicals). The
brain lysates are then centrifuged at 15,000.times.g for 5 min at
4.degree. C. to clear and remove insoluble debris, snap-frozen, and
stored at -80.degree. C. until used.
[0084] For gel electrophoresis and electroblotting, cleared CSF
samples (7 .mu.l) are prepared for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a
2.times. 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. Twenty micrograms (20 .mu.g) of protein per lane are
routinely resolved by SDS-PAGE on 10-20% Tris/glycine gels
(Invitrogen, Cat #EC61352) at 130 V for 2 hours. Following
electrophoresis, separated proteins are laterally transferred to
polyvinylidene fluoride (PVDF) membranes in a transfer buffer
containing 39 mM glycine, 48 mM Tris-HCl (pH 8.3), and 5% methanol
at a constant voltage of 20 V for 2 hours at ambient temperature in
a semi-dry transfer unit (Bio-Rad). After electro-transfer, the
membranes are blocked for 1 hour at ambient temperature in 5%
non-fat milk in TBS and 0.05% Tween-2 (TBST) then are incubated
with the primary polyclonal UCH-L1 antibody in TBST with 5% non-fat
milk at 1:2000 dilution as recommended by the manufacturer at
4.degree. C. overnight. This is followed by three washes with TBST,
a 2 hour incubation at ambient temperature with a biotinylated
linked secondary antibody (Amersham, Cat #RPN1177v1), and a 30 min
incubation with Streptavidin-conjugated alkaline phosphatase
(BCIP/NBT reagent: KPL, Cat #50-81-08). Molecular weights of intact
biomarker proteins are assessed using rainbow colored molecular
weight standards (Amersham, Cat #RPN800V). Semi-quantitative
evaluation of intact GFAP, UCH-L1, or SBDP protein levels is
performed via computer-assisted densitometric scanning (Epson
XL3500 scanner) and image analysis with ImageJ software (NIH).
EXAMPLE 6
UCH-L1 is Increased in CSF Following MCAO Challenge
[0085] Subjects are subjected to MCAO challenge and CSF samples
analyzed by quantitative western blotting. UCH-L1 protein is
readily detectable after injury at statically significant levels
above the amounts of UCH-L1 in sham treated samples (FIGS. 1A, B).
These UCH-L1 levels are transiently elevated (at 6 h) after mild
ischemia (30 min MCAO) followed by reperfusion, while levels are
sustained from 6 to 72 h after a more severe (2 h MCAO) ischemia
(FIGS. 1A, B).
EXAMPLE 7
ELISA Readily Identifies UCH-L1 Levels in Both CSF and Serum
[0086] ELISA is used to more rapidly and readily detect and
quantitate UCH-L1 in biological samples. For a UCH-L1 sandwich
ELISA (swELISA), 96-well plates are coated with 100 .mu.l/well
capture antibody (500 ng/well purified rabbit anti-UCH-L1, made
in-house by conventional techniques) in 0.1 M sodium bicarbonate,
pH 9.2. Plates are incubated overnight at 4.degree. C., emptied and
300 .mu.l/well blocking buffer (Startingblock T20-TBS) is added and
incubated for 30 min at ambient temperature with gentle shaking.
This is followed by either the addition of the antigen standard
(recombinant UCH-L1) for standard curve (0.05-50 ng/well) or
samples (3-10 .mu.l CSF) in sample diluent (total volume 100
.mu.l/well). The plate is incubated for 2 hours at room
temperature, then washed with automatic plate washer (5.times.300
.mu.l/well with wash buffer, TBST). Detection antibody mouse
anti-UCH-L1-HRP conjugated (made in-house, 50 .mu.g/ml) in blocking
buffer is then added to wells at 100 .mu.L/well and incubated for
1.5 h at room temperature, followed by washing. If amplification is
needed, biotinyl-tyramide solution (Perkin Elmer Elast
Amplification Kit) is added for 15 min at room temperature, washed
then followed by 100 .mu.l/well streptavidin-HRP (1:500) in PBS
with 0.02% Tween-20 and 1% BSA for 30 min and then followed by
washing. Lastly, the wells are developed with 100 .mu.l/well TMB
substrate solution (Ultra-TMB ELISA, Pierce #34028) with incubation
times of 5-30 minutes. The signal is read at 652 nm with a 96-well
spectrophotometer (Molecular Device Spectramax 190).
[0087] UCH-L1 levels of the TBI group (percussive injury) are
significantly higher than the sham controls (p<0.01, ANOVA
analysis) and the naive controls as measured by a swELISA
demonstrating that UCH-L1 is elevated early in CSF (2 h after
injury) then declines at around 24 h after injury before rising
again 48 h after injury (FIG. 2).
[0088] Following MCAO challenge the magnitude of UCH-L1 in CSF is
dramatically increased with severe (2 h) challenge relative to a
more mild challenge (30 min). (FIG. 3) The more severe 2 h MCAO
group UCH-L1 protein levels are 2-5 fold higher than 30 min MCAO
(p<0.01, ANOVA analysis). UCH-L1 protein levels for shams are
virtually indistinguishable from naive controls.
[0089] Similar results are obtained for UCH-L1 in serum. Blood (3-4
ml) is collected at the end of each experimental period directly
from the heart using syringe equipped with 21 gage needle placed in
a polypropylene tube and allowed to stand for 45 min to 1 hour at
room temperature to form clot. Tubes are centrifuged for 20 min at
3,000.times.g and the serum removed and analyzed by ELISA (FIGS. 4,
5).
[0090] UCH-L1 levels of the TBI group are significantly higher than
the sham group (p<0.001, ANOVA analysis) and for each time point
tested 2 h through 24 h respective to the same sham time points
(p<0.005, Student's T-test). UCH-L1 is significantly elevated
after injury as early as 2 h in serum. Severe MCAO challenge
produces increased serum UCH-L1 relative to mild challenge. Both
mild and severe challenge are statistically higher than sham
treated animals indicating that serum detection of UCH-L1 is a
robust diagnostic and the levels are able to sufficiently
distinguish mild from severe injury.
EXAMPLE 8
Analysis of Spectrin Breakdown Products
[0091] Spectrin breakdown products are analyzed following rat MCAO
challenge by procedures similar to those described in U.S. Pat. No.
7,291,710, the contents of which are incorporated herein by
reference. FIG. 6 demonstrates that levels of SBDP145 in both serum
and CSF are significantly (p<0.05) increased at all time points
studied following severe (2 hr) MCAO challenge relative to mild (30
min) challenge. Similarly, SBDP120 demonstrates significant
elevations following severe MCAO challenge between 24 and 72 hours
after injury in CSF (FIG. 7). However, levels of SBDP120 in serum
are increased following severe challenge relative to mild challenge
at all time points between 2 and 120 hours. In both CSF and Serum
both mild and severe MCAO challenge produces increased SPBP120 and
140 relative to sham treated subjects.
EXAMPLE 9
Analysis of MAP2
[0092] Microtubule Associated Protein 2 (MAP2) is assayed as a
biomarker in both CSF and serum following mild (30 min) and severe
(2 hr) MCAO challenge in subjects by ELISA or western blotting
essentially as described herein. Antibodies to MAP2 (MAP-2 (E-12))
are obtained from Santa Cruz Biotechnology, Santa Cruz, Calif.
These antibodies are suitable for both ELISA and western blotting
procedures and are crossreactive to murine and human MAP2. Levels
of MAP2 are significantly (p<0.05) increased in subjects
following mild MCAO challenge relative to naive animals in both CSF
and serum (FIG. 8). Similar to UCH-L1 and SBDPs, severe challenge
(2 hr) produces much higher levels of MAP2 in both samples than
mild challenge (30 min).
EXAMPLE 10
Severe Traumatic Brain Injury Study
[0093] A study was conducted that included 46 human subjects
suffering severe traumatic brain injury. Each of these subjects is
characterized by being over age 18, having a GCS of less than or
equal to 8 and required ventriculostomy and neuromonitoring as part
of routine care. A control group A, synonymously detailed as CSF
controls, included 10 individuals also being over the age of 18 or
older and no injuries. Samples are obtained during spinal
anesthesia for routine surgical procedures or access to CSF
associated with treatment of hydrocephalus or meningitis. A control
group B, synonymously described as normal controls, totaled 64
individuals, each age 18 or older and experiencing multiple
injuries without brain injury. Further details with respect to the
demographics of the study are provided in Table 1.
TABLE-US-00001 TABLE 1 Subject Demographics for Severe Traumatic
Brain Injury Study TBI CSF Controls Normal Controls Number 46 10 64
Males 34 (73.9%) 29 (65.9%) 26 (40.6%) Females 12 (26.1%) 15
(34.1%) 38 (59.4% Age: Average 50.2 58.2 1, 2 30.09 2, 3 Std Dev
19.54 20.52 15.42 Minimum 19 23 18 Maximum 88 82 74 Race: Caucasian
Black 45 38 (86.4%) 52 (81.2%) Asian 1 6 (13.6) 4 (6.3%) Other 7
(10.9%) 1 (1.6%) GCS in Emergency Department Average 5.3 Std Dev
1.9
[0094] The level of biomarkers found in the first available CSF and
serum samples obtained in the study are provided in FIGS. 9 and 10,
respectively. The average first CSF sample collected as detailed in
FIG. 9 was 11.2 hours while the average time for collection of a
serum sample subsequent to injury event as per FIG. 10 is 10.1
hours. The quantity of each of the biomarkers of UCH-L1, MAP2,
SBDP145, SBDP120, and GFAP are provided for each sample for the
cohort of traumatic brain injury sufferers as compared to a control
group. The diagnostic utility of the various biomarkers within the
first 12 hours subsequent to injury based on a compilation of CSF
and serum data is provided in FIG. 11 and indicates in particular
the value of GFAP as well as that of additional markers UCH-L1 and
the spectrin breakdown products. Elevated levels of UCH-L1 are
indicative of the compromise of neuronal cell body damage while an
increase in SPDP145 with a corresponding decrease in SBDP120 is
suggestive of acute axonal necrosis.
[0095] One subject from the traumatic brain injury cohort was a 52
year old Caucasian woman who had been involved in a motorcycle
accident while not wearing a helmet. Upon admission to an emergency
room her GCS was 3 and during the first 24 hours subsequent to
trauma her best GCS was 8. After 10 days her GCS was 11. CT
scanning revealed SAH and facial fractures with a Marshall score of
11 and a Rotterdam score of 2. Ventriculostomy was removed after 5
years and an overall good outcome was obtained. Arterial blood
pressure (MABP), intracranial pressure (ICP) and cerebral profusion
pressure (CPP) for this sufferer of traumatic brain injury as a
function of time is depicted in FIG. 12. A possible secondary
insult is noted at approximately 40 hours subsequent to the injury
as noted by a drop in MABP and CPP. The changes in concentration of
inventive biomarkers per CSF and serum samples from this individual
are noted in FIG. 13. These results include a sharp increase in
GFAP in both the CSF and serum as well as the changes in the other
biomarkers depicted in FIG. 13 and provide important clinical
information as to the nature of the injury and the types of cells
involved, as well as modes of cell death associated with the
spectrin breakdown products.
[0096] Another individual of the severe traumatic brain injury
cohort included a 51 year old Caucasian woman who suffered a crush
injury associated with a horse falling on the individual. GCS on
admission to emergency room was 3 with imaging analysis initially
being unremarkable with minor cortical and subcortical contusions.
MRI on day 5 revealed significant contusions in posterior fossa.
The Marshall scale at that point was indicated to be 11 with a
Rotterdam scale score of 3. The subject deteriorated and care was
withdrawn 10 days after injury. The CSF and serum values for this
individual during a period of time are provided in FIG. 14.
[0097] Based on the sandwich ELISA testing, GFAP values as a
function of time are noted to be markedly elevated relative to
normal controls (control group B) as a function of time.
[0098] The concentration of spectrin breakdown products, MAP2 and
UCH-L1 as a function of time subsequent to traumatic brain injury
has been reported elsewhere as exemplified in U.S. Pat. Nos.
7,291,710 and 7,396,654 each of which is incorporated herein by
reference.
[0099] An analysis was performed to evaluate the ability of
biomarkers measured in serum to predict TBI outcome, specifically
GCS. Stepwise regression analysis was the statistical method used
to evaluate each of the biomarkers as an independent predictive
factor, along with the demographic factors of age and gender, and
also interactions between pairs of factors. Interactions determine
important predictive potential between related factors, such as
when the relationship between a biomarker and outcome may be
different for men and women, such a relationship would be defined
as a gender by biomarker interaction.
[0100] The resulting analysis identified biomarkers UCH-L1, MAP2,
and GFAP as being statistically significant predictors of GCS
(Table 2, 3). Furthermore, GFAP was shown to have improved
predictability when evaluated in interaction with UCH-L1 and gender
(Table 4, 5).
TABLE-US-00002 TABLE 2 Stepwise Regression Analysis 1 - Cohort
includes: All Subjects >= 18 Years Old Summary of Stepwise
Selection - 48 Subjects Variable Parameter Model Step Entered
Estimate R-Square F Value p-value Intercept 13.02579 2 SEXCD
-2.99242 0.1580 7.29 0.0098 1 CSF_UCH_L1 -0.01164 0.2519 11.54
0.0015 3 Serum_MAP_2 0.96055 0.3226 4.59 0.0377
TABLE-US-00003 TABLE 3 Stepwise Regression Analysis 2 - Cohort
includes: TBI Subjects >= 18 Years Old Summary of Stepwise
Selection - 39 Subjects Variable Parameter Model Step Entered
Estimate R-Square F Value p-value Intercept 5.73685 1 Serum_UCH_L1
-0.30025 0.0821 8.82 0.0053 2 Serum_GFAP 0.12083 0.1973 5.16
0.0291
TABLE-US-00004 TABLE 4 Stepwise Regression Analysis 1 - Cohort
includes: TBI and A Subjects >= 18 Years Old Summary of Stepwise
Selection - 57 Subjects Variable Parameter Model Step Entered
Estimate R-Square F Value p-value Intercept 8.04382 1 Serum_UCH_L
-0.92556 0.1126 12.90 0.0007 2 Serum_MAP_2 1.07573 0.2061 5.79
0.0197 3 Serum_UCH-L1 + 0.01643 0.2663 4.35 0.0419 Serum_GFAP
TABLE-US-00005 TABLE 5 Stepwise Regression Analysis 2 - Cohort
includes: TBI Subjects >= 18 Years Old Summary of Stepwise
Selection - 44 Subjects Variable Parameter Model Step Entered
Estimate R-Square F Value p-value Intercept 5.50479 1 Serum_UCH_L1
-0.36311 0.0737 11.95 0.0013 2 SEX_Serum_GFAP 0.05922 0.1840 5.09
0.0296 3 Serum_MAP_2 0.63072 0.2336 2.59 0.1157
EXAMPLE 11
[0101] The study of Example 10 was repeated with a moderate
traumatic brain injury cohort characterized by GCS scores of
between 9 and 11, as well as a mild traumatic brain injury cohort
characterized by GCS scores of 12-15. Blood samples were obtained
from each patient on arrival to the emergency department of a
hospital within 2 hours of injury and measured by ELISA for levels
of GFAP in nanograms per milliliter. The results were compared to
those of a control group who had not experienced any form of
injury. Secondary outcomes included the presence of intracranial
lesions in head CT scans.
[0102] Over 3 months 53 patients were enrolled: 35 with GCS 13-15,
4 with GCS 9-12 and 14 controls. The mean age was 37 years (range
18-69) and 66% were male. The mean GFAP serum level was 0 in
control patients, 0.107 (0.012) in patients with GCS 13-15 and
0.366 (0.126) in GCS 9-12 (P<0.001). The difference between GCS
13-15 and controls was significant at P<0.001. In patients with
intracranial lesions on CT GFAP levels were 0.234 (0.055) compared
to 0.085 (0.003) in patients without lesions (P<0.001). There is
a significant increase in GFAP in serum following a MTBI compared
to uninjured controls in both the mild and moderate groups. GFAP
was also significantly associated with the presence of intracranial
lesions on CT.
[0103] FIG. 16 shows GFAP concentration for controls as well as
individuals in the mild/moderate traumatic brain injury cohort as a
function of CT scan results upon admission and 24 hours thereafter.
Simultaneous assays were performed in the course of this study for
UCH-L1 biomarker. The UCH-L1 concentration derived from the same
samples as those used to determine GFAP is provided FIG. 17. The
concentration of UCH-L1 and GFAP as well as a biomarker not
selected for diagnosis of neurological condition, S100b, is
provided as a function of injury magnitude between control, mild,
and moderate traumatic brain injury as shown in FIG. 18. The
simultaneous analyses of UCH-L1 and GFAP from these patients
illustrates the synergistic effect of the inventive process in
allowing an investigator to simultaneously diagnose traumatic brain
injury as well as discern the level of traumatic brain injury
between mild and moderate levels of severity. FIG. 19 shows the
concentration of the same markers as depicted in FIG. 18 with
respect to initial evidence upon hospital admission as to lesions
in tomography scans illustrating the high confidence in predictive
outcome of the inventive process. FIG. 20 shows that both NSE and
MAP2 are elevated in subjects with MTBI in serum both at admission
and at 24 hours of follow up. These data demonstrate a synergistic
diagnostic effect of measuring multiple biomarkers such as GFAP,
UCH-L1, NSE, and MAP2 in a subject.
[0104] Through the simultaneous measurement of multiple biomarkers
such as UCH-L1, GFAP, NSE, and MAP2, rapid and quantifiable
determination as to the severity of the brain injury is obtained
consistent with GSC scoring and CT scanning yet in a surprisingly
more quantifiable, expeditious and economic process. Additionally,
with a coupled assay for biomarkers indicative of neurological
condition, the nature of the neurological abnormality is assessed
and in this particular study suggestive of neuronal cell body
damage. As with severe traumatic brain injury, gender variations
are noted suggesting a role for hormonal anti-inflammatories as
therapeutic candidates. A receiver operating characteristic (ROC)
modeling of UCH-L1, GFAP and SBDP145 post TBI further supports the
value of simultaneous measurement of these biomarkers, as shown in
FIGS. 21, 22.
[0105] In addition, FIG. 22 showed that several brain biomarkers
(GFAP, UCH-L1 and MAP2) in stroke patients' plasma. Samples were
collected with an average post-injury time 24.2 hr (range 18-30 h).
Top panel shows GFAP, UCH-L1 and MAP2 levels in stroke (n=11)
versus normal controls (n=30). Bottom panel further shows that
UCH-L1 is elevated with both hemorrhagic and ischemic stroke
populations when compared to normal control plasma.
[0106] Patent documents and publications mentioned in the
specification are indicative of the levels of those skilled in the
art to which the invention pertains. These documents and
publications are incorporated herein by reference to the same
extent as if each individual document or publication was
specifically and individually incorporated herein by reference.
[0107] The foregoing description is illustrative of particular
embodiments of the invention, but is not meant to be a limitation
upon the practice thereof. The following claims, including all
equivalents thereof, are intended to define the scope of the
invention.
* * * * *