U.S. patent application number 15/989120 was filed with the patent office on 2018-11-29 for detection of biomarkers on vesicles for the diagnosis and prognosis of diseases and disorders.
The applicant listed for this patent is NANOSOMIX, INC.. Invention is credited to Masato Mitsuhashi.
Application Number | 20180340945 15/989120 |
Document ID | / |
Family ID | 64397085 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180340945 |
Kind Code |
A1 |
Mitsuhashi; Masato |
November 29, 2018 |
DETECTION OF BIOMARKERS ON VESICLES FOR THE DIAGNOSIS AND PROGNOSIS
OF DISEASES AND DISORDERS
Abstract
The present invention relates to methods, compositions, and kits
for detecting and quantitating biomarkers on vesicles without lysis
or permeabilization of the vesicles and the use of biomarkers
identified on vesicles in diagnostic and prognostic methods for
various diseases and disorders. Disease and disorders of the
present invention include neurological disorders, immunological
disorders, placental diseases, and cancer.
Inventors: |
Mitsuhashi; Masato; (Irvine,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NANOSOMIX, INC. |
Aliso Viejo |
CA |
US |
|
|
Family ID: |
64397085 |
Appl. No.: |
15/989120 |
Filed: |
May 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62547024 |
Aug 17, 2017 |
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62510726 |
May 24, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/28 20130101;
A61P 25/00 20180101; A61P 25/28 20180101; A61P 35/00 20180101; G01N
2800/2835 20130101; G01N 33/6896 20130101; G01N 2800/2821
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A method comprising: a) providing a biological sample comprising
vesicles; b) contacting a solid support comprising capture agents
associated therewith with the biological sample under conditions
wherein the capture agents selectively bind to a first biomarker,
if present, on the vesicles, thereby capturing said vesicles having
said first biomarker on the solid support; c) separating the solid
support from the biological sample; and d) detecting a second
biomarker on the vesicles captured on the solid support using a
detection agent that selectively binds to the second biomarker,
wherein the vesicles are not lysed or permeabilized.
2. The method of claim 1, wherein the first and second biomarkers
are membrane-bound proteins or adsorbed proteins on the
vesicles.
3. The method of claim 1, wherein the first or second biomarker is
an exosome surface marker.
4. The method of claim 3, wherein the exosome surface marker is
CD81, CD63, CD171, SNAP25, EAAT1, DAT, CD11b or OMG.
5. The method of claim 1, wherein the first or second biomarker is
selected from the group consisting of a neuron-specific protein, an
astrocyte-specific protein, a microglia-specific protein, and an
oligodendrocyte-specific protein.
6. The method of claim 5, wherein the neuron-specific protein is
selected from the group consisting of NRGN, Tau, phosphorylated
Tau, synaptophysin, .alpha..beta.-42, alpha-synuclein (SNCA), AchE,
LAMP1, REST, SYT, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR,
DR6, HSP, IL12b, A.beta., and BACE.
7. The method of claim 5, wherein the astrocyte-specific proteins
is selected from the group consisting of glial fibrillary acidic
protein (GFAP) and excitatory amino acid transporter 1 (EAAT1).
8. The method of claim 5, wherein the oligodendrocyte-specific
protein is selected from the group consisting of myelin basic
protein (MBP) and oligodendrocyte myelin glycoprotein (OMG).
9. The method of claim 5, wherein the vesicles captured on the
solid support are selected from the group consisting of
neuron-derived exosomes, astrocyte-derived exosomes,
oligodendrocyte-derived exosomes, and microglia-derived
exosomes.
10. The method of claim 2, wherein the membrane-bound proteins or
adsorbed proteins on the vesicles is dopamine transporter (DAT) or
tyrosine hydroxygenase (TH).
11. The method of claim 1, further comprising detecting a cytosolic
protein on the vesicles captured on the solid support.
12. The method of claim 11, wherein the cytosolic protein is GAPDH,
CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau (e.g., T181),
synaptophysin, SNCA, .alpha..beta.-42, AchE, LAMP1, REST, SYT, TH,
SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase,
ubiquitin, PSEN1, GSK, PLAP, CSH1, or PSG1.
13. The method of claim 12, wherein the cytosolic protein is a
pathological form, including aggregates and/or mutated form of
GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau (e.g., T181),
synaptophysin, SNCA, .alpha..beta.-42, AchE, LAMP1, REST, SYT, TH,
SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase,
ubiquitin, PSEN1, GSK, PLAP, CSH1, or PSG1.
14. The method of claim 1, further comprising detecting a secretory
protein on the vesicles captured on the solid support.
15. The method of claim 14, wherein the secretory protein is
selected from the group consisting of cytokines, growth factors,
chemokines, interleukins, nociceptin (opioid peptide), and
GnRH.
16. The method of claim 15, wherein the cytokine is selected from
the group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32,
IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, TNFSF13, IL12B, and
FasL.
17. The method of claim 1, further comprising detecting a receptor
protein, a transporter protein, or a membrane protein on the
vesicles captured on the solid support.
18. The method of claim 17, wherein the receptor protein is a
neurotransmitter receptor, a dopamine receptor, a serotonin
receptor, a GABA receptor, a glutamate receptor, an insulin
receptor, a tumor necrosis factor receptor, or a neuropeptide
receptor.
19. The method of claim 17, wherein the membrane protein is EpCAM,
PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, or
NCAM.
20. The method of claim 17, wherein the transporter protein is a
neurotransmitter transporter, a dopamine transporter (DAT), a
serotonin transporter, a GABA transporter, a glutamate transporter,
an insulin transporter, a tumor necrosis factor transporter, or a
neuropeptide transporter.
21. The method of claim 1, wherein the solid support is a plate, a
non-magnetic bead, a magnetic bead, a filter, a slide, a wafer, a
rod, a particle, a strand, a disc, a membrane, or a surface of a
tube, channel, column, flow cell device, or microfluidic
device.
22. The method of claim 1, wherein the capture agents comprise
antibodies, antibody fragments, antibody mimetics, or aptamers that
specifically bind to the first biomarker on the vesicles.
23. The method of claim 1, wherein the detection agent comprises an
antibody, an antibody fragment, an antibody mimetic, or an aptamer
that specifically binds to the second biomarker on the
vesicles.
24. The method of claim 1, wherein said detecting comprises
performing an immunoassay.
25. The method of claim 24, wherein the immunoassay is selected
from the group consisting of an enzyme-linked immunosorbent assay
(ELISA), an immunofluorescent assay (IFA), an immune-polymerase
chain reaction assay, an electro-chemiluminescence immunoassay
(ECLIA), and a radioimmunoassay (RIA).
Description
RELATED APPLICATIONS
[0001] This application claims priority to the U.S. Provisional
Patent Application Ser. No. 62/510,726, filed on May 24, 2017, and
U.S. Provisional Patent Application Ser. No. 62/547,024, filed on
Aug. 17, 2017, which is hereby incorporated by reference herein in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods, compositions, and
kits for detecting and quantitating biomarkers on vesicles without
lysis or permeabilization of the vesicles and the use of biomarkers
identified on vesicles in diagnostic and prognostic methods for
various diseases and disorders. Disease and disorders of the
present invention include neurological disorders, immunological
disorders, placental diseases, and cancer.
BACKGROUND OF THE INVENTION
[0003] Exosomes in biological fluids are potentially useful
diagnostically for various diseases, because exosomes carry
physiological and pathological materials (proteins, metabolites,
RNAs, small molecules, etc.) of the mother cells from which they
originate and the microenvironment near their mother cells.
However, even though exosomes are recognized as valuable resources
for diagnostics, current analytical methods of analyzing exosomes
are too complicated and expensive for routine use in diagnostic
testing.
[0004] More than 5.4 million Americans and 35 million people
worldwide have Alzheimer's disease, the most common form of
dementia. Currently, the only definitive way to diagnose
Alzheimer's disease is by direct examination of brain tissue after
a patient dies. Doctors use brain imaging, evaluation of behavior,
psychiatric tests, and other means to diagnose the disease in the
patients suspected of having Alzheimer's disease, but none are
highly accurate, and many are costly or not practical.
[0005] In 2017, there will be an estimated 1,688,780 new cancer
cases diagnosed and 600,920 cancer deaths in the US. The most
common cancers are breast cancer, lung and bronchus cancer,
prostate cancer, colon and rectum cancer, bladder cancer, melanoma
of the skin, non-Hodgkin lymphoma, thyroid cancer, kidney and renal
pelvis cancer, leukemia, endometrial cancer, and pancreatic cancer.
Similarly, millions of Americans suffer from immunological
disorders and diseases caused by a dysfunction of the immune
system.
[0006] Therefore, there is a need in the art for biomarkers and
methods for diagnosing diseases and other disorders, such as, for
example, neurological disorders, cancer, immunological disorders,
and placental disease. Additionally, there is a need in the art for
compositions for detecting the biomarkers as well as compositions
and methods useful for treating diseases and other disorders. The
present invention meets this need by providing accurate,
noninvasive methods for diagnosing diseases and other disorders.
The present invention further provides novel methods, assays,
biomarkers, kits, and compositions for diagnosing, prognosing,
predicting, and treating various diseases and disorders.
SUMMARY OF THE INVENTION
[0007] The present invention relates to methods, compositions, and
kits for detecting and quantitating biomarkers on vesicles. In
particular, the invention provides a method for selectively
capturing vesicles carrying surface markers of interest on a solid
support and detecting membrane-bound and adsorbed biomarkers on the
captured vesicles without the need for lysis or permeabilization of
the vesicles. Measurement of biomarkers on vesicles is useful in
diagnostic and prognostic methods for various diseases. In
particular, the present invention provides methods of diagnosing or
prognosing a disease or disorder in a subject, identifying a
subject at risk of a disease or a disorder, or prescribing a
therapeutic regimen or predicting benefit from therapy in a subject
having a disease or a disorder. In certain embodiments, the present
invention provides methods of diagnosing or prognosing a
neurological disorder in a subject, identifying a subject at risk
of a neurological disorder, or prescribing a therapeutic regimen or
predicting benefit from therapy in a subject having a neurological
disorder. In other embodiments, the present invention provides
methods of diagnosing or prognosing an immunological disorder in a
subject, identifying a subject at risk of an immunological
disorder, or prescribing a therapeutic regimen or predicting
benefit from therapy in a subject having an immunological disorder.
In other embodiments, the present invention provides methods of
diagnosing or prognosing cancer in a subject, identifying a subject
at risk of cancer, or prescribing a therapeutic regimen or
predicting benefit from therapy in a subject having cancer. In
other embodiments, the present invention provides methods of
diagnosing or prognosing placental disease in a subject,
identifying a subject at risk of a placental disease, or
prescribing a therapeutic regimen or predicting benefit from
therapy in a subject having a placental disease.
[0008] In one aspect, the invention provides a method comprising:
a) providing a biological sample comprising vesicles; b) contacting
a solid support comprising capture agents associated therewith with
the biological sample under conditions wherein the capture agents
selectively bind to a first biomarker, if present, on the vesicles,
thereby capturing said vesicles having said first biomarker on the
solid support; c) separating the solid support from the biological
sample; and d) detecting a second biomarker on the vesicles
captured on the solid support using a detection agent that
selectively binds to the second biomarker, wherein the vesicles are
not lysed or permeabilized.
[0009] In some embodiments, the methods of the invention further
comprise detecting a secretory protein on the vesicles captured on
the solid support. In certain aspects, the secretory protein is
selected from the group consisting of cytokines, growth factors,
chemokines, and interleukins. In other aspects, the cytokine is
selected from the group consisting of IL1b, IL34, IL6, IL8, IL16,
IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B,
nociceptin, GnRH, FasL, and TNFSF13.
[0010] In certain embodiments, the vesicles are selected from the
group consisting of exosomes, microparticles, microvesicles,
nanosomes, extracellular vesicles, ectosomes, and apoptotic
bodies.
[0011] In certain embodiments, the first and second biomarkers are
membrane-bound proteins or adsorbed proteins on the vesicles. For
example, the first or second biomarker may be an exosome surface
marker (e.g., CD81, CD63, CD171), a neuron-specific protein (e.g.,
synaptosome associated protein 25 (SNAP25), neurogranin (NRGN),
tau, phosphorylated tau, .alpha..beta.-42, and synaptophysin), an
astrocyte-specific protein (e.g., glial fibrillary acidic protein
(GFAP) and excitatory amino acid transporter 1 (EAAT1)), a
microglia-specific protein (CD11b), an oligodendrocyte-specific
protein (e.g., myelin basic protein (MBP), an oligodendrocyte
myelin glycoprotein (OMG), or a chemokine (CX3CL1) or cytokine
(IL1b, IL34, FasL, or IL12B). In another embodiment, the first or
second biomarker is a metabolite. In another embodiment, the method
further comprises detecting a cytosolic protein (e.g.,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein
(SNCA), tyrosine hydroxygenase (TH), cathepsin D (CTSD), AchE,
LAMP1, REST, SYT, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO,
NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL) on
the vesicles captured on the solid support. In another embodiment,
the method further comprises detecting one or more additional
biomarkers on the vesicles captured on the solid support. In some
embodiments, the biomarker is selected from the group consisting of
CD81, acetylcholinesterase (AchE), Lysosomal Associated Membrane
Protein 1 (LAMP1), CTSD, RE1 Silencing Transcription Factor (REST),
synaptotagmin (SYT), synaptophysin (SYP), synaptopodin (SYNPO),
postsynaptic density protein 95 (PSD95), synaptic vesicle
glycoprotein 2A (SV2A), NGRN, monocyte chemotactic protein-1
(CCL2), IL34, glycogen synthase (GYS), (OR), death receptor 6
(DR6), heat shock protein (HSP), IL12beta, alpha-beta (A.beta.),
beta-secretase (BACE), dopamine receptors (D1 and D2), serotonin
receptors (2A, 2C, and 3B), GABA receptors (1-6, 5. B1, B2),
glutamate receptors (1 and 2), insulin receptors, tumor necrosis
factor receptors superfamily (TRAL, TNF receptor, death receptor 5
and 6), neuropeptide receptors (orexin receptor, opioid receptor
KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a,
TREM2, and NCAM. In some embodiments, the first biomarker is SNAP25
and the second biomarker is selected from the group consisting of
CD81, tau, NEFL, TNFa, and IL8. In some embodiments, the first
biomarker is EAAT1 and the second biomarker is CD81 or GFAP. In
some embodiments, the first biomarker is OMG and the second
biomarker is CD81 or MBP. In some embodiments, the first biomarker
is CD11b and the second biomarker is SYP. In some embodiments, the
first biomarker is DAT and the second biomarker is CD81, SNCA, or
TH. In some embodiments, the first biomarker is CD11b and the
second biomarker is SYP.
[0012] In another embodiment, the vesicles captured on the solid
support are selected from the group consisting of neuron-derived
exosomes, astrocyte-derived exosomes, oligodendrocyte-derived
exosomes, and microglia-derived exosomes.
[0013] In certain embodiments, the solid support is a plate, a
non-magnetic bead, a magnetic bead, a filter, a slide, a wafer, a
rod, a particle, a strand, a disc, a membrane, or a surface of a
tube, channel, column, flow cell device, or microfluidic device.
The solid support can comprise, for example, glass, quartz,
silicon, metal, ceramic, plastic, nylon, polyacrylamide, a
hydrogel, or a resin.
[0014] In certain embodiments, the solid support comprises more
than one type of capture agent associated therewith, for example,
at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more different
capture agents that selectively bind to different biomarkers on the
vesicles.
[0015] In certain embodiments, detection of biomarker on vesicles
captured on the solid support comprises using more than one type of
detection agent, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, or more different detection agents that selectively bind to
different biomarkers on the vesicles.
[0016] In certain embodiments, the capture agents comprise
antibodies, antibody fragments, antibody mimetics, or aptamers that
specifically bind to the first biomarker on the vesicles. In other
embodiments, the detection agents comprise antibodies, antibody
fragments, antibody mimetics, or aptamers that specifically bind to
the second biomarker on the vesicles. Capture agents and detection
agents may comprise monoclonal antibodies, polyclonal antibodies,
chimeric antibodies, nanobodies, recombinant fragments of
antibodies, Fab fragments, Fab' fragments, F(ab').sub.2 fragments,
F.sub.v fragments, or scF.sub.v fragments.
[0017] In certain embodiments, the detection agent further
comprises a detectable label, for example, a fluorescent,
chemiluminescent, electrochemiluminescent, bioluminescent,
isotopic, or radioactive label.
[0018] In certain embodiments, detecting a biomarker on a vesicle
comprises performing an immunoassay, such as an enzyme-linked
immunosorbent assay (ELISA), an immunofluorescent assay (IFA), an
immune-polymerase chain reaction assay, an
electro-chemiluminescence immunoassay (ECLIA), and a
radioimmunoassay (RIA).
[0019] The biological sample comprising vesicles may be from a
subject or a cell culture supernatant. In certain embodiments, the
biological sample is selected from the group consisting of whole
blood, serum, plasma, urine, interstitial fluid, peritoneal fluid,
cervical swab, tears, saliva, buccal swab, skin, brain tissue, and
cerebrospinal fluid.
[0020] In certain embodiments, a biological sample is obtained from
a subject who has been diagnosed or is suspected of having a
neurological disorder, such as Alzheimer's disease (AD), vascular
disease dementia, frontotemporal dementia (FTD), corticobasal
degeneration (CBD), progressive supranuclear palsy (PSP), Lewy body
dementia, tangle-predominant senile dementia, Pick's disease (PiD),
argyrophilic grain disease, amyotrophic lateral sclerosis (ALS),
other motor neuron diseases, Guam parkinsonism-dementia complex,
FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain
injury (TBI), stroke, depression, bipolar disease, epilepsy,
autism, schizophrenia, brain tumor, white matter disease, brain
atrophy, mental retardation, cerebellar ataxia, multiple system
atrophy, concussion, subconcussive impacts, or Parkinson's
disease.
[0021] In some embodiments, a biological sample is obtained from a
subject who has been diagnosed or is suspected of having cancer,
such as breast cancer, pancreatic cancer, lung cancer, colon
cancer, colorectal cancer, rectal cancer, kidney cancer, bladder
cancer, stomach cancer, prostate cancer, liver cancer, ovarian
cancer, brain cancer, vaginal cancer, vulvar cancer, uterine
cancer, oral cancer, penic cancer, testicular cancer, esophageal
cancer, skin cancer, cancer of the fallopian tubes, head and neck
cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous
or intraocular melanoma, cancer of the anal region, cancer of the
small intestine, cancer of the endocrine system, cancer of the
thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, cancer of the urethra, cancer of the renal pelvis,
cancer of the ureter, cancer of the endometrium, cancer of the
cervix, cancer of the pituitary gland, neoplasms of the central
nervous system (CNS), primary CNS lymphoma, brain stem glioma, or
spinal axis tumors.
[0022] In some embodiments, a biological sample is obtained from a
subject who has been diagnosed or is suspected of having an
immunological disorder. In other embodiments, a biological sample
is obtained from a subject who has been diagnosed or is suspected
of having a placental disease.
[0023] In one aspect, the invention provides a method of diagnosing
and treating a disease or a disorder in a subject, the method
comprising: a) obtaining a biological sample comprising exosomes
from the subject; b) contacting a solid support comprising capture
agents associated therewith with the biological sample under
conditions wherein the capture agents selectively bind to a first
biomarker on the exosomes, wherein exosomes are captured on the
solid support; c) separating the solid support from the biological
sample; d) measuring a level of a second biomarker on the exosomes
captured on the solid support, wherein the exosomes are not lysed
or permeabilized; e) diagnosing the subject with the disease or
disorder by comparing the level of the second biomarker to a
control level of the second biomarker; and f) treating the subject
for the disease or disorder if the subject is diagnosed as having
the disease or disorder. In some embodiments, the first biomarker
is a neuron-specific protein, an astrocyte-specific protein, a
microglia-specific protein, or an oligodendrocyte-specific protein.
In other embodiments, the second biomarker is a cytosolic protein,
a secretory protein, a receptor protein, a transporter protein, or
a membrane protein. In some embodiments, the cytosolic protein is
selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP,
Tau, phosphorylated Tau (e.g., T181), synaptophysin, SNCA,
.alpha..beta.-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95,
SV2A, GYS, HSP70, BACE, NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP,
CSH1, PSG1, or FasL. In other embodiments, the secretory protein is
selected from the group consisting of cytokines, growth factors,
chemokines, and interleukins. In certain aspects, the cytokine is
selected from the group consisting of IL1b, IL34, IL6, IL8, IL16,
IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B,
nociceptin, GnRH, FasL and TNFSF13. In other embodiments, the
neurotransmitter receptor is selected from the group consisting of
dopamine receptors (D1 and D2), serotonin receptors (2A, 2C, and
3B), GABA receptors (1-6, 5. B1, B2), glutamate receptors (1 and
2), insulin receptors, tumor necrosis factor receptors superfamily
(TRAL, TNF receptor, death receptor 5 and 6), and neuropeptide
receptors (orexin receptor, opioid receptor KOR). In other
embodiments, the membrane protein is selected from the group
consisting of EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32,
CD79a, TREM2, and NCAM. In some embodiments, the transporter
protein is a neurotransmitter transporter, a dopamine transporter
(e.g., DAT), a serotonin transporter, a GABA transporter, a
glutamate transporter, an insulin transporter, a tumor necrosis
factor transporter, or a neuropeptide transporter. In some
embodiments, the disease or disorder is a neurological disorder, an
immunological disorder, a placental disease, or cancer.
[0024] In another aspect, the invention provides a method of
diagnosing and treating a neurological disorder in a subject, the
method comprising: a) obtaining a biological sample comprising
exosomes from the subject; b) contacting a solid support comprising
capture agents associated therewith with the biological sample
under conditions wherein the capture agents selectively bind to a
first biomarker on the exosomes selected from the group consisting
of a neuron-specific protein, an astrocyte-specific protein, a
microglia-specific protein, and an oligodendrocyte-specific
protein, wherein neuron-derived exosomes, astrocyte-derived
exosomes, oligodendrocyte-derived exosomes, or microglia-derived
exosomes are captured on the solid support; c) separating the solid
support from the biological sample; d) measuring a level of a
second biomarker selected from the group consisting of a
neuron-specific protein, an astrocyte-specific protein, a
microglia-specific protein on the exosomes captured on the solid
support, wherein the exosomes are not lysed or permeabilized; e)
diagnosing the subject with the neurological disorder by comparing
the level of the second biomarker to a control level of the second
biomarker; and f) treating the subject for the neurological
disorder if the subject is diagnosed as having the neurological
disorder.
[0025] In other embodiments, the present invention provides a
method of prognosing or monitoring the progression of a disease or
disorder in a subject, the method comprising: a) obtaining a
biological sample comprising exosomes from the subject; b)
contacting a solid support comprising capture agents associated
therewith with the biological sample under conditions wherein the
capture agents selectively bind to a first biomarker on the
exosomes, wherein exosomes are captured on the solid support; c)
separating the solid support from the biological sample; d)
measuring a level of a second biomarker protein on the exosomes
captured on the solid support, wherein the exosomes are not lysed
or permeabilized, thereby prognosing or monitoring the progression
of the disease or disorder in the subject. In some embodiments, the
method further comprises obtaining a second biological sample
comprising exosomes from the subject and measuring the level of a
biomarker of the present invention and comparing the level of the
biomarker in the second biological samples to the level of the
biomarker in the first biological sample. In some embodiments, the
first biomarker is a neuron-specific protein, an astrocyte-specific
protein, a microglia-specific protein, or an
oligodendrocyte-specific protein. In other embodiments, the second
biomarker is a cytosolic protein, a secretory protein, a receptor
protein, a transporter protein, or a membrane protein. In some
embodiments, the cytosolic protein is selected from the group
consisting of GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau
(e.g., T181), synaptophysin, SNCA, .alpha..beta.-42, AchE, LAMP1,
REST, SYT, TH, SYP, SYNPO, PSD-95, SV2A, GYS, HSP70, BACE, SYMPO,
NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL. In
other embodiments, the secretory protein is selected from the group
consisting of cytokines, growth factors, chemokines, and
interleukins. In certain aspects, the cytokine is selected from the
group consisting of IL1b, IL34, IL6, IL8, IL16, IL23A, IL32, IL33,
CX3CL1, CCL2, CXCL12, TNFalpha, TNFSF10, IL12B, nociceptin, GnRH,
FasL and TNFSF13. In other embodiments, the neurotransmitter
receptor is selected from the group consisting of dopamine
receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA
receptors (1-6, 5. B1, B2), glutamate receptors (1 and 2), insulin
receptors, tumor necrosis factor receptors superfamily (TRAL, TNF
receptor, death receptor 5 and 6), and neuropeptide receptors
(orexin receptor, opioid receptor KOR). In other embodiments, the
membrane protein is selected from the group consisting of EpCAM,
PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM.
In some embodiments, the transporter protein is a neurotransmitter
transporter, a dopamine transporter, a serotonin transporter, a
GABA transporter, a glutamate transporter, an insulin transporter,
a tumor necrosis factor transporter, or a neuropeptide transporter.
In some embodiments, the disease or disorder is a neurological
disorder, an immunological disorder, a placental disease, or
cancer.
[0026] In other embodiments, the present invention provides a
method of prognosing or monitoring the progression of a
neurological disorder in a subject, the method comprising: a)
obtaining a biological sample comprising exosomes from the subject;
b) contacting a solid support comprising capture agents associated
therewith with the biological sample under conditions wherein the
capture agents selectively bind to a first biomarker on the
exosomes selected from the group consisting of a neuron-specific
protein, an astrocyte-specific protein, a microglia-specific
protein, and an oligodendrocyte-specific protein, wherein
neuron-derived exosomes, astrocyte-derived exosomes,
oligodendrocyte-derived exosomes, or microglia-derived exosomes are
captured on the solid support; c) separating the solid support from
the biological sample; d) measuring a level of a second biomarker
selected from the group consisting of a neuron-specific protein, an
astrocyte-specific protein, a microglia-specific protein on the
exosomes captured on the solid support, wherein the exosomes are
not lysed or permeabilized, thereby prognosing or monitoring the
progression of the neurological disease in the subject. In some
embodiments, the method further comprises obtaining a second
biological sample comprising exosomes from the subject and
measuring the level of a biomarker of the present invention and
comparing the level of the biomarker in the second biological
samples to the level of the biomarker in the first biological
sample.
[0027] In yet another aspect, the invention provides a method of
diagnosing and treating a disease or disorder in a subject, the
method comprising: a) obtaining a biological sample comprising
exosomes from the subject; b) contacting a solid support comprising
capture agents associated therewith with the biological sample
under conditions wherein the capture agents selectively bind to a
first biomarker on the exosomes, wherein exosomes are captured on
the solid support; c) separating the solid support from the
biological sample; d) detecting a second biomarker on the exosomes
captured on the solid support, wherein the exosomes are not lysed
or permeabilized; e) diagnosing the subject with the disease or
disorder by comparing the level of the second biomarker to a
control level of the second biomarker; and f) treating the subject
for the disease or disorder if the subject is diagnosed as having
the disease or disorder. In some embodiments, the first biomarker
is a neuron-specific protein, an astrocyte-specific protein, a
microglia-specific protein, or an oligodendrocyte-specific protein.
In other embodiments, the second biomarker is a cytosolic protein,
a secretory protein, a receptor protein, a transporter protein, or
a membrane protein. In some embodiments, the cytosolic protein is
selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP,
Tau, phosphorylated Tau (e.g., T181), synaptophysin, SNCA,
.alpha..beta.-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95,
SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1,
GSK, PLAP, CSH1, PSG1, or FasL. In other embodiments, the secretory
protein is selected from the group consisting of cytokines, growth
factors, chemokines, and interleukins. In certain aspects, the
cytokine is selected from the group consisting of IL1b, IL34, IL6,
IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha,
TNFSF10, IL12B, nociceptin, GnRH, FasL and TNFSF13. In other
embodiments, the neurotransmitter receptor is selected from the
group consisting of dopamine receptors (D1 and D2), serotonin
receptors (2A, 2C, and 3B), GABA receptors (1-6, 5. B1, B2),
glutamate receptors (1 and 2), insulin receptors, tumor necrosis
factor receptors superfamily (TRAL, TNF receptor, death receptor 5
and 6), and neuropeptide receptors (orexin receptor, opioid
receptor KOR). In other embodiments, the membrane protein is
selected from the group consisting of EpCAM, PD-L1, ErbB2, CK19,
TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM. In some embodiments,
the transporter protein is a neurotransmitter transporter, a
dopamine transporter, a serotonin transporter, a GABA transporter,
a glutamate transporter, an insulin transporter, a tumor necrosis
factor transporter, or a neuropeptide transporter. In some
embodiments, the disease or disorder is a neurological disorder, an
immunological disorder, a placental disease, or cancer.
[0028] In yet another aspect, the invention provides a method of
diagnosing and treating a neurological disorder in a subject, the
method comprising: a) obtaining a biological sample comprising
exosomes from the subject; b) contacting a solid support comprising
capture agents associated therewith with the biological sample
under conditions wherein the capture agents selectively bind to a
first biomarker on the exosomes selected from the group consisting
of a neuron-specific protein, an astrocyte-specific protein, a
microglia-specific protein, and an oligodendrocyte-specific
protein, wherein neuron-derived exosomes, astrocyte-derived
exosomes, oligodendrocyte-derived exosomes, or microglia-derived
exosomes are captured on the solid support; c) separating the solid
support from the biological sample; d) detecting a second biomarker
selected from the group consisting of a neuron-specific protein, an
astrocyte-specific protein, a microglia-specific protein on the
exosomes captured on the solid support, wherein the exosomes are
not lysed or permeabilized; e) diagnosing the subject with the
neurological disorder by comparing the level of the second
biomarker to a control level of the second biomarker; and f)
treating the subject for the neurological disorder if the subject
is diagnosed as having the neurological disorder.
[0029] In certain embodiments, the neurological disorder is
selected from the group consisting of: Alzheimer's disease (AD),
vascular disease dementia, frontotemporal dementia (FTD),
corticobasal degeneration (CBD), progressive supranuclear palsy
(PSP), Lewy body dementia, tangle-predominant senile dementia,
Pick's disease (PiD), argyrophilic grain disease, amyotrophic
lateral sclerosis (ALS), other motor neuron diseases, Guam
parkinsonism-dementia complex, FTDP-17, Lytico-Bodig disease,
multiple sclerosis, traumatic brain injury (TBI), stroke,
depression, bipolar disease, epilepsy, autism, schizophrenia, brain
tumor, white matter disease, brain atrophy, mental retardation,
cerebellar ataxia, multiple system atrophy, concussion,
subconcussive impacts, and Parkinson's disease.
[0030] In certain embodiments, the first and second biomarkers are
membrane-bound proteins or adsorbed proteins on the vesicles,
including but not limited to, a neuron-specific protein selected
from the group consisting of synaptosome associated protein 25
(SNAP25), neurogranin (NRGN), tau, phosphorylated tau, and
synaptophysin, an astrocyte-specific proteins selected from the
group consisting of glial fibrillary acidic protein (GFAP) and
excitatory amino acid transporter 1 (EAAT1), a microglia-specific
protein (CD11b), a cytokine selected from the group consisting of
IL1b, IL34, FasL, or IL12B, a chemokine (CX3CL1), CD81, CD171,
CTSD, CD63, 4-42, an oligodendrocyte-specific protein selected from
the group consisting of myelin basic protein (MBP) and
oligodendrocyte myelin glycoprotein (OMG). In some embodiments, the
biomarker is selected from the group consisting of CD81,
acetylcholinesterase (AchE), Lysosomal Associated Membrane Protein
1 (LAMP1), CTSD, RE1 Silencing Transcription Factor (REST),
synaptotagmin (SYT), NGRN, monocyte chemotactic protein-1 (CCL2),
IL34, glycogen synthase (GYS), olfactory receptor (OR), death
receptor 6 (DR6), heat shock protein (HSP), IL12beta, alpha-beta
(A.beta.), beta-secretase (BACE), dopamine receptors (D1 and D2),
serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5, B1,
B2), glutamate receptors (1 and 2), insulin receptors, tumor
necrosis factor receptors superfamily (TRAL, TNF receptor, death
receptor 5 and 6), neuropeptide receptors (orexin receptor, opioid
receptor KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32,
CD79a, TREM2, and NCAM. In some embodiments, the first biomarker is
SNAP25 and the second biomarker is selected from the group
consisting of CD81, tau, NEFL, TNFa, and IL8. In some embodiments,
the first biomarker is EAAT1 and the second biomarker is CD81 or
GFAP. In some embodiments, the first biomarker is OMG and the
second biomarker is CD81 or MBP. In some embodiments, the first
biomarker is CD11b and the second biomarker is SYP.
[0031] In some embodiments, the level of one or more biomarkers on
exosomes in the biological sample is compared to the level of one
or more biomarkers in a control sample, wherein the level of the
one or more biomarkers of the biological sample is elevated
compared to the control sample. In some embodiments, the level of
the one or more biomarkers in the biological sample is compared to
the level of one or more biomarkers in a control sample, wherein
the level of the one or more biomarkers of the biological sample is
decreased compared to the control sample. In some embodiments, the
biomarker level determines the disease or disorder status of the
subject with at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
99%, or 100% specificity. In some embodiments, the disease or
disorder is a neurological disorder, an immunological disorder, a
placental disease, or cancer. In some embodiments, the neurological
disorder is selected from the group consisting of: Alzheimer's
disease (AD), vascular disease dementia, frontotemporal dementia
(FTD), corticobasal degeneration (CBD), progressive supranuclear
palsy (PSP), Lewy body dementia, tangle-predominant senile
dementia, Pick's disease (PiD), argyrophilic grain disease,
amyotrophic lateral sclerosis (ALS), other motor neuron diseases,
Guam parkinsonism-dementia complex, FTDP-17, Lytico-Bodig disease,
multiple sclerosis, traumatic brain injury (TBI), stroke,
depression, bipolar disease, epilepsy, autism, schizophrenia, brain
tumor, white matter disease, brain atrophy, mental retardation,
cerebellar ataxia, multiple system atrophy, concussion,
subconcussive impacts, and Parkinson's disease. In other
embodiments, the biological sample is selected from the group
consisting of whole blood, serum, plasma, urine, interstitial
fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab,
skin, brain tissue, and cerebrospinal fluid. In some embodiments,
the cancer is breast cancer, pancreatic cancer, lung cancer, colon
cancer, colorectal cancer, rectal cancer, kidney cancer, bladder
cancer, stomach cancer, prostate cancer, liver cancer, ovarian
cancer, brain cancer, vaginal cancer, vulvar cancer, uterine
cancer, oral cancer, penic cancer, testicular cancer, esophageal
cancer, skin cancer, cancer of the fallopian tubes, head and neck
cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous
or intraocular melanoma, cancer of the anal region, cancer of the
small intestine, cancer of the endocrine system, cancer of the
thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, cancer of the urethra, cancer of the renal pelvis,
cancer of the ureter, cancer of the endometrium, cancer of the
cervix, cancer of the pituitary gland, neoplasms of the central
nervous system (CNS), primary CNS lymphoma, brain stem glioma, or
spinal axis tumors.
[0032] In another embodiment, the invention includes a method of
diagnosing and treating a disease or disorder in a subject, the
method comprising: a) obtaining a biological sample comprising
exosomes from the subject; b) contacting a solid support comprising
capture agents associated therewith with the biological sample
under conditions wherein the capture agents selectively bind to a
membrane marker on the exosomes wherein the membrane marker is
selected from the group consisting of SNAP25, OMG, CD11b, EAAT1 and
CD171, wherein exosomes having the membrane marker are captured on
the solid support; c) separating the solid support from the
biological sample; d) measuring levels of one or more biomarkers
selected from the group consisting of CD81, GAPDH, CTSD, NRGN, MBP,
GFAP, Tau, phosphorylated Tau, CD63, .alpha..beta.-42, CX3CL1,
IL1b, and IL34 on the exosomes captured on the solid support,
wherein the exosomes are not lysed or permeabilized; e) diagnosing
the subject with the disease or disorder by comparing the levels of
the one or more biomarkers to control levels of the biomarkers in a
control sample, wherein increased levels of one or more biomarkers
and/or decreased levels of one or more biomarkers compared to the
control levels of the one or more biomarkers in the control sample
indicate that the subject has the disease or disorder; and f)
treating the subject for the disease or disorder if the subject is
diagnosed as having the disease or disorder. In some embodiments,
the biomarker is selected from the group consisting of CD81,
acetylcholinesterase (AchE), Lysosomal Associated Membrane Protein
1 (LAMP1), CTSD, RE1 Silencing Transcription Factor (REST),
synaptotagmin (SYT), NGRN, monocyte chemotactic protein-1 (CCL2),
IL34, glycogen synthase (GYS), olfactory receptor (OR), death
receptor 6 (DR6), heat shock protein (HSP), IL12beta, alpha-beta
(A.beta.), beta-secretase (BACE), dopamine receptors (D1 and D2),
serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5. B1,
B2), glutamate receptors (1 and 2), insulin receptors, tumor
necrosis factor receptors superfamily (TRAL, TNF receptor, death
receptor 5 and 6), neuropeptide receptors (orexin receptor, opioid
receptor KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32,
CD79a, TREM2, and NCAM. In some embodiments, the disease or
disorder is a neurological disorder, an immunological disorder, a
placental disease, or cancer.
[0033] In another aspect, the invention includes a kit for
detecting biomarkers on vesicles, the kit comprising: a) a solid
support comprising capture agents associated therewith, wherein the
capture agents selectively bind to a first biomarker on the surface
of the vesicles; and b) at least one detection agent that
selectively binds to a second biomarker on the surface of the
vesicles. In certain embodiments, the vesicles are selected from
the group consisting of exosomes, microparticles, microvesicles,
nanosomes, extracellular vesicles, and ectosomes. In yet other
aspects, the invention provides a kit for detecting biomarkers on
vesicles, the kit comprising: a) a solid support comprising capture
agents associated therewith, wherein at least one capture agent
selectively binds to a neuron-specific protein, an
astrocyte-specific protein, a microglia-specific protein, or an
oligodendrocyte-specific protein on exosomes; and b) one or more
detection agents, wherein the one or more detection agents
selectively binds to cytosolic proteins, secretory proteins,
neurotransmitter receptors or membrane proteins on the surface of
the exosomes. In some embodiments, the cytosolic proteins are
selected from the group consisting of GAPDH, CTSD, NRGN, MBP, GFAP,
Tau, phosphorylated Tau (e.g., T181), synaptophysin, SNCA,
.alpha..beta.-42, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95,
SV2A, GYS, HSP70, BACE, SYMPO, NEFL, caspase, ubiquitin, PSEN1,
GSK, PLAP, CSH1, PSG1, or FasL. In other embodiments, the secretory
protein is selected from the group consisting of cytokines, growth
factors, chemokines, and interleukins. In certain aspects, the
cytokine is selected from the group consisting of IL1b, IL34, IL6,
IL8, IL16, IL23A, IL32, IL33, CX3CL1, CCL2, CXCL12, TNFalpha,
TNFSF10, IL12B, nociceptin, GnRH, FasL and TNFSF13. In other
embodiments, the neurotransmitter receptor is selected from the
group consisting of dopamine receptors (D1 and D2), serotonin
receptors (2A, 2C, and 3B), GABA receptors (1-6, 5. B1, B2),
glutamate receptors (1 and 2), insulin receptors, tumor necrosis
factor receptors superfamily (TRAL, TNF receptor, death receptor 5
and 6), and neuropeptide receptors (orexin receptor, opioid
receptor KOR). In other embodiments, the membrane protein is
selected from the group consisting of EpCAM, PD-L1, ErbB2, CK19,
TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM.
[0034] In certain embodiments, the capture agents and detection
agents in the kit comprise antibodies, antibody fragments, antibody
mimetics, or aptamers that specifically bind to the first biomarker
on the vesicles. The antibodies may include, for example,
monoclonal antibodies, polyclonal antibodies, chimeric antibodies,
nanobodies, recombinant fragments of antibodies, Fab fragments,
Fab' fragments, F(ab').sub.2 fragments, F.sub.v fragments, and an
scF.sub.v fragments. In another embodiment, the capture agents or
detection agents comprise antibodies selected from the group
consisting of an anti-CD171 antibody, an anti-synaptosome
associated protein 25 (SNAP25) antibody, an anti-neurogranin (NRGN)
antibody, an anti-tau antibody, an anti-synaptophysin antibody, and
anti-CD63 antibody, an anti-.alpha..beta.-42 antibody, an anti-CD81
antibody, an anti-CTD antibody, an anti-GAPDH antibody, an
anti-IL1b antibody, an anti-IL34 antibody, an anti-CX3CL1 antibody,
an anti-glial fibrillary acidic protein (GFAP) antibody, an
anti-excitatory amino acid transporter 1 (EAAT1) antibody, an
anti-myelin basic protein (MBP) antibody, an anti-SNCA antibody, an
anti-TH antibody, an anti-CD11b antibody, and an
anti-oligodendrocyte myelin glycoprotein (OMG) antibody. In some
embodiments, the at least one capture agent selectively binds to
CD171, CD63, CD81, SNAP25, EAAT1, CD11b, or OMG on exosomes. In
other embodiments, the one or more detection agents selectively
binds to CD81, GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated
Tau (e.g., T181), CD63, .alpha..beta.-42, CX3CL1, IL1b, or IL34 on
the surface of the exosomes. In some embodiments, the at least one
or more detection agents selectively binds to CD81,
acetylcholinesterase (AchE), Lysosomal Associated Membrane Protein
1 (LAMP1), CTSD, RE1 Silencing Transcription Factor (REST),
synaptotagmin (SYT), NGRN, monocyte chemotactic protein-1 (CCL2),
IL34, glycogen synthase (GYS), olfactory receptor (OR), death
receptor 6 (DR6), heat shock protein (HSP), IL12beta, alpha-beta
(A.beta.), beta-secretase (BACE), dopamine receptors (D1 and D2),
serotonin receptors (2A, 2C, and 3B), GABA receptors (1-6, 5. B1,
B2), glutamate receptors (1 and 2), insulin receptors, tumor
necrosis factor receptors superfamily (TRAL, TNF receptor, death
receptor 5 and 6), neuropeptide receptors (orexin receptor, opioid
receptor KOR), EpCAM, PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32,
CD79a, TREM2, or NCAM.
[0035] In certain embodiments, the detection agent in the kit
further comprises a detectable label, for example, a fluorescent,
chemiluminescent, electrochemiluminescent, bioluminescent,
isotopic, or radioactive label.
[0036] In another embodiment, the kit further comprises reagents
for performing an immunoassay. Exemplary immunoassays include an
enzyme-linked immunosorbent assay (ELISA), an immunofluorescent
assay (IFA), an immune-polymerase chain reaction assay, an
electro-chemiluminescence immunoassay (ECLIA), and a
radioimmunoassay (RIA).
[0037] In another embodiment, the invention includes a kit for
diagnosing or prognosing a disease or disorder in a subject,
identifying a subject at risk of a disease or disorder, or
prescribing a therapeutic regimen or predicting benefit from
therapy in a subject having a disease or disorder, the kit
comprising: a) a solid support comprising capture agents associated
therewith, wherein at least one capture agent selectively binds to
a CD171 membrane marker on exosomes; and b) at least two detection
agents, wherein at least one detection agent selectively binds to
phosphorylated tau T181 and at least one detection agent
selectively binds to neurogranin on the surface of the exosomes. In
certain embodiments, at least one capture agent or detection agent
comprises an antibody, an antibody fragment, an antibody mimetic,
or an aptamer that specifically binds to CD171, phosphorylated tau
T181, or neurogranin. In certain embodiments, the antibody is
selected from the group consisting of a monoclonal antibody, a
polyclonal antibody, a chimeric antibody, a nanobody, a recombinant
fragment of an antibody, an Fab fragment, an Fab' fragment, an
F(ab').sub.2 fragment, an F.sub.v fragment, and an scF.sub.v
fragment. In another embodiment, the kit comprises an
anti-neurogranin antibody, an anti-phosphorylated tau T181
antibody, and an anti-CD171 antibody. In one embodiment, the
disease or disorder is a neurological disorder, an immunological
disorder, a placental disease, or cancer.
[0038] In some embodiments, the invention provides a method of
prognosing or monitoring the progression of a disease or disorder
in a subject, the method comprising: a) obtaining a biological
sample comprising vesicles or exosomes from the subject; b)
contacting a solid support comprising capture agents associated
therewith with the biological sample under conditions wherein the
capture agents selectively bind to a first biomarker on the
vesicles or exosomes, wherein vesicles or exosomes are captured on
the solid support; c) separating the solid support from the
biological sample; d) measuring a level of a second biomarker
protein on the vesicles or exosomes captured on the solid support,
wherein the vesicles or exosomes are not lysed or permeabilized,
thereby prognosing or monitoring the progression of the disease or
disorder in the subject. In other embodiments, the method further
comprises repeating (a), (b), (c), and (d) at a second time point
with a second biological sample wherein an increase in the levels
of the second biomarker relative to its level at the first time
point indicates that the disease or disorder is progressing, and a
decrease in the level of the second biomarker relative to its level
at the first time point indicates that the disease or disorder is
regressing. In other embodiments, the methods further comprise
treating the subject for the disease or disorder if the disease or
disorder is progressing. In still other embodiments, the method
further comprises reducing treatment or stopping treatment for the
disease or disorder if the disease or disorder is regressing. In
some embodiment, the disease or disorder is a neurological
disorder, an immunological disorder, a placental disease, or
cancer. In other embodiments, the neurological disorder is selected
from the group consisting of: Alzheimer's disease (AD), vascular
disease dementia, frontotemporal dementia (FTD), corticobasal
degeneration (CBD), progressive supranuclear palsy (PSP), Lewy body
dementia, tangle-predominant senile dementia, Pick's disease (PiD),
argyrophilic grain disease, amyotrophic lateral sclerosis (ALS),
other motor neuron diseases, Guam parkinsonism-dementia complex,
FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain
injury (TBI), stroke, depression, bipolar disease, epilepsy,
autism, schizophrenia, brain tumor, white matter disease, brain
atrophy, mental retardation, cerebellar ataxia, multiple system
atrophy, concussion, subconcussive impacts, and Parkinson's
disease.
[0039] These and other embodiments of the present invention will
readily occur to those of skill in the art in light of the
disclosure herein, and all such embodiments are specifically
contemplated.
[0040] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description. The invention is capable of
other embodiments and of being practiced or of being carried out in
various ways. Also, the phraseology and terminology used herein is
for the purpose of description and should not be regarded as
limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIGS. 1A and 1B shows methods for quantifying cell-specific
biomarkers of exosomes. FIG. 1A shows a schematic depicting the
generation of exosomes containing proteins of the cell from which
they originated (mother cell). FIG. 1B shows methods of screening
exosomes for membrane markers and adsorbed markers.
[0042] FIGS. 2A-2F show exemplary detection of cytosolic
(non-membrane) proteins on the surface of exosomes. Neuron-specific
anti-SNAP25 (FIGS. 2A-2C) or control mouse IgG (FIGS. 2D-2F) were
immobilized on white ELISA plates. Various volumes (10, 2.5, and 0
uL) of plasma was suspended in PBS (.tangle-solidup.), 0.1%
tween-20 PBS (o), or 0.1% triton-X100 PBS (I) in a final volume of
40 uL, and applied to the ELISA plates. After overnight incubation
at 4.degree. C., unbound materials were removed. Then,
biotin-labeled antibodies against the exosome surface marker, CD81
(FIGS. 2A and 2D), and 2 different soluble cytosolic proteins,
GAPDH (FIGS. 2B and 2E)) and CTSD (FIGS. 2C and 2F), were applied
to ELISA plates. For PBS samples (.tangle-solidup.), antibodies
were suspended in PBS without tween-20. For samples with tween-20
(.smallcircle.) and triton-X100 (|), labeled antibodies were
suspended in 0.1% tween-20 PBS. Conventional chemiluminescent ELISA
procedure was carried out, and relative light unit (RLU) was
determined.
[0043] FIGS. 3A-3D show the specificity of marker proteins of the
present invention. Antibodies against (FIG. 3A) the exosome surface
marker CD81, (FIG. 3B) neuron surface marker SNAP25, (FIG. 3C)
astrocyte surface marker (EAAT1), or (FIG. 3D) oligodendrocyte
surface marker (OMG) were immobilized onto white ELISA plates. Ten
.mu.L of plasma suspended in 40 .mu.L of PBS were applied to the
ELISA plates. After overnight incubation at 4.degree. C., unbound
materials were removed. Biotin-labeled antibodies against the
exosome surface marker CD81, general cytosolic marker GAPDH,
cytosolic proteins in neuron (NRGN), oligodendrocyte (MBP), and
astrocyte (GFAP) were applied to the ELISA plates. A no antibody
control (tPBS) was also included. Conventional chemiluminescent
ELISA was carried out, and the relative light unit (RLU) was
determined.
[0044] FIGS. 4A-4D show detection and quantification of exosome
biomarkers of the present invention. Anti-SNAP25 (FIG. 4A and FIG.
4B), anti-EAAT1 (FIG. 4C), or anti-OMG (FIG. 4D) were immobilized
to ELISA plate. Various volume of standard plasma was suspended in
PBS, and applied to ELISA wells in a final volume of 40 mL.
Antibodies against NRGN (FIG. 4A), tau (FIG. 4B), GFAP (FIG. 4C),
and MBP (FIG. 4D) were used as detection antibodies.
[0045] FIGS. 5A-5C show screening of exosome adsorbed markers.
Control mouse IgG (open columns), mouse anti-human CD81 (light gray
columns), or mouse anti-human synaptosomal-associated protein 25
(SNAP25) (dark gray columns) were immobilized on an ELISA plate,
then pooled human plasma was applied to all of ELISA wells. The
various detection antibodies listed were used to carry out the
ELISA. Detection antibodies (all biotinylated) were against not
only exosome membrane proteins (CD81, CD171, CD63, and SNAP25), but
also various non-membrane proteins (glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), cathepsin D (CTSD), neurogranin (NRGN),
myelin basic protein (MBP), glial fibrillary acidic protein (GFAP),
tau, microtubule associated protein tau (tau) and phosphorylated
tau T181, and amyloid b.sub.1-42 peptide (ABETA42). As a negative
control, PBS without any biotinylated antibody was used. FIG. 5A
shows the detection of the membrane proteins, CD81 and CD171, and
the non-membrane proteins, GAPDH and CTSD. FIG. 5B shows the
detection of the membrane proteins, CD63, and SNAP25. FIG. 5C shows
the detection of the non-membrane proteins, NRGN, MBP, GFAP, tau,
T181, and ABETA42.
[0046] FIG. 6A-6E shows clinical applications for diagnosis of
Alzheimer's disease. Anti-CD171 antibodies were immobilized on an
ELISA plate as shown in the assay schematic in FIG. 6A. Twelve EDTA
plasma samples from subjects with Alzheimer's disease and an age,
gender-matched control were applied to ELISA wells, and antibodies
against (FIG. 6B) CD81, (FIG. 6C) CTSD. (FIG. 6D) NRGN, and (FIG.
6E) p-tau T181 were used as detection antibodies.
[0047] FIGS. 7A-7L show the stability of plasma levels of various
exosome biomarkers of the present invention. Anti-CD81, anti-CD171,
anti-SNAP25, anti-EAAT1, and anti-OMG were immobilized to ELISA
plate. EDTA plasma was obtained from 7 control subjects every week
for 2-3 weeks, and applied to ELISA wells. FIG. 7A shows assay
schematic for detecting exosome membrane targets. FIGS. 7B-7F show
detection with (FIG. 7B) the anti-CD81 probe on the CD81 plate for
total exosome (TE), (FIG. 7C) the anti-CD171 probe on the CD81
plate for CD171-based NDE (cNDE), (FIG. 7D) the anti-SNAP25 probe
on the CD81 plate for SNAP25-based NDE (sNDE), (FIG. 7E) the
anti-CD81 probe on the EAAT1 plate (ADE), and (FIG. 7F) the
anti-CD81 probe on the OMG plate for ODE. FIG. 7G shows assay
schematic for exosome surface protein targets. FIGS. 7H-7L show
detection with (FIG. 7H) the GFAP probe on the EAAT1 plate, (FIG.
7I) the MBP probe on the OMG plate, (FIG. 7J) the NRGN probe on the
SNAP25 plate, (FIG. 7K) the tau probe on the SNAP25 plate, and
(FIG. 7L) the NRGN probe on the CD171 plate, respectively.
[0048] FIGS. 8A and 8B show detection and quantification of exosome
biomarkers of the present invention in plasma using an
anti-NRGN-immobilized ELISA plate. FIG. 8A shows an exemplary assay
schematic. FIG. 8B shows the assay results. Anti-NRGN (medium gray
and dark gray columns) or control mouse IgG (black and light gray
columns) were immobilized on an ELISA plate. EDTA plasma (light
gray and dark gray columns) or PBS alone (black and medium gray
columns) were applied to ELISA wells. Antibodies against NRGN,
CD171, SNAP25, CD81, EAAT1, OMG and PBS control were used as
detection antibodies.
[0049] FIGS. 9A-9D show microglia targeting of exosome biomarkers
of the present invention. FIG. 9A shows an assay schematic. FIGS.
9B-9D show the assay results. Anti-CD11b (medium gray and dark gray
columns) or control mouse IgG (black and light gray columns) were
immobilized to ELISA plate. EDTA plasma from 7 different donors
(IR1-IR7) (light gray and dark gray columns) or PBS alone (black
and medium gray columns) were applied to ELISA wells. Antibodies
against GFAP (FIG. 9B), MBP (FIG. 9C), or NRGN (FIG. 9D) were used
as detection antibodies
[0050] FIGS. 10A-10F set forth data showing detection of secretory
proteins (cytokines (IL1b and IL34) and chemokines (CX3CL1)) on the
surface of brain-derived exosomes.
[0051] FIG. 11 sets forth data showing dilution of standard plasma
as an ELISA quantification standard.
[0052] FIG. 12 sets forth data showing duplicate variation for
plasma levels of 16 exosomal biomarkers of the present
invention.
[0053] FIG. 13 sets forth data showing standard curves for plasma
levels of 16 exosomal biomarkers of the present invention.
[0054] FIG. 14 sets forth data showing quantification of target
exosomal surface protein biomarkers in plasma.
[0055] FIG. 15 sets forth data showing plasma exosomal biomarker
levels normalized to CD81.
[0056] FIG. 16 sets forth data showing plasma exosomal biomarker
levels normalized to SYT.
[0057] FIG. 17 sets forth data showing the principle of an
exemplary assay of the present invention. NDE, ADE, and ODE are
specifically captured on ELISA plates, where anti-SNAP25 (neuron
marker), anti-EAAT1 (astrocyte marker), and anti-OMG
(oligodedrocyte marker) are immobilized. Captured exosomes were
further probed with biotinylated anti-CD81 (exosome common
marker).
[0058] FIGS. 18A-18C set forth data showing the specificity of an
exemplary assay of the present invention. Anti-SNAP25 (500 ng/mL)
(A.cndot.), anti-EAAT1 (830 ng/mL) (B.cndot.), anti-OMG (830 ng/mL)
(C.cndot.), and control mouse IgG (500 ng/mL) (4) were immobilized
onto ELISA plates as described in the Methods. Serial dilution of a
single donor plasma sample was prepared and applied to ELISA
plates, then probed with biotinylated anti-CD81. ELISA results were
expressed as relative light units (RLU).
[0059] FIGS. 19A-19C set forth data showing intra-assay precision
of an exemplary assay of the present invention. NDE (A), ADE (B),
and ODE (C) were enumerated in 52 samples (15 each of control, PD,
MSA, and PSP) in duplicate, and each replicate was analyzed in x-y
plot to show the intra-assay variation. Dotted lines are 45.degree.
line and solid lines are regression lines.
[0060] FIGS. 20A-20C set forth data showing inter-assay precision
for an exemplary assay of the present invention. NDE (A), ADE (B),
and ODE (C) were enumerated in 3 different control plasma samples 9
separate times. In each experiment, 7 dilutions of standard plasma
(arbitrary assigned 100 units/mL) and buffer control were analyzed
simultaneously, then ELISA readings (RLU) were converted to
units/mL. Average.+-.standard deviation (CV %) of control plasma 1
(.DELTA.), 2 (.quadrature.), and 3 (.cndot.) were 64.6.+-.9.2
units/mL (14.2%), 1.6.+-.0.8 units/mL (52.9%), and 44.4.+-.5.5
units/mL (12.4%) for NDE, 7.5.+-.1.3 units/mL (16.8%), 2.6.+-.0.5
units/mL (20.3%), and 0.2.+-.0.1 units/mL (63.33%) for ADE, and
12.6.+-.1.3 units/mL (10.1%), 2.6.+-.0.3 units/mL (11.45%), and
0.2.+-.0.1 units/mL (34.5%) for ODE, respectively.
[0061] FIGS. 21A-21C set forth data showing dilutional linearity
for an exemplary assay of the present invention. After screening of
various control plasma samples, we found 3 plasma samples
(.quadrature., .cndot., .tangle-solidup.), showing a wide range of
NDE (A), ADE (B), and ODE (C). Then, plasma dilution study was
carried out to analyze the linearity and the slope among 3 plasma
samples.
[0062] FIGS. 22A-22C set forth data showing enumeration of
brain-derived exosomes in human subjects with Parkinson's Disease.
NDE (A), ADE (B), and ODE (C) were enumerated in 15 each of
control, PD, MSA, and 7 PSP patients. Each dot represents a single
individual, and p values were against controls.
[0063] FIGS. 23A-23C set forth data showing Receiver Operating
Characteristic (ROC) of NDE and ODE for the differential diagnosis
of Parkinson's Disease and controls. ROC was calculated using NDE
(A) and ODE (B) data of Parkinson's Disease and control. Area under
curve (AUC) was 88.9% and 88% for NDE and ODE, respectively.
[0064] FIGS. 24A-24F set forth data showing the correlation between
the levels of plasma BDE and disease severity in plasma samples
from subjects with Parkinson's Disease. NDE (A, D), ADE (B, E), and
ODE (C, F) of PD patients were classified with the severity scores
of mRS (A-C) and Hoehn-Yahr (D-F) and shown with those of controls.
Each dot represents a single individual, and statistical p-values
were between each severity group and control.
[0065] FIGS. 25A-25D set forth data showing correlation of
Brain-Derived Exosomes and Parkinson's Disease severity. A: ADE/NDE
ratio and disease duration. B: ADE/NDE ratio and mRS score. C:
ADE/NDE ratio and UPDRS part 3 (motor). D: ODE/NDE ratio and UPDRS
part 3 (motor).
[0066] FIGS. 26A-26D set forth data showing correlation between the
levels of plasma BDE and disease severity in MSA. A: ODE (A) of MSA
patients was classified with the severity scores of mRS and shown
with those of controls. Each dot represents a single individual. B:
Same data as shown in FIG. 3C. C-E: ODE of each MSA patient was
compared with disease duration in months (C), and scores of
International Cooperative Ataxia Rating Scale (ICARS) (D) and
Unified Parkinson's Disease Rating Scale (UPDRS) 3 (motor) (E). The
r.sup.2 and p values were shown in E.
[0067] FIGS. 27A-27F set forth data showing detection of
pathological form of .alpha.-synuclein on the surface of neuron-,
astrocyte-, and oligodendrocyte-derived exosomes.
[0068] FIGS. 28A-28B set forth data showing detection of dopamine
receptor D2 (DRD2) on the surface of neuron-derived exosomes
(NDE).
[0069] FIGS. 29A-29C set forth data showing plasma dilution study
results for DRD2.
[0070] FIGS. 30A-30G set forth data showing DRD2 detection in
neuron-derived exosomes in plasma samples from humans with
neurological disease.
[0071] FIG. 31 sets forth data showing ROC curve and area under the
curve (AUC) for DRD2 levels between PD and control samples.
[0072] FIGS. 32A-32D set forth data showing detection of CD81,
SNCA, SNCA oligomer F1, and TH on the surface of DAT+ vesicles.
[0073] FIG. 33 sets forth data showing detection of SNAP25, EAAT1,
and OMG on the surface of vesicles in plasma.
[0074] FIG. 34 sets forth data showing particle size for captured
DAT+CD81+ vesicles in plasma.
[0075] FIG. 35 sets forth data showing detection of various
biomarkers on the surface of vesicles in plasma.
DESCRIPTION OF THE INVENTION
[0076] It is to be understood that the invention is not limited to
the particular methodologies, protocols, cell lines, assays, and
reagents described herein, as these may vary. It is also to be
understood that the terminology used herein is intended to describe
particular embodiments of the present invention, and is in no way
intended to limit the scope of the present invention as set forth
in the appended claims.
[0077] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
references unless context clearly dictates otherwise. Thus, for
example, a reference to "a fragment" includes a plurality of such
fragments, a reference to an "antibody" is a reference to one or
more antibodies and to equivalents thereof known to those skilled
in the art, and so forth.
[0078] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications cited herein are incorporated herein by
reference in their entirety for the purpose of describing and
disclosing the methodologies, reagents, and tools reported in the
publications that might be used in connection with the invention.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0079] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, cell biology, genetics, immunology
and pharmacology, within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., Gennaro, A. R., ed.
(1990) Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology,
Academic Press, Inc.; Handbook of Experimental Immunology, Vols.
I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell
Scientific Publications); Maniatis, T. et al., eds. (1989)
Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds.
(1999) Short Protocols in Molecular Biology, 4th edition, John
Wiley & Sons; Ream et al., eds. (1998) Molecular Biology
Techniques: An Intensive Laboratory Course, Academic Press); PCR
(Introduction to Biotechniques Series), 2nd ed. (Newton &
Graham eds., 1997, Springer Verlag).
[0080] The present invention relates, in part, to the development
of an efficient method for detecting and quantitating biomarkers on
vesicles. In particular, vesicles are selectively captured on a
solid support, and membrane-bound and adsorbed biomarkers on the
captured vesicles are detected without lysis or permeabilization of
the vesicles. The invention further relates to the use of such
biomarkers identified on vesicles in diagnostic and prognostic
methods for various diseases.
[0081] Additionally, the present invention provides compositions
for use in the methods described herein. Such compositions may
include a solid support, capture agents which specifically bind to
a surface marker (i.e., membrane protein or adsorbed protein) on
vesicles to capture vesicles on the solid support, detection agents
that specifically bind to biomarkers on vesicles, reagents for
performing immunoassays, and other reagents for performing the
methods described herein.
[0082] The present invention further provides kits for detecting
biomarkers on vesicles. The kits may comprise a solid support, one
or more capture agents which specifically bind and capture vesicles
on the solid support, one or more detection agents which
specifically bind a biomarker on vesicles, and optionally,
immunoassay reagents and other reagents for performing the methods
described herein, one or more containers for collecting and or
holding the biological sample, and instructions for using the
kits.
[0083] The present invention further relates to the discovery that
exosomal biomarkers can be assayed to identify subjects having or
likely to develop neurological disorders, including, for example,
Alzheimer's disease (AD), multiple sclerosis (MS), and
frontotemporal dementia (FTD).
[0084] The present invention is based, in part, on the discovery of
unexpected decreases or increases in certain biomarkers in
neuron-derived exosomes present in the circulation of subjects
having neurological disease (e.g., Alzheimer's disease). The
present invention demonstrates that exosomal levels of these
biomarkers may be assayed to diagnose a neurological disorder in a
subject having a neurological disease. The present invention
further shows that measurement of certain biomarkers in
neuron-derived exosomes from a subject may be used to predict the
subsequent development of a neurological disease (e.g., identify a
subject at risk of developing a neurological disorder).
[0085] The section headings are used herein for organizational
purposes only, and are not to be construed as in any way limiting
the subject matter described herein.
Biological Sample
[0086] A biological sample comprising vesicles (e.g., exosomes) may
be obtained from a subject. The biological sample obtained from the
subject is typically blood, but can be any sample from bodily
fluids, tissue or cells comprising the vesicles to be analyzed. The
biological sample may include, but is not limited to, whole blood,
serum, plasma, urine, interstitial fluid, peritoneal fluid,
cerebrospinal fluid, a cervical swab, tears, saliva, a buccal swab,
skin, organs, and biopsies. Alternatively, exosomes can be obtained
from cultured cells by collection of secreted exosomes from the
surrounding culture media.
[0087] In some embodiments, the biological sample of the invention
is obtained from blood. In some embodiments, about 1-10 mL of blood
is drawn from a subject. In other embodiments, about 10-50 mL of
blood is drawn from a subject. Blood can be drawn from any suitable
area of the body, including an arm, a leg, or blood accessible
through a central venous catheter. In some embodiments, blood is
collected following a treatment or activity. For example, blood can
be collected following a medical exam. The timing of collection can
also be coordinated to increase the number and/or composition of
vesicles (e.g., exosomes) present in the sample. For example, blood
can be collected following exercise or a treatment that induces
vascular dilation.
[0088] Blood may be combined with various components following
collection to preserve or prepare samples for subsequent
techniques. For example, in some embodiments, blood is treated with
an anticoagulant, a cell fixative, a protease inhibitor, a
phosphatase inhibitor, a protein, a DNA, or an RNA preservative
following collection. In some embodiments, blood is collected via
venipuncture using vacuum collection tubes containing an
anticoagulant such as EDTA or heparin. Blood can also be collected
using a heparin-coated syringe and hypodermic needle. Blood can
also be combined with components that will be useful for cell
culture. For example, in some embodiments, blood is combined with
cell culture media or supplemented cell culture media (e.g.,
cytokines).
Enrichment or Isolation of Vesicles (Exosomes, Microparticles,
Microvesicles, Nanosomes, Extracellular Vesicles, and
Ectosomes)
[0089] Samples can be enriched for vesicles through positive
selection, negative selection, or a combination of positive and
negative selection. In some embodiments, vesicles are directly
captured. In other embodiments, blood cells are captured and
vesicles are collected from the remaining biological samples. In
some embodiments, the vesicles enriched in the biological samples
are exosomes, microparticles, microvesicles, nanosomes,
extracellular vesicles, or ectosomes. In some embodiments, the
vesicles enriched in the biological samples are neuron-derived
exosomes, astrocyte-derived exosomes, oligodendrocyte-derived
exosomes, or microglia-derived exosomes.
[0090] Samples can also be enriched for vesicles based on
differences in the biochemical properties of vesicles. For example,
samples can be enriched for vesicles based on antigen, nucleic
acid, metabolic, gene expression, or epigenetic differences. In
some of the embodiments based on antigen differences,
antibody-conjugated magnetic or paramagnetic beads in magnetic
field gradients or fluorescently labeled antibodies with flow
cytometry are used. In some of the embodiments based on nucleic
acid differences, flow cytometry is used. In some of the
embodiments based on metabolic differences, dye uptake/exclusion
measured by flow cytometry or another sorting technology is used.
In some of the embodiments based on gene expression, cell culture
with cytokines is used. Samples can also be enriched for vesicles
based on other biochemical properties known in the art. For
example, samples can be enriched for vesicles based on pH or
motility. Further, in some embodiments, more than one method is
used to enrich for vesicles. In other embodiments, samples are
enriched for vesicles using antibodies, ligands, or soluble
receptors.
[0091] In other embodiments, surface markers are used to positively
enrich vesicles in the sample. In some embodiments, the vesicles
are exosomes, microparticles, microvesicles, nanosomes,
extracellular vesicles, or ectosomes. In other embodiments, NCAM,
CD171, CD9, CD63, CD81, SNAP25, EAAT1, OMG, neuron-specific
enolase, diverse neuron or astrocyte adhesive proteins, microglial
CD18/11, or CD3 T cell membrane cell surface markers are used to
enrich for exosomes. In some embodiments, cell surface markers that
are not found on vesicles populations are used to negatively enrich
vesicles by depleting cell populations. Flow cytometry sorting may
also be used to further enrich for exosomes using cell surface
markers or intracellular or extracellular markers conjugated to
fluorescent labels. Intracellular and extracellular markers may
include nuclear stains or antibodies against intracellular or
extracellular proteins preferentially expressed in vesicles. Cell
surface markers may include antibodies against cell surface
antigens that are preferentially expressed on exosomes (e.g.,
NCAM). In some embodiments, the cell surface marker is a
neuron-derived exosome surface marker, including, for example, NCAM
or CD171. In some embodiments, a monoclonal NCAM, CD9, CD63, CD81,
neuron-specific enolase or CD171 antibody is used to enrich or
isolate exosomes from the sample. In certain aspects, the NCAM,
CD9, CD63, CD81, neuron-specific enolase or CD171 antibody is
biotinylated. In this embodiment, biotinylated NCAM or CD171
antibody can form an antibody-exosome complex that can be
subsequently isolated using streptavidin-agarose resin or beads. In
other embodiments, the NCAM, CD9, CD63, CD81, neuron-specific
enolase or CD171 antibody is a monoclonal anti-human NCAM, CD9,
CD63, CD81, neuron-specific enolase or CD171 antibody. In other
embodiments, the cell surface marker is a neuron-specific protein
(e.g., synaptosome associated protein 25 (SNAP25), neurogranin
(NRGN), tau, phosphorylated tau, .alpha..beta.-42, and
synaptophysin), an astrocyte-specific protein (e.g., glial
fibrillary acidic protein (GFAP) and excitatory amino acid
transporter 1 (EAAT1)), a microglia-specific protein (CD11b), an
oligodendrocyte-specific protein (e.g., myelin basic protein (MBP),
an oligodendrocyte myelin glycoprotein (OMG), a cytosolic protein
(e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1, REST, SYT,
TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL,
caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL), or a
chemokine (CX3CL1) or cytokine (IL1b, IL34, FasL, or IL12B).
[0092] In some embodiments, enriched vesicles from the biological
sample are subsequently enriched for a specific type of vesicle.
For example, the biological sample is enriched for exosomes and
then the enriched exosomes are subsequently enriched for
neural-derived exosomes. In some embodiments, the biological sample
is enriched for individual neural cell sources of vesicles. In
certain aspects, the neural cell sources of vesicles are microglia,
neurons, or astrocytes.
[0093] In other embodiments, vesicles are isolated or enriched from
a biological sample by a method comprising: contacting a biological
sample with an agent under conditions wherein a vesicle present in
said biological sample binds to said agent to form a vesicle-agent
complex; and isolating said vesicle from said vesicle-agent complex
to obtain a sample containing said vesicle, wherein the purity of
vesicles present in said sample is greater than the purity of
vesicles present in said biological sample. In certain embodiments,
the agent is an antibody or a lectin. Lectins useful for forming a
vesicle-lectin complex are described in U.S. Patent Application
Publication No. 2012/0077263. In some embodiments, the vesicle is
an exosome, a microparticle, a microvesicle, nanosomes,
extracellular vesicles, or an ectosome. In some embodiments, the
exosomes are neuron-derived exosomes, astrocyte-derived exosomes,
oligodendrocyte-derived exosomes, or microglia-derived exosomes. In
some embodiments, multiple isolating or enriching steps are
performed. In certain aspects of the present embodiment, a first
isolating step is performed to isolate exosomes from a blood sample
and a second isolating step is performed to isolate neural-derived
exosomes from other exosomes.
[0094] In yet other embodiments, the methods further comprise
releasing the vesicle from the vesicle-agent complex. In other
embodiments, the vesicle is released by exposing the vesicle-agent
complex to low pH between 3.5 and 1.5. In other embodiments, the
vesicle is released using a competing peptide that competes for the
binding of the selection antibody used in the methods of the
present invention. In yet other embodiments, the released vesicle
is neutralized by adding a high pH solution. In other embodiments,
the released vesicle is lysed by incubating the released vesicles
with a lysis solution. In still other embodiments, the lysis
solution contains inhibitors for proteases and phosphatases.
Capture and Detection of Vesicles on a Solid Support
[0095] In other embodiments, a subset of vesicles is separated from
other vesicles in a biological sample using capture agents
immobilized on a solid support. Such capture agents bind
selectively to a surface marker (e.g., membrane protein or adsorbed
protein) on vesicles such that the capture agent can "capture"
vesicles having the surface marker. By "capture" is meant that the
target vesicle can be separated from other vesicles in the sample
by virtue of the binding of the capture agent to the surface marker
on the vesicle.
[0096] The specificity of the capture agent determines the subset
of vesicles from a biological sample that are captured on the solid
support. One or more capture agents can be used in combination in
order to capture vesicles having different surface markers. For
example, the solid support may comprise more than one type of
capture agent associated therewith, for example, at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, or more different capture agents that
selectively bind to different biomarkers on the vesicles. In some
embodiments, the vesicles targeted by a capture agent are exosomes,
microparticles, microvesicles, nanosomes, extracellular vesicles,
or ectosomes.
[0097] In some embodiments, the capture agent selectively binds to
neuron-derived exosomes, astrocyte-derived exosomes,
oligodendrocyte-derived exosomes, or microglia-derived exosomes.
For example, a capture agent can be chosen that selectively binds
to an exosome surface marker (e.g., CD81) to capture exosomes
generally, a neuron-specific protein (e.g., synaptosome associated
protein 25 (SNAP25), neurogranin (NRGN), tau, phosphorylated tau,
.alpha..beta.-42, and synaptophysin) to capture neuron-derived
exosomes, an astrocyte-specific protein (e.g., glial fibrillary
acidic protein (GFAP) and excitatory amino acid transporter 1
(EAAT1) to capture astrocyte-derived exosomes, a microglia-specific
protein (CD11b) to capture microglia-derived exosomes, an
oligodendrocyte-specific protein (e.g., myelin basic protein (MBP)
or oligodendrocyte myelin glycoprotein (OMG)) to capture
oligodendrocyte-derived exosomes, a cytosolic protein (e.g.,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-synuclein
(SNCA) or cathepsin D (CTSD)), or an a chemokine (CX3CL1) or
cytokine (IL1b, IL34, FasL, or IL12B) protein to capture a
brain-derived exosome. In some embodiments, a capture agent can be
chosen that selectively binds to an extracellular vesicle (EV)
surface marker (e.g., DAT) to capture extracellular vesicles
generally.
[0098] Typically, the capture agent is associated with a solid
support, either directly or indirectly. Capture agents may be
immobilized on the surface of a solid support, such as, but not
limited to, a plate, slide, wafer, non-magnetic bead, magnetic
bead, rod, particle, strand, disc, membrane, film, or the inner
surface of a tube, channel, column, flow cell device, or
microfluidic device. A solid support may comprise various
materials, including, but not limited to glass, quartz, silicon,
metal, ceramic, plastic, nylon, polyacrylamide, agarose, resin,
porous polymer monoliths, hydrogels, and composites thereof.
Additionally, a substrate may be added to the surface of a solid
support to facilitate attachment of a capture agent.
[0099] Once captured on the solid support, the vesicles can be
screened for one or more membrane-bound and adsorbed biomarkers
using detection agents without the need for lysis or
permeabilization of the vesicles. Such detection agents bind
selectively to membrane-bound or adsorbed biomarkers on the
vesicles. In certain embodiments, the detection agent selectively
binds to a neuron-specific protein (e.g., synaptosome associated
protein 25 (SNAP25), neurogranin (NRGN), .alpha..beta.-42, tau,
phosphorylated tau, and synaptophysin), an astrocyte-specific
protein (e.g., glial fibrillary acidic protein (GFAP) and
excitatory amino acid transporter 1 (EAAT1)), a microglia-specific
protein (CD11b), an oligodendrocyte-specific protein (e.g., myelin
basic protein (MBP), an oligodendrocyte myelin glycoprotein (OMG),
a cytosolic protein (e.g., glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1,
REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO,
NEFL, caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL), a
chemokine (CX3CL1) or cytokine (IL1b, IL34, FasL, or IL12B), CTSD,
GAPDH, CD81, CD63, or CD171, AchE, LAMP1, REST, SYT, TH, SYP,
SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12beta,
A.beta., or BACE. In certain embodiments, detection of biomarkers
on vesicles captured on the solid support comprises using more than
one type of detection agent, for example, at least 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, or more different detection agents that
selectively bind to different biomarkers on the vesicles.
[0100] Capture agents and detection agents may comprise, for
example, antibodies, antibody fragments, antibody mimetics, or
aptamers that specifically bind to a surface marker (e.g.,
membrane-bound or adsorbed protein) on a vesicle. The phrase
"specifically (or selectively) binds" refers to a binding reaction
that is determinative of the presence of the surface marker on a
vesicle in a heterogeneous population of proteins and other
biologics. Thus, under designated assay conditions, the specified
capture agents or detection agents bind to a particular surface
marker on a vesicle at least two times the background and do not
substantially bind in a significant amount to other proteins
present in the sample.
[0101] In certain embodiments, the capture agent or detection agent
comprises an antibody that specifically binds to a surface marker
(e.g., membrane protein or adsorbed protein) on a vesicle. Any type
of antibody may be used, including polyclonal and monoclonal
antibodies, hybrid antibodies, altered antibodies, chimeric
antibodies and, humanized antibodies, as well as: hybrid (chimeric)
antibody molecules (see, for example, Winter et al. (1991) Nature
349:293-299; and U.S. Pat. No. 4,816,567); F(ab').sub.2 and F(ab)
fragments; F.sub.v molecules (noncovalent heterodimers, see, for
example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662;
and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv
molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci
USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb)
(see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303,
Vincke et al. (2012) Methods Mol Biol 911:15-26; dimeric and
trimeric antibody fragment constructs; minibodies (see, e.g., Pack
et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J
Immunology 149B:120-126); humanized antibody molecules (see, e.g.,
Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988)
Science 239:1534-1536; and U.K. Patent Publication No. GB
2,276,169, published 21 Sep. 1994); and, any functional fragments
obtained from such molecules, wherein such fragments retain
specific-binding properties of the parent antibody molecule (i.e.,
specifically binds to a target surface marker on a vesicle).
[0102] In other embodiments, the capture agent or detection agent
comprises an aptamer that specifically binds to the target surface
marker on a vesicle. Any type of aptamer may be used, including a
DNA, RNA, xeno-nucleic acid (XNA), or peptide aptamer that
specifically binds to the target antibody isotype. Such aptamers
can be identified, for example, by screening a combinatorial
library. Nucleic acid aptamers (e.g., DNA or RNA aptamers) that
bind selectively to a target antibody isotype can be produced by
carrying out repeated rounds of in vitro selection or systematic
evolution of ligands by exponential enrichment (SELEX). Peptide
aptamers that bind to a target antibody isotype may be isolated
from a combinatorial library and improved by directed mutation or
repeated rounds of mutagenesis and selection. For a description of
methods of producing aptamers, see, e.g., Aptamers: Tools for
Nanotherapy and Molecular Imaging (R. N. Veedu ed., Pan Stanford,
2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols
(Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009),
Nucleic Acid Aptamers: Selection, Characterization, and Application
(Methods in Molecular Biology, G. Mayer ed., Humana Press, 2016),
Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang
eds., Springer, 2015), Cox et al. (2001) Bioorg. Med. Chem.
9(10):2525-2531; Cox et al. (2002) Nucleic Acids Res. 30(20): e108,
Kenan et al. (1999) Methods Mol Biol. 118:217-231; Platella et al.
(2016) Biochim. Biophys. Acta November 16 pii:
S0304-4165(16).sub.30447-0, and Lyu et al. (2016) Theranostics
6(9):1440-1452; herein incorporated by reference in their
entireties. In yet other embodiment, the capture agent or detection
agent comprises an antibody mimetic. Any type of antibody mimetic
may be used, including, but not limited to, affibody molecules
(Nygren (2008) FEBS J. 275 (11):2668-2676), affilins (Ebersbach et
al. (2007) J. Mol. Biol. 372 (1):172-185), affimers (Johnson et al.
(2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al.
(2008) J. Mol. Biol. 383 (5):1058-1068), alphabodies (Desmet et al.
(2014) Nature Communications 5:5237), anticalins (Skerra (2008)
FEBS J. 275 (11):2677-2683), avimers (Silverman et al. (2005) Nat.
Biotechnol. 23 (12):1556-1561), darpins (Stumpp et al. (2008) Drug
Discov. Today 13 (15-16):695-701), fynomers (Grabulovski et al.
(2007) J. Biol. Chem. 282 (5):3196-3204), and monobodies (Koide et
al. (2007) Methods Mol. Biol. 352:95-109).
[0103] Detection agents may further comprise a detectable label to
facilitate detection and/or quantitation of biomarkers on vesicles.
Detectable labels include fluorescent, chemiluminescent,
electrochemiluminescent, or bioluminescent tags, metals, dyes,
radionuclides, and the like, attached to the specific binding agent
(e.g., antibody, antibody fragment, antibody mimetic, or aptamer
that specifically binds to a membrane-bound or adsorbed biomarker
on vesicles).
Neurological Disorders
[0104] The present invention provides methods for diagnosing or
prognosing a neurological disorder in a subject, identifying a
subject at risk of a neurological disorder, or prescribing a
therapeutic regimen or predicting benefit from therapy in a subject
having a neurological disorder. In some embodiments, the present
invention provides methods for differential diagnosis of a
neurological disorder in a subject.
[0105] In some embodiments the neurological disorder is selected
from the group consisting of: Alzheimer's disease (AD), vascular
disease dementia, frontotemporal dementia (FTD), corticobasal
degeneration (CBD), progressive supranuclear palsy (PSP), Lewy body
dementia, tangle-predominant senile dementia, Pick's disease (PiD),
argyrophilic grain disease, amyotrophic lateral sclerosis (ALS),
other motor neuron diseases, Guam parkinsonism-dementia complex,
FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain
injury (TBI), stroke, depression, bipolar disease, epilepsy,
autism, schizophrenia, brain tumor, white matter disease, brain
atrophy, mental retardation, cerebellar ataxia, concussion,
subconcussive impacts, and Parkinson's disease.
[0106] In some embodiments, the present invention enables a medical
practitioner to diagnose or prognose one or more neurological
disorders in a subject. In other embodiments, the present invention
enables a medical practitioner to rule out or eliminate one or more
neurological diseases as a diagnostic possibility. In other
embodiments, the methods of the present invention allow a medical
practitioner to distinguish some forms of FTD from Alzheimer's
disease. In yet other embodiments, the present invention enables a
medical practitioner to identify a subject at risk of developing a
neurological disorder. In other embodiments, the present invention
enables a medical practitioner to predict whether a subject will
later develop a neurological disorder. In further embodiments, the
present invention enables a medical practitioner to prescribe a
therapeutic regimen or predict benefit from therapy in a subject
having a neurological disorder.
Cancer
[0107] The present invention provides methods for diagnosing or
prognosing cancer in a subject, identifying a subject at risk of
developing cancer, or prescribing a therapeutic regimen or
predicting benefit from therapy in a subject having cancer.
Generally, a cancer is characterized by the uncontrolled growth of
abnormal cells anywhere in a body. The abnormal cells may be termed
cancer cells, malignant cells, or tumor cells. Cancer is not
confined to humans; animals and other living organisms can get
cancer.
[0108] In some instances, the cancer may be malignant.
Alternatively, the cancer may be benign. The cancer may be a
recurrent and/or refractory cancer. Most cancers can be classified
as a carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a central
nervous system cancer.
[0109] The cancer may be a sarcoma. Sarcomas are cancers of the
bone, cartilage, fat, muscle, blood vessels, or other connective or
supportive tissue. Sarcomas include, but are not limited to, bone
cancer, fibrosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
hemangioendothelioma, malignant schwannoma, bilateral vestibular
schwannoma, osteosarcoma, soft tissue sarcomas (e.g. alveolar soft
part sarcoma, angiosarcoma, cystosarcoma phylloides,
dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma,
extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma,
hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma,
neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).
[0110] Alternatively, the cancer may be a carcinoma. Carcinomas are
cancers that begin in the epithelial cells, which are cells that
cover the surface of the body, produce hormones, and make up
glands. By way of non-limiting example, carcinomas include breast
cancer, pancreatic cancer, lung cancer, colon cancer, colorectal
cancer, rectal cancer, kidney cancer, bladder cancer, stomach
cancer, prostate cancer, liver cancer, ovarian cancer, brain
cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer,
penic cancer, testicular cancer, esophageal cancer, skin cancer,
cancer of the fallopian tubes, head and neck cancer,
gastrointestinal stromal cancer, adenocarcinoma, cutaneous or
intraocular melanoma, cancer of the anal region, cancer of the
small intestine, cancer of the endocrine system, cancer of the
thyroid gland, cancer of the parathyroid gland, cancer of the
adrenal gland, cancer of the urethra, cancer of the renal pelvis,
cancer of the ureter, cancer of the endometrium, cancer of the
cervix, cancer of the pituitary gland, neoplasms of the central
nervous system (CNS), primary CNS lymphoma, brain stem glioma, and
spinal axis tumors. In some instances, the cancer is a skin cancer,
such as a basal cell carcinoma, squamous, melanoma, nonmelanoma, or
actinic (solar) keratosis. Preferably, the cancer is a prostate
cancer. Alternatively, the cancer may be a thyroid cancer, bladder
cancer, or pancreatic cancer.
[0111] In some instances, the cancer is a lung cancer. Lung cancer
can start in the airways that branch off the trachea to supply the
lungs (bronchi) or the small air sacs of the lung (the alveoli).
Lung cancers include non-small cell lung carcinoma (NSCLC), small
cell lung carcinoma, and mesotheliomia. Examples of NSCLC include
squamous cell carcinoma, adenocarcinoma, and large cell carcinoma.
The mesothelioma may be a cancerous tumor of the lining of the lung
and chest cavity (pleura) or lining of the abdomen (peritoneum).
The mesothelioma may be due to asbestos exposure. The cancer may be
a brain cancer, such as a glioblastoma.
[0112] Alternatively, the cancer may be a central nervous system
(CNS) tumor. CNS tumors may be classified as gliomas or nongliomas.
The glioma may be malignant glioma, high grade glioma, diffuse
intrinsic pontine glioma. Examples of gliomas include astrocytomas,
oligodendrogliomas (or mixtures of oligodendroglioma and astocytoma
elements), and ependymomas. Astrocytomas include, but are not
limited to, low-grade astrocytomas, anaplastic astrocytomas,
glioblastoma multiforme, pilocytic astrocytoma, pleomorphic
xanthoastrocytoma, and subependymal giant cell astrocytoma.
Oligodendrogliomas include low-grade oligodendrogliomas (or
oligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomas
include meningiomas, pituitary adenomas, primary CNS lymphomas, and
medulloblastomas. In some instances, the cancer is a
meningioma.
[0113] The cancer may be a leukemia. The leukemia may be an acute
lymphocytic leukemia, acute myelocytic leukemia, chronic
lymphocytic leukemia, or chronic myelocytic leukemia. Additional
types of leukemias include hairy cell leukemia, chronic
myelomonocytic leukemia, and juvenile myelomonocytic-leukemia.
[0114] In some instances, the cancer is a lymphoma. Lymphomas are
cancers of the lymphocytes and may develop from either B or T
lymphocytes. The two major types of lymphoma are Hodgkin's
lymphoma, previously known as Hodgkin's disease, and non-Hodgkin's
lymphoma. Hodgkin's lymphoma is marked by the presence of the
Reed-Sternberg cell. Non-Hodgkin's lymphomas are all lymphomas
which are not Hodgkin's lymphoma. Non-Hodgkin lymphomas may be
indolent lymphomas and aggressive lymphomas. Non-Hodgkin's
lymphomas include, but are not limited to, diffuse large B cell
lymphoma, follicular lymphoma, mucosa-associated lymphatic tissue
lymphoma (MALT), small cell lymphocytic lymphoma, mantle cell
lymphoma, Burkitt's lymphoma, mediastinal large B cell lymphoma,
Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma
(NMZL), splenic marginal zone lymphoma (SMZL), extranodal marginal
zone B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, and lymphomatoid granulomatosis.
[0115] In some embodiments, the present invention enables a medical
practitioner to diagnose or prognose one or more cancers in a
subject. In other embodiments, the present invention enables a
medical practitioner to rule out or eliminate one or more cancers
as a diagnostic possibility. In other embodiments, the methods of
the present invention allow a medical practitioner to identify the
origin of a cancer. In yet other embodiments, the present invention
enables a medical practitioner to identify a subject at risk of
developing cancer. In other embodiments, the present invention
enables a medical practitioner to predict whether a subject will
later develop cancer. In further embodiments, the present invention
enables a medical practitioner to prescribe a therapeutic regimen
or predict benefit from therapy in a subject having cancer.
Exemplary biomarkers of the present invention that are useful in
cancer diagnosis and prognosis include, but are not limited to,
EpCAM, PD-L1, ErbB2, CK19.
Immunological Disorders
[0116] The present invention provides methods for diagnosing or
prognosing an immunological disorder in a subject, identifying a
subject at risk of developing an immunological disorder, or
prescribing a therapeutic regimen or predicting benefit from
therapy in a subject having an immunological disorder.
Immunological disorders are diseases or conditions caused by a
dysfunction of the immune system and include allergy, asthma,
autoimmune diseases, autoinflammatory syndromes and immunological
deficiency syndromes.
[0117] In some embodiments, the present invention enables a medical
practitioner to diagnose or prognose one or more immunological
disorders in a subject. In other embodiments, the present invention
enables a medical practitioner to rule out or eliminate one or more
immunological disorders as a diagnostic possibility. In yet other
embodiments, the present invention enables a medical practitioner
to identify a subject at risk of developing an immunological
disorder. In other embodiments, the present invention enables a
medical practitioner to predict whether a subject will later
develop an immunological disorder. In further embodiments, the
present invention enables a medical practitioner to prescribe a
therapeutic regimen or predict benefit from therapy in a subject
having an immunological disorder. Exemplary biomarkers of the
present invention that are useful in immunological disorder
diagnosis and prognosis include, but are not limited to, TCR, CD16,
CD28, CD32, CD79a, and TREM2.
Placental Disease and Fetal Assessment
[0118] The present invention provides methods for diagnosing or
prognosing placental disease in a subject, identifying a subject at
risk of developing a placental disease, or prescribing a
therapeutic regimen or predicting benefit from therapy in a subject
having a placental disease. Generally, a placental disease is any
disease, disorder, or pathology of the placenta. The methods and
biomarkers of the present invention may also be used for fetal
assessment or diagnosis of fetal disorders, such as, for example,
fetal alcohol syndrome or fetal genetic abnormalities. Exemplary
biomarkers of the present invention that are useful in placental
disease or fetal assessment diagnosis and prognosis include, but
are not limited to, PLAP, CSH1, and PSG1.
Biomarkers
[0119] Biomarker levels on vesicles are assayed in a biological
sample obtained from a subject having or at-risk of having a
disease. In some embodiments, biomarker levels on vesicles are
assayed in a biological sample obtained from a subject having or
at-risk of having a neurological disorder (e.g., Alzheimer's
disease). In other embodiments, biomarker levels on vesicles are
assayed in a biological sample obtained from a subject having or
at-risk of having cancer. In yet other embodiments, biomarker
levels on vesicles are assayed in a biological sample obtained from
a subject having or at-risk of having an immunological disorder. In
still other embodiments, biomarker levels on vesicles are assayed
in a biological sample obtained from a subject having or at-risk of
having a placental disorder. In some embodiments, one or more
biomarkers are selected from the group consisting of a
neuron-specific protein (e.g., synaptosome associated protein 25
(SNAP25), neurogranin (NRGN), tau, and synaptophysin), an
astrocyte-specific protein (e.g., glial fibrillary acidic protein
(GFAP) and excitatory amino acid transporter 1 (EAAT1)), a
microglia-specific protein (CD11b), an oligodendrocyte-specific
protein (e.g., myelin basic protein (MBP), an oligodendrocyte
myelin glycoprotein (OMG)), and an extracellular vesicle-specific
protein (dopamine transporter, DAT). In another embodiment, the
biomarkers are CD171, phosphorylated tau T181, SNCA, and NRGN. In
other embodiments, the biomarkers are acetylcholinesterase (AchE),
Lysosomal Associated Membrane Protein 1 (LAMP1), CTSD, RE1
Silencing Transcription Factor (REST), synaptotagmin (SYT),
monocyte chemotactic protein-1 (CCL2), IL34, glycogen synthase
(GYS), (OR), death receptor 6 (DR6), heat shock protein (HSP),
IL12beta, alpha-beta (A.beta.), and beta-secretase (BACE). In some
embodiments, one or more biomarkers are selected from the group
consisting of cytosolic proteins, secretory proteins, membrane
proteins and receptors and their pathological forms, including
aggregates and mutated ones. Biomarkers of the present invention
include neurotransmitter receptors, such as, for example, dopamine
receptors (D1 and D2), serotonin receptors (2A, 2C, and 3B), GABA
receptors (1-6, 5. B1, B2), and glutamate receptors (1 and 2).
Other receptor biomarkers of the present invention include, insulin
receptors, tumor necrosis factor receptors superfamily (TRAL, TNF
receptor, death receptor 5 and 6), and neuropeptide receptors
(orexin receptor, opioid receptor KOR). Biomarkers of the present
invention include membrane proteins, such as, for example, EpCAM,
PD-L1, ErbB2, CK19, TCR, CD16, CD28, CD32, CD79a, TREM2, and NCAM.
Other known neurological disorder biomarkers may be used in
combination with the biomarkers of the present invention. Examples
of such biomarkers are provided in US Patent Application Pub. No.
2015/0119278, the contents of which are hereby incorporated by
reference.
[0120] One of ordinary skill in the art has several methods and
devices available for the detection and analysis of the markers of
the instant invention. With regard to polypeptides or proteins on
vesicles in patient test samples, immunoassay devices and methods
are often used. These devices and methods can utilize labeled
molecules in various sandwich, competitive, or non-competitive
assay formats, to generate a signal that is related to the presence
or amount of an analyte of interest. Additionally, certain methods
and devices, such as biosensors and optical immunoassays, may be
employed to determine the presence or amounts of analytes without
the need for a labeled molecule.
[0121] Preferably the markers are analyzed using an immunoassay,
although other methods are well known to those skilled in the art
(for example, the measurement of marker RNA levels). The presence
or amount of a marker is generally determined using antibodies
specific for each marker and detecting specific binding. Any
suitable immunoassay may be utilized, for example, an enzyme-linked
immunosorbent assay (ELISA), immunofluorescent assay (IFA),
immune-polymerase chain reaction assay, electro-chemiluminescence
immunoassay (ECLIA), radioimmunoassay (RIA), competitive binding
assay, planar waveguide technology, and the like. Specific
immunological binding of the antibody to the marker can be detected
directly or indirectly. Direct labels include fluorescent or
luminescent tags, metals, dyes, radionuclides, and the like,
attached to the antibody. Indirect labels include various enzymes
well known in the art, such as alkaline phosphatase, horseradish
peroxidase and the like.
[0122] The use of immobilized antibodies specific for the surface
markers on vesicles is also contemplated by the present invention.
The antibodies could be immobilized onto a variety of solid
supports, such as magnetic or chromatographic matrix particles, the
surface of an assay place (such as microtiter wells), pieces of a
solid substrate material (such as plastic, nylon, paper), and the
like. An assay strip could be prepared by coating the antibody or a
plurality of antibodies in an array on solid support. This strip
could then be dipped into the test sample to capture vesicles
through binding to surface markers, and then processed quickly
through washes and detection steps with detection reagents, as
described above, to generate a measurable signal, such as a colored
spot.
[0123] The analysis of a plurality of markers may be carried out
separately or simultaneously with one test sample. Several markers
on vesicles may be captured and/or detected using a combination of
multiple capture agents and/or detection agents in one test for
efficient processing of multiple of samples. In addition, one
skilled in the art would recognize the value of testing multiple
samples (for example, at successive time points) from the same
individual. Such testing of serial samples will allow the
identification of changes in marker levels over time. Increases or
decreases in marker levels, as well as the absence of change in
marker levels, would provide useful information about the disease
status that includes, but is not limited to identifying the
approximate time from onset of the event, the presence and amount
of salvageable tissue, the appropriateness of drug therapies, the
effectiveness of various therapies, identification of the severity
of the event, identification of the disease severity, and
identification of the patient's outcome, including risk of future
events.
[0124] An assay consisting of a combination of the markers
referenced in the instant invention may be constructed to provide
relevant information related to differential diagnosis. Such a
panel may be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, or more or individual markers. The analysis of a single marker
or subsets of markers comprising a larger panel of markers could be
carried out methods described within the instant invention to
optimize clinical sensitivity or specificity in various clinical
settings.
[0125] The analysis of markers could be carried out in a variety of
physical formats as well. For example, the use of microtiter plates
or automation could be used to facilitate the processing of large
numbers of test samples. Alternatively, single sample formats could
be developed to facilitate immediate treatment and diagnosis in a
timely fashion, for example, in ambulatory transport or emergency
room settings. Particularly useful physical formats comprise
surfaces having a plurality of discrete, addressable locations for
the detection of a plurality of different analytes. Such formats
include protein microarrays, or "protein chips" and capillary
devices.
[0126] Biomarkers of the present invention serve an important role
in the early detection and monitoring of neurological disorders
(e.g., Alzheimer's disease). Markers of such disorders are
typically substances found in a bodily sample that can be measured.
The measured amount can correlate to underlying disorder or disease
pathophysiology, presence or absence of a neurological disorder,
probability of a neurological disorder in the future. In patients
receiving treatment for their condition the measured amount will
also correlate with responsiveness to therapy. In some embodiments,
a decrease or increase in the level of one or more biomarkers of
the present invention is indicative of a neurological disorder. For
example, an increase in phosphorylated tau T181 levels and/or a
decrease in NRGN levels on exosomes having the CD171 membrane
marker is indicative of Alzheimer's disease. Accordingly, the
methods of the present invention are useful for the differential
diagnosis of Alzheimer's disease.
[0127] In some embodiments, a biomarker is measured by a method
selected from the group consisting of immunohistochemistry,
immunocytochemistry, immunofluorescence, immunoprecipitation,
electro-chemiluminescence immunoassay, radioimmunoassay,
immune-polymerase chain reaction, western blotting, and ELISA.
Clinical Assay Performance
[0128] The methods of the present invention may be used in clinical
assays to diagnose or prognose a neurological disorder in a
subject, identify a subject at risk of a neurological disorder,
and/or for prescribing a therapeutic regimen or predicting benefit
from therapy in a subject having a neurological disorder. Clinical
assay performance can be assessed by determining the assay's
sensitivity, specificity, area under the ROC curve (AUC), accuracy,
positive predictive value (PPV), and negative predictive value
(NPV). Disclosed herein are assays for diagnosing or prognosing a
neurological disorder in a subject, identifying a subject at risk
of a neurological disorder, or for prescribing a therapeutic
regimen or predicting benefit from therapy in a subject having a
neurological disorder.
[0129] The clinical performance of the assay may be based on
sensitivity. The sensitivity of an assay of the present invention
may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 99%, or 100%. The clinical performance of the assay
may be based on specificity. The specificity of an assay of the
present invention may be at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. The clinical
performance of the assay may be based on area under the ROC curve
(AUC). The AUC of an assay of the present invention may be at least
about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95. The
clinical performance of the assay may be based on accuracy. The
accuracy of an assay of the present invention may be at least about
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or
100%.
Compositions
[0130] Compositions useful in the methods of the present invention
include compositions that specifically recognize a biomarker
associated with a neurological disorder. Such compositions may
include capture agents and/or detection agents that recognize, for
example, a neuron-specific protein biomarker, such as synaptosome
associated protein 25 (SNAP25), .alpha..beta.-42, neurogranin
(NRGN), tau, phosphorylated tau, and synaptophysin, an
astrocyte-specific protein biomarker, such as glial fibrillary
acidic protein (GFAP) and excitatory amino acid transporter 1
(EAAT1), an oligodendrocyte-specific protein biomarker, such as
myelin basic protein (MBP) and oligodendrocyte myelin glycoprotein
(OMG), a microglia-specific protein (CD11b), a cytosolic protein
(e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
alpha-synuclein (SNCA), cathepsin D (CTSD), AchE, LAMP1, REST, SYT,
TH, SYP, SYNPO, PSD95, SV2A, GYS, HSP70, BACE, SYMPO, NEFL,
caspase, ubiquitin, PSEN1, GSK, PLAP, CSH1, PSG1, or FasL), or a
chemokine (CX3CL1) or cytokine (IL1b, IL34, FasL, or IL12B).
[0131] In yet other embodiments, the composition is selected from
the group consisting of a peptide, a nucleic acid, an antibody, and
a small molecule.
[0132] In certain embodiments, the present invention relates to
compositions that specifically detect a biomarker associated with a
neurological disorder. As detailed elsewhere herein, the present
invention is based upon the finding that GAPDH, CTSD, NRGN, MBP,
GFAP, Tau, phosphorylated Tau, synaptophysin, .alpha..beta.-42,
CX3CL1, IL1b, IL34, CD81, CD63, CD171, SNAP25, EAAT1, SNCA, CD11b,
OMG, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2,
IL34, GYS, OR, DR6, HSP, IL12b, A.beta., and BACE can be used as
biomarkers for AD and other neurological disorders. In some
embodiments, the compositions of the present invention specifically
bind to and detect such biomarkers. For example, a composition may
comprise a solid support comprising capture agents associated
therewith that selectively bind to CD81, CD63, CD171, SNAP25,
EAAT1, CD11b, or OMG. In another example, a composition may
comprise detection agents that selectively bind to GAPDH, CTSD,
NRGN, MBP, GFAP, Tau, phosphorylated Tau, synaptophysin,
.alpha..beta.-42, SNCA, CX3CL1, IL1b, IL34, OMG, AchE, LAMP1, REST,
SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP,
IL12b, A.beta., and/or BACE.
[0133] In some embodiments, the composition comprises an antibody,
where the antibody specifically binds to a biomarker or vesicles of
the invention. The term "antibody" as used herein and further
discussed below is intended to include fragments thereof which are
also specifically reactive with a biomarker or vesicle (e.g.,
exosome). Antibodies can be fragmented using conventional
techniques and the fragments screened for utility in the same
manner as described above for whole antibodies. For example,
F(ab).sub.2 fragments can be generated by treating antibody with
pepsin. The resulting F(ab).sub.2 fragment can be treated to reduce
disulfide bridges to produce Fab fragments. Antigen-binding
portions may also be produced by recombinant DNA techniques or by
enzymatic or chemical cleavage of intact antibodies.
Antigen-binding portions include, inter alia, Fab, Fab',
F(ab').sub.2, Fv, dAb, and complementarity determining region (CDR)
fragments, single-chain antibodies (scFv), single domain
antibodies, bispecific antibodies, chimeric antibodies, humanized
antibodies, diabodies and polypeptides that contain at least a
portion of an immunoglobulin that is sufficient to confer specific
antigen binding to the polypeptide. In certain embodiments, the
antibody further comprises a label attached thereto and able to be
detected (e.g., the label can be a radioisotope, fluorescent
compound, enzyme or enzyme co-factor).
[0134] In certain embodiments, an antibody of the invention is a
monoclonal antibody, and in certain embodiments, the invention
makes available methods for generating novel antibodies that
specifically bind the biomarker or the exosome of the invention.
For example, a method for generating a monoclonal antibody that
specifically binds a biomarker or exosome, may comprise
administering to a mouse an amount of an immunogenic composition
comprising the biomarker or exosome, or fragment thereof, effective
to stimulate a detectable immune response, obtaining
antibody-producing cells (e.g., cells from the spleen) from the
mouse and fusing the antibody-producing cells with myeloma cells to
obtain antibody-producing hybridomas, and testing the
antibody-producing hybridomas to identify a hybridoma that produces
a monocolonal antibody that binds specifically to the biomarker or
exosome. Once obtained, a hybridoma can be propagated in a cell
culture, optionally in culture conditions where the
hybridoma-derived cells produce the monoclonal antibody that binds
specifically to the biomarker or exosome. The monoclonal antibody
may be purified from the cell culture.
[0135] The term "specifically reactive with" as used in reference
to an antibody is intended to mean, as is generally understood in
the art, that the antibody is sufficiently selective between the
antigen of interest (e.g., a biomarker or exosome) and other
antigens that are not of interest. In certain methods employing the
antibody, such as therapeutic applications, a higher degree of
specificity in binding may be desirable. Monoclonal antibodies
generally have a greater tendency (as compared to polyclonal
antibodies) to discriminate effectively between the desired
antigens and cross-reacting polypeptides. One characteristic that
influences the specificity of an antibody:antigen interaction is
the affinity of the antibody for the antigen. Although the desired
specificity may be reached with a range of different affinities,
generally preferred antibodies will have an affinity (a
dissociation constant) of about 10.sup.-6, 10.sup.-7, 10.sup.-8,
10.sup.-9 or less.
[0136] Antibodies can be generated to bind specifically to an
epitope of an exosome or a biomarker of the present invention,
including, for example, neuron-derived exosomes, astrocyte-derived
exosomes, oligodendrocyte-derived exosomes, and microglia-derived
exosomes, or neuron-specific proteins selected from the group
consisting of synaptosome associated protein 25 (SNAP25),
neurogranin (NRGN), tau, phosphorylated tar, and synaptophysin,
astrocyte-specific proteins selected from the group consisting of
glial fibrillary acidic protein (GFAP) and excitatory amino acid
transporter 1 (EAAT1), and oligodendrocyte-specific proteins
selected from the group consisting of myelin basic protein (MBP)
and oligodendrocyte myelin glycoprotein (OMG), a microglia-specific
protein (CD11b), and chemokine (CX3CL1) or cytokine (IL1b, IL34,
FasL, or IL12B). In another embodiment, the antibody generated is
an anti-CD171 antibody, an anti-synaptosome associated protein 25
(SNAP25) antibody, an anti-neurogranin (NRGN) antibody, an anti-tau
antibody, an anti-synaptophysin antibody, and anti-CD63 antibody,
an anti-4-42 antibody, an anti-CD81 antibody, an anti-CTD antibody,
an anti-GAPDH antibody, an anti-IL1b antibody, an anti-IL34
antibody, an anti-CX3CL1 antibody, an anti-glial fibrillary acidic
protein (GFAP) antibody, an anti-excitatory amino acid transporter
1 (EAAT1) antibody, an anti-SNCA antibody, an anti-TH antibody, and
anti-CD11b antibody, an anti-myelin basic protein (MBP) antibody,
an anti-oligodendrocyte myelin glycoprotein (OMG) antibody, an
anti-dopamine transporter (DAT) antibody, an anti-AchE antibody
AchE, an anti-LAMP1 antibody LAMP1, an anti-REST antibody REST, an
anti-SYT antibody SYT, an anti-SYP antibody, an anti-SYNPO
antibody, an anti-PSD95 antibody, an anti-SV2A antibody, an
anti-CCL2 antibody CCL2, an anti-IL34 antibody IL34, an anti-GYS
antibody GYS, an anti-OR antibody OR, an anti-DR6 antibody DR6, an
anti-HSP antibody HSP, an anti-IL12b antibody IL12b, an
anti-A.beta. antibody A.beta., or an anti-BACE antibody BACE.
[0137] In addition, the techniques used to screen antibodies in
order to identify a desirable antibody may influence the properties
of the antibody obtained. A variety of different techniques are
available for testing interaction between antibodies and antigens
to identify particularly desirable antibodies. Such techniques
include ELISAs, surface plasmon resonance binding assays (e.g., the
Biacore binding assay, Biacore AB, Uppsala, Sweden), sandwich
assays (e.g., the paramagnetic bead system of IGEN International,
Inc., Gaithersburg, Md.), western blots, immunoprecipitation
assays, immunocytochemistry, and immunohistochemistry.
[0138] In some embodiments, the present invention relates to
compositions used for treating or preventing a neurological
disorder. As detailed elsewhere herein, the present invention is
based upon the findings that the levels of CD81, GAPDH, CTSD, NRGN,
MBP, GFAP, Tau, phosphorylated Tau (e.g., T181), synaptophysin,
CD63, .alpha..beta.-42, SNCA, CX3CL1, IL1b, IL34, AchE, LAMP1,
REST, SYT, TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6,
HSP, IL12b, A.beta., and/or BACE are implicated in the pathology of
a variety of neurological disorders, such as, for example,
Alzheimer's disease.
[0139] In some embodiments, biomarkers inside vesicles are analyzed
in addition to the surface biomarkers. In certain embodiments, the
present invention relates to compositions for lysing vesicles
(e.g., exosomes) in biological samples obtained from a subject.
Lytic agents useful in the methods of the present invention
include: RIPA buffer; Tris-HCl (pH 6.8); glycerol; SDS;
2-mercaptoethanol; Triton-X 100; M-PER Reagent; T-PER solution; and
CHAPS. Lytic agents may be incubated with biological samples to
disrupt the membrane of the vesicles of the present invention and
release vesicle cargo (e.g., exosomal proteins) for subsequent
analysis.
Methods of Treatment
[0140] The present invention provides methods of treating a
neurological disorder in a subject, comprising administering to the
subject an effective amount of a composition, wherein the
composition increases, decreases, or maintains the level of CD81,
GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated Tau (e.g., T181),
synaptophysin, CD63, .alpha..beta.-42, SNCA, CX3CL1, IL1b, or IL34
in the subject. In yet other embodiments, the composition prevents
increases or decreases in CD81, GAPDH, CTSD, NRGN, MBP, GFAP, Tau,
phosphorylated Tau (e.g., T181), synaptophysin, CD63,
.alpha..beta.-42, SNCA, CX3CL1, IL1b, IL34, AchE, LAMP1, REST, SYT,
TH, SYP, SYNPO, PSD95, SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b,
A.beta., or BACE levels. In other embodiments, the present
invention provides methods of treating a neurological disorder in a
subject, comprising administering to the subject an effective
amount of a composition, wherein the composition normalizes the
level of CD81, GAPDH, CTSD, NRGN, MBP, GFAP, Tau, phosphorylated
Tau (e.g., T181), synaptophysin, CD63, .alpha..beta.-42, SNCA,
CX3CL1, IL1b, IL34, AchE, LAMP1, REST, SYT, TH, SYP, SYNPO, PSD95,
SV2A, CCL2, IL34, GYS, OR, DR6, HSP, IL12b, A.beta., and/or BACE to
a reference level.
Kits
[0141] The above-described assay reagents, including a solid
support with bound capture agents, detection agents, and optionally
reagents for performing immunoassays, such as by ELISA, IFA,
immune-polymerase chain reaction assay, ECLIA, or RIA, can be
provided in kits, with suitable instructions and other necessary
reagents, in order to conduct the assays for detecting biomarkers
on vesicles, as described above. The kit will normally contain in
separate containers the solid support with bound capture agents,
detection agents, control formulations (positive and/or negative),
and other reagents that the assay format requires. Instructions
(e.g., written, CD-ROM, DVD, Blu-ray, flash drive, digital
download, etc.) for carrying out the assay usually will be included
in the kit. The kit can also contain, depending on the particular
assay used, other packaged reagents and materials (i.e., wash
buffers, and the like). Assays, such as those described above, can
be conducted using these kits.
[0142] In another embodiment, the invention encompasses kits for
detecting or monitoring a neurological disorder in a subject. A
variety of kits having different components are contemplated by the
current invention. Generally speaking, the kit will include the
means for quantifying one or more biomarkers in a subject. In
another embodiment, the kit will include means for collecting a
biological sample, means for quantifying one or more biomarkers in
the biological sample, and instructions for use of the kit
contents. In certain embodiments, the kit comprises a means for
enriching or isolating exosomes in a biological sample. In further
aspects, the kit comprises a solid support with bound capture
agents for isolating exosomes from a biological sample. In certain
aspects, the kit comprises a means for quantifying the amount of a
biomarker. In further aspects, the means for quantifying the amount
of a biomarker comprises reagents necessary to detect the amount of
a biomarker.
[0143] In another embodiment, the invention includes a kit for
diagnosing or prognosing a neurological disorder in a subject,
identifying a subject at risk of a neurological disorder, or
prescribing a therapeutic regimen or predicting benefit from
therapy in a subject having a neurological disorder, the kit
comprising: a) a solid support comprising capture agents associated
therewith, wherein at least one capture agent selectively binds to
CD171, CD63, CD81, SNAP25, EAAT1, or OMG; and b) one or more
detection agents, wherein the one or more detection agents
selectively binds to CD81, GAPDH, CTSD, NRGN, MBP, GFAP, Tau,
phosphorylated Tau (e.g., T181), CD63, .alpha..beta.-42, CX3CL1,
IL1b, IL34, AchE, LAMP1, REST, SYT, SYP, SYNPO, PSD95, SV2A, CCL2,
IL34, GYS, OR, DR6, HSP, IL12b, A.beta., or BACE on the surface of
the exosomes. In certain embodiments, at least one capture agent or
detection agent comprises an antibody, an antibody fragment, an
antibody mimetic, or an aptamer that specifically binds to CD171,
phosphorylated tau T181, or neurogranin. In certain embodiments,
the antibody is selected from the group consisting of a monoclonal
antibody, a polyclonal antibody, a chimeric antibody, a nanobody, a
recombinant fragment of an antibody, an Fab fragment, an Fab'
fragment, an F(ab').sub.2 fragment, an F.sub.v fragment, and an
scF.sub.v fragment. In another embodiment, the kit comprises an
anti-neurogranin antibody, an anti-phosphorylated tau T181
antibody, and an anti-CD171 antibody.
[0144] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
EXAMPLES
[0145] The invention will be further understood by reference to the
following examples, which are intended to be purely exemplary of
the invention. These examples are provided solely to illustrate the
claimed invention. The present invention is not limited in scope by
the exemplified embodiments, which are intended as illustrations of
single aspects of the invention only. Any methods that are
functionally equivalent are within the scope of the invention.
Various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are intended to
fall within the scope of the appended claims.
Example 1: Methods for Quantifying Cell-Specific Biomarkers of
Exosomes
[0146] Exosomes in biological fluids are a new resource of
diagnostics in various diseases, because they carry physiological
and pathological materials (proteins, metabolites, RNAs, small
molecules, etc.) of the mother cells from which they originate and
the microenvironment near the mother cells (FIG. 1A). Thus,
quantification of such pathological materials in exosomes will be a
foundation of the next generation of diagnostics.
[0147] However, current methods for analysis of exosomes are
complicated and expensive limiting their use in routine diagnostic
testing. Quantification of intra-exosomal materials typically
involves isolation of target exosomes, lysis or permeabilization of
the exosomes to release exosome cargo, and transfer to suitable
assay vessels for analysis. The multiple steps involved in current
procedures for exosome analysis are labor-intensive and produce
large sample-to-sample and assay-to-assay variation.
[0148] Here we describe a method in which exosome materials derived
from the original mother cell are measured on the surface of
exosomes; hence, eliminating the need for a lysis or
permeabilization step. Exosome isolation and analysis are conducted
on the same solid support without transferring the samples to other
assay vessels, which avoids loss of material.
[0149] As shown in FIG. 1B, the method is performed using
antibodies against at least two different markers on the surface of
an exosome, which may include exosome membrane proteins and/or
adsorbed markers. For example, a first antibody against an exosome
marker, such as a membrane marker (CD81, SNAP25, CD171, EAAT1, OMG,
etc.) or adsorbed marker (Tau, NRGN, GFAP, MBP, etc.) is
immobilized on an ELISA plate to allow capture of exosomes having
that marker. A second antibody against another exosome marker of
interest, which can also be a membrane marker (CD81, SNAP25, CD171,
EAAT1, OMG, etc.) or adsorbed marker (Tau, NRGN, GFAP, MBP, etc.),
is subsequently used to screen the exosomes captured by the first
antibody.
Example 2: Detection of Cytosolic Non-Membrane Proteins on the
Surface of Exosomes
[0150] Cytosolic non-membrane proteins were detected on the surface
of exosomes as follows. Neuron-specific anti-SNAP25 (FIGS. 2A-2C)
or control mouse IgG (FIGS. 2D-2F) were immobilized onto white
ELISA plates. Various volume (10, 2.5, and 0 .mu.L) of plasma was
suspended in PBS, 0.1% tween-20, or 0.1% triton-X100 PBS in a final
volume of 40 .mu.L, and applied to the ELISA plates. After
overnight incubation at 4.degree. C., unbound materials were
removed, then biotin-labeled antibodies against the exosome surface
marker CD81, and 2 different soluble cytosolic proteins (GAPDH and
CTSD) were applied to ELISA plates. For PBS samples, antibodies
were suspended in PBS without tween-20. For samples with tween-20
and triton-X100, labeled antibodies were suspended in 0.1% tween-20
PBS. Then, conventional chemiluminescent ELISA procedure was
carried out, and relative light unit (RLU) was determined.
[0151] Because GAPDH and CTSD are cytosolic proteins, we
hypothesized that these would be detected only after
permeabilization of exosomes by tween-20 or triton-X100, whereas
membrane protein CD81 would show up in both cases. However, as
shown in FIGS. 2A-2C, all 3 markers were detected in a plasma
volume dependent manner, whereas no signal was detected on the
mouse IgG-immobilized control ELISA plates. These results clearly
showed that soluble cytosolic proteins such as GAPDH and CTSD may
be detected on the surface of exosomes. These results further
showed that the methods and compositions of the present invention
are useful for detecting biomarkers on vesicles captured on a solid
support using a detection agent that selectively binds to the
biomarkers, wherein the vesicles are not lysed or
permeabilized.
Example 3: Origin of Cytosolic Non-Membrane Proteins on the Surface
of Exosomes
[0152] The origin of cytosolic non-membrane proteins on the surface
of exosomes was determined as follows. Antibodies against exosome
surface marker CD81, neuron surface marker SNAP25, astrocyte
surface marker (EAAT1), or oligodendrocyte surface marker (OMG)
were immobilized onto white ELISA plates. Ten .mu.L of plasma
suspended in 40 .mu.L of PBS were applied to the ELISA plates.
After overnight incubation at 4.degree. C., unbound materials were
removed, then biotin-labeled antibodies against exosome surface
marker CD81, general cytosolic marker GAPDH, cytosolic proteins in
neuron (NRGN), oligodendrocyte (MBP), and astrocyte (GFAP) were
applied to ELISA plates. No antibody control (tPBS) was also
included. Then, conventional chemiluminescent ELISA procedure was
carried out, and relative light unit (RLU) was determined (FIGS.
3A-3D).
[0153] Both CD81 and GAPDH were detected on all ELISA plates
(left), indicating that each ELISA plate captured exosomes with
GAPDH attached on the surface. In CD81 plate (FIG. 3A) which
captured whole exosomes, MBP and GFAP was not detected. Although
NRGN was detected, values were very low. In the SNAP25 plate (FIG.
3B) where neuron-derived exosomes were captured, neuron specific
NRGN was detected, whereas MBP and GFAP were very low. In the EAAT1
plate (FIG. 3C), where astrocyte-derived exosomes were captured,
astrocyte specific GFAP was detected, although NRGN and MBP were
also detected. In the OMG plate (FIG. 3D), where
oligodendrocyte-derived exosomes were captured, oligodendrocyte
specific MBP was detected, though NRGN and GFAP were also
detected.
[0154] These results showed that soluble markers on the surface of
exosomes are derived from mother cells before releasing to outside
or from microenvironment shortly after releasing from mother cells.
These results further showed that the methods and compositions of
the present invention are useful for detecting biomarkers on
vesicles, wherein the vesicles are not lysed or permeabilized.
Example 4: Quantification of Marker Proteins
[0155] Marker proteins of the present invention were quantified as
follows. Since immobilized antibody and biotin-labeled detection
antibody bind to 2 different target molecules (such as CD81 and
SNAP25), detectable targets standards should have both antigens in
the single molecules. Thus, unlike conventional ELISAs, purified
proteins or recombinant proteins are not applicable as a
quantification standard. Thus, we first screened various plasma
samples and found appropriate ones, which contained a large
quantity of target exosomes. By assigning 100 units/mL to this
plasma, dilution studies were carried out on SNAP25, EAAT1, and OMG
plates (FIGS. 4A-4D).
[0156] As shown in FIGS. 4A and 4B, the ELISA readings RLU (x-axis)
of NRGN (FIG. 4A) and tau proteins (FIG. 4B) on
anti-SNAP25-immobilized ELISA (neuron-derived exosome, NDE) plates
showed linear changes. Similarly, GFAP (FIG. 4C) and MBP (FIG. 4D)
on anti-EAAT1 (astrocyte-derived exosome, ADE) and anti-OMG
(oligodendrocyte-derived exosome, ODE) immobilized plates,
respectively showed linear changes. Thus, RLU can be successfully
converted to units/mL. These results showed that the methods and
compositions of the present invention are useful for detecting and
measuring biomarkers on vesicles, wherein the vesicles are not
lysed or permeabilized.
Example 5: Screening of Exosome Adsorbed Biomarkers
[0157] Exosome adsorbed biomarkers were identified as follows.
Control mouse IgG, mouse anti-human CD81, or mouse anti-human
synaptosomal-associated protein 25 (SNAP25) were immobilized to
ELISA plate, then pooled human plasma was applied to all of ELISA
wells (FIGS. 5A-5C). Various detection antibodies (all
biotinylated) were used to carry out the ELISAs, which included not
only antibodies against exosome membrane proteins (CD81, CD171,
CD63, and SNAP25), but also, antibodies against various
non-membrane proteins (glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), cathepsin D (CTSD), neurogranin (NRGN), myelin basic
protein (MBP), glial fibrillary acidic protein (GFAP), tau,
microtubule associated protein tau (tau) and phosphorylated tau
T181, and amyloid b.sub.1-42 peptide). As a negative control, PBS
without any biotinylated antibody was used.
[0158] As shown in FIG. 5A, ELISA signals (RLU) were clearly
positive for both the anti-CD81 and anti-SNAP25-immobilized plate,
whereas very few signal was detected on control mouse
IgG-immobilized plate, indicating that the system was CD81 or
SNAP25 specific. As shown in PBS, non-specific signal without
detection antibodies were very low, indicating that the assay was
detection antibody specific. The detection of membrane proteins
(CD81, CD171, CD63, and SNAP25) confirmed that the assay captured
exosomes (FIGS. 5A and 5B). Surprisingly, non-membrane proteins
such as GAPDH, CTSD, NRGN, MBP, and GFAP were also detected (FIG.
5C). These results showed that the methods and compositions of the
present invention are useful for identifying, detecting, and
measuring biomarkers on vesicles, wherein the vesicles are not
lysed or permeabilized.
Example 6: Diagnosis of Alzheimer's Disease in Humans
[0159] The methods and compositions of the present invention were
used to diagnose Alzheimer's disease in biological samples from
humans as follows. Anti-CD171 were immobilized on an ELISA plate.
Twelve samples of EDTA plasma from subjects having Alzheimer's
disease (AD) and age, gender-matched controls were applied to ELISA
wells, and antibodies against CD81, CTSD. NRGN, p-tau T181 were
used as detection antibodies (FIGS. 6A-6E).
[0160] As shown in Table 1 below, NRGN values were lower in AD
samples than the control, and T181 values were higher in AD samples
than the control. A chi square test showed that these 2 groups were
significantly different (p=0.002 for NRGN, p=0.0001 for T181), as
shown in Table 1. These results showed that the methods and
compositions of the present invention could be used to diagnose
Alzheimer's disease.
TABLE-US-00001 TABLE 1 NRGN on CD171 High Low AD 1 9 Cont 9 3 p =
0.002 T181 on CD171 High Low AD 9 1 Cont 1 11 p = 0.0001
Example 7: Stability of Plasma Exosomal Biomarker Levels
[0161] The stability of plasma exosomal biomarker levels was
determined as follows. Anti-CD81, anti-CD171, anti-SNAP25,
anti-EAAT1, and anti-OMG were immobilized on separate ELISA plates.
EDTA plasma was obtained from 7 control subjects every week for 2-3
weeks, and applied to the ELISA wells. For detection of exosome
membrane targets (FIGS. 7A-7F), an anti-CD81 probe was used on the
CD81 plate (total exosome, TE); an anti-CD171 probe was used on the
CD81 plate (CD171-based NDE, cNDE); an anti-SNAP25 probe was used
on the CD81 plate (SNAP25-based NDE, sNDE); an anti-CD81 probe was
used on the EAAT1 plate (ADE); and an anti-CD81 probe was used on
the OMG plate (ODE). For detection of exosome surface protein
targets (FIGS. 7G-7L), GFAP probe on EAAT1 plate, MBP probe on OMG
plate, NRGN probe on SNAP25 plate, tau probe on SNAP25 plate, and
NRGN probe on CD171 plate, respectively.
[0162] Although each value was largely spread among tested subjects
(except tau on SNAP25 plate), the values were quite constant within
the same individual except NRGN on SANP25 plate. These results
showed that the methods and compositions of the present invention
are useful for identifying, detecting, and measuring biomarkers on
vesicles, wherein the vesicles are not lysed or permeabilized.
These results further suggested that the methods and compositions
of the present invention would be useful for diagnosing a
neurological disorder in a subject. These results further suggested
that the methods and compositions of the present invention would be
useful for diagnosing a disease or disorder.
Example 8: Plasma on Anti-NRGN-Immobilized ELISA Plate
[0163] Plasma exosomal biomarkers were identified as follows.
Anti-NRGN or control mouse IgG were immobilized on an ELISA plate.
EDTA plasma or PBS alone were applied to the ELISA wells.
Antibodies against NRGN, CD171, SNAP25, CD81, EAAT1, OMG were used
for detection, and a PBS control was used for comparison.
[0164] As shown in FIG. 8B, plasma applied on the anti-NRGN
immobilized ELISA plate tested positive for NRGN, CD171, SNAP25,
and CD81, but not EAAT1 and OMG, indicating specificity for neurons
of NRGN on the SNAP25 plate, but not astrocytes or oligodendrocytes
(FIG. 8B). These results showed that the methods and compositions
of the present invention are useful for identifying, detecting, and
measuring biomarkers on vesicles, wherein the vesicles are not
lysed or permeabilized. These results further suggested that the
methods and compositions of the present invention would be useful
for diagnosing a neurological disorder in a subject. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
Example 9: Microglia Targeting of Exosome Biomarkers
[0165] Microglia targeting of exosome biomarkers was performed as
follows. An anti-CD11b antibody or a control mouse IgG were
immobilized on ELISA plates. EDTA plasma from 7 different donors
(IR1-IR7) or PBS alone were applied to ELISA wells. Antibodies
against GFAP (FIG. 9B), MBP (FIG. 9C), or NRGN (FIG. 9D) were used
as detection antibodies.
[0166] As shown in FIGS. 9B-9D, although no microglia-specific
antibody is available, and anti-CD11b binds to both microglia and
peripheral macrophages, microglia analysis can be done by assessing
brain-derived proteins on the surface of CD11b.sup.+ exosomes. Four
(GFAP) to five (MBP) donors showed positive signals higher than the
3 controls (PBS on IgG plate, plasma on IgG plate and PBS on CD1b
plate), indicating the interaction of microglia with astrocytes and
oligodendrocytes in these donors. None of the seven subjects,
however, failed to show NRGN (FIG. 9D).
[0167] These results showed that the methods of the present
invention are useful for detecting biomarkers (GFAP, MBP, and NGRN)
on microglia-derived exosomes. These results showed that the
methods and compositions of the present invention are useful for
identifying, detecting, and measuring biomarkers on vesicles,
wherein the vesicles are not lysed or permeabilized. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing neurological disease in a
subject. These results further suggested that the methods and
compositions of the present invention would be useful for
diagnosing a disease or disorder.
Example 10: Detection of Secretory Proteins (Cytokines IL1b and
IL34 and Chemokines CX3CL1) on the Surface of Brain-Derived
Exosomes
[0168] Secretory proteins (cytokines and chemokines) were detected
on the surface of brain-derived exosomes as follows. Various
concentrations of standard plasma (FIGS. 10A, 10C, and 10E) and
fixed dilution of seven control plasma samples (1/8 dilution for
IL1b and IL34 and 1/4 dilution for CX3CL1) and PBS control was
applied to ELISA plates containing immobilized antibodies
(anti-SNAP25, anti-EAAT1, anti-OMG, and control mouse IgG) to
capture NDE, ADE, and ODE, respectively. Control mouse IgG was used
to evaluate non-specific bindings.
[0169] As shown in FIGS. 10A, 10C, and 10E, standard plasma showed
volume dependent increases in IL1b, IL34 and CX3CL1 levels on
SNAP25, EAAT1, and OMG ELISA plates compared to control
IgG-immobilized plates. IL1b and IL34 on anti-OMG-immobilized
plates were saturated at 2.5% plasma (see FIGS. 10A and 10C).
[0170] Plasma samples (IR1, IR4, IR6) were negative for IL1b, IL34,
and CX3CL1, similar to those of negative control PBS alone.
However, IR2 was IL1b positive on the SNAP25 ELISA plate, and IR3
was CX3CL1 positive on EAAT1 and OMG ELISA plates. IRS showed
positive IL1b levels and IL34 on SNAP25 plates, and detectable
levels of CX3CL1 on EAAT1 and OMG ELISA plates. IR7 showed positive
IL1b on all ELISA plates, IL34 on SNAP25 ELISA plate, and
detectable levels of CX3CL1 on both EAAT1 and OMG ELISA plates.
[0171] These results showed that methods of the present invention
are useful for detecting cytokines (IL1b and IL34) and chemokines
(CX3CL1) on the surface of exosomes (SNAP25+ NDE, EAAT1+ ADE, and
OMG+ ODE). These results also showed that cytokine and chemokine
profiles are unique to each subset of brain-derived exosomes (NDE,
ADE, and ODE). These results showed that the methods and
compositions of the present invention are useful for identifying,
detecting, and measuring biomarkers on vesicles, wherein the
vesicles are not lysed or permeabilized. These results further
suggested that the methods and compositions of the present
invention would be useful for diagnosing neurological disease in a
subject. These results further suggested that the methods and
compositions of the present invention would be useful for
diagnosing a disease or disorder.
Example 11: Detection of Membrane Proteins and Receptors on the
Surface of Exosomes
[0172] Multiple membrane proteins and receptors were detected on
the surface of exosomes as follows. Various antibodies were
suspended in a coating buffer, and applied to an ELISA plate. After
an hour of incubation, each well was washed once, then blocking
solution (0.5% BSA in Blocker casein) was added, and incubation was
continued for an additional hour. After washing each well twice,
plasma or buffer control (phosphate buffered saline, PBS) was
added, then incubation was continued in a refrigerator overnight.
After washing each well twice, biotinylated anti-CD81 or
anti-SNAP25 was added and incubation was continued for an hour.
After washing each well twice, streptavidin-horseradish peroxidase
was added, and incubation was continued for another 30 minutes.
After washing each well three times, SuperSignal substrate was
added and chemiluminescent signals were measured in a
luminometer.
[0173] As shown in Table 2 below, multiple cytosolic proteins,
secretary proteins, and membrane proteins and receptors were
detected on the surface of exosomes.
TABLE-US-00002 TABLE 2 Antibody immobilization, probed with
CD81/SNAP25. samples applied Experiment Categories Targets
(immobilization) Probes PBS (RLU) Plasma (RLU) Positive (O)
Clinical area Exp-1160 Control IgG CD81 6.5 36 CD81 3.7 1,443 O
Cytoslic proteins CTSD Cathepsin D 9.9 226 O Neurology, oncology
NRGN (R&D) neurogranin 6.4 91 Neurology NRGN (BioLegend) 6.4
592 O Neurology Membrane proteins PD-L1 Programmed death-ligand 1
7.2 453 O Immunology, oncology ErbB2 v-erb-b2 erythroblastic
leukemia viral oncogene homolog 2 185.4 193 Immunology, oncology
RBC Red blood cells 3.5 674 O Hematology NCAM Neural cell adhesion
molecule 3.8 179 O Immunology, oncology CD11b 4.8 415 O Immunology,
oncology CD11a 2.4 745 O Immunology, oncology CD16 4.5 40
Immunology, oncology CD28 3.8 392 O Immunology, oncology CD79a 3.2
351 O Immunology, oncology TCR ab T cell receptor 20.8 47
Immunology, oncology TCR .gamma.d 5.3 815 O Immunology, oncology
CD32 5.5 36 Immunology, oncology Exp-1193 Control IgG CD81 4.0 20
SNAP 1.4 1,618 O CD81 1.5 1,075 O Cytosolic proteins REST 2.6 1,724
O Neurology SYP synaptophysin 1.4 1,963 O Neurology SYT
synaptotagmin 2.0 1,309 O Neurology SYMPO synaptopodin 1.8 3,051 O
Neurology SYN synapsin 1.6 85 Neurology NEFL neurofilamant light
chain 1.3 2,711 O Neurology NRGN (Santa Cruz) 4.0 1,275 O Neurology
Exp-1203-1 Control SNAP25 SNAP25 2.8 375 O IgG 3.8 5 Cytosolic
proteins Caspase 3 3.0 364 O Neurology, oncology GYS Glycogen
synthase 1 3.5 111 O Neurology Ubiquitin 3.0 388 O Neurology HSP70
8.8 289 O Neurology Beta amyloid 2.8 394 O Neurology Secretary
proteins CCL2 2.8 208 O Neurology, immunology IL8 interleukin 8 2.5
344 O Neurology, immunology IL12B 2.8 163 O Neurology, immunology
IL16 3.4 185 O Neurology, immunology Orexin 2.2 2 Neurology
Membrane proteins Insulin Receptor 3.2 364 O Neurology, metabolic
TRAIL Tumor necrosis factor ligand superfamily member 10 3.3 346 O
Neurology, oncology TNF Receptor 1 2.5 214 O Neurology, oncology
Death Receptor 5 3.1 309 O Neurology, oncology Death Receptor 6 3.8
82 O Neurology, oncology Cannabinoid receptor-1 2.3 2 Neurology
Orexin receptor 2.4 547 O Neurology Dopamin receptor D1 2.4 280 O
Neurology Dopamin receptor D2 2.1 255 O Neurology Serotonin
receptor 2A 2.8 267 O Neurology Exp-1203-2 Control SNAP25 SNAP25
4.1 389 O IgG 5.2 8 Cytosolic proteins Dopa decarboxylase 6.8 7
Neurology Secretary proteins Enkephalin 6.5 174 O Neurology
Nociceptin 5.5 123 O Neurology Membrane proteins Serotonin receptor
2C 4.6 366 O Neurology Serotonin receptor 3B 3.9 432 O Neurology
GABA receptor 1-6 5.1 349 O Neurology GABA Receptor-5 5.4 130 O
Neurology GABA Receptor B1 4.5 423 O Neurology GABA Receptor B2 4.8
384 O Neurology Opioid receptor KOR 6.1 349 O Neurology Glutamate
receptor-1 6.0 256 O Neurology Glutamate receptor-2 7.4 232 O
Neurology Exp-1206 Control SNAP SNAP25 1.9 417 O IgG 2.4 2
Cytosolic proteins LAMP1 Lysosome-associated membrane glycoprotein
1 0.9 310 O Neurology PSEN1 Presenilin-1 1.0 320 O Neurology BACE
be-ta secretase 2.5 299 O Neurology TREM2 Triggering receptor
expressed on myloid cells 2 3.0 319 O Neurology AchE
Acetylcholinesterase 3.6 267 O Neurology GSK Glycogen synthase
kinase 1.6 258 O Neurology IL6 3.2 365 O Neurology, immunology TNFa
tumor necrosis factor 0.8 404 O Neurology, immunology CX3CL1 0.9
360 O Neurology, immunology Exp-1220 Control IgG CD81 6.1 120 O
CD81 (1.3.3.22) 3.0 2,773 O CD81 (5A6) (BioL) 5.0 886 O CD81 (D4)
3.2 1,938 O CD81 (5A6) SC 6.3 3,162 O CD81 (B11) 2.1 3,280 O CD81
(BD) 4.7 3,064 O CD61 (Sino) 10.8 1,858 O CD63 (MX49.129) 5.7 2,629
O SNAP25 2.8 3,712 O Cytosolic proteins ACTB beta-actin 16.1 1,968
O SNCA alpha synuclein 3.3 2,608 O Neurology Secretary proteins
GnRH Gonadotropin-releasing hormone 2.3 3,424 O Neurology,
endocrinology Membrane proteins CD171 UJ127.11 2.0 3,331 O CD171 D5
2.9 2,111 O CD171 (eBio) 4.2 49 O CTSD (SC) cathepsin D 10.6 349
CTSD (R&D) 6.4 350 O Neurology oncology EpCAM Epithelial cell
adhesion molecule 1.9 3,655 O Oncology CK19 cytokeratin 19 2.4
3,255 O Oncology IgG SNAP25 3.2 4 CD81 (1.3.3.22) 1.7 159 O CD81
(5A6) (BioL) 1.5 71 O CD81 (D4) 1.5 157 O CD81 (5A6) SC 4.0 340 O
CD81 (B11) 1.6 332 O CD81 (JS81) (BD) 1.7 361 O CD61 (Sino) 1.4 108
O CD63 (MX49.129) 2.0 243 O SNAP25 1.1 277 O Exp-1225 IgG CD81 3.0
11 O CD81 2.1 315 O PLAP Alkaline phosphatase, placental 2.7 19 O
OBGYN CSH1 Chorionic somatomammotropin hormone 1 2.5 32 O OBGYN
PSG1 Pregnancy-specific beta-1-glycoprotein 1 2.3 16 O OBGYN FasL
Tumor necrosis factor superfamily 6 2.9 67 O OBGYN, oncology
[0174] To confirm the proteins detected in Table 2 above, a second
series of experiments were performed as follow. Positive antibodies
were biotinylated, and used on the ELISA where PBS or plasma was
applied to anti-SNAP25-, or control IgG-immobilized plates. As
shown in Table 3 below, all positive antibodies in Table 2 were
also positive in this series of experiments.
TABLE-US-00003 TABLE 3 Anti-SNAP25 or control IgG-immobilized
plate, probed with various biotinylated antibodies. samples applied
Target PBS Plasma Positive Experiment Categories (biotinylated
probes) Immobilized (RLU) (RLU) (O) Exp-1197 Control SNAP SNAP25
2.5 405 O CD81 3.2 4503 O Cytosolic proteins REST 2.9 30 O SYP 2.9
226 O SYT 3.5 92 O SYMPO 2.7 28 O NEFL 2.6 82 O NRGN 2.3 50 O
Exp-1205 Control PBS IgG 0.3 0.3 Cytosolic proteins Tau 4.7 11.1
Abeta 7.1 10.6 Caspase 1.2 2.8 HSP 1.2 7.3 ubiquitin 1.3 5.2 GYS
7.7 15.6 Secretary proteins CCL2 3.4 7.3 IL8 1.0 8.7 IL12B 0.8 6.1
IL16 10.1 12.3 Membrane proteins DRD1 5.9 7.1 SR2A 4.8 6.1 SR2C 1.0
4.3 TNFR1 0.9 2.3 DR6 12.2 12.3 Control PBS SNAP25 0.2 0 Cytosolic
proteins Tau 2.5 1085 O Abeta 2.1 1259 O Caspase 1.0 396 O HSP 0.9
1403 O ubiquitin 1.2 692 O GYS 2.0 1338 O Secretary proteins CCL2
2.0 580 O IL8 0.5 1256 O IL12B 0.4 670 O IL16 3.7 724 O Membrane
proteins DRD1 2.1 489 O SR2A 1.6 495 O SR2C 0.9 672 O TNFR1 0.4 499
O DR6 3.6 953 O Exp-1207 Control PBS IgG 1.5 1.1 CD81 0.9 7.7
SNAP25 1.3 1.1 Cytosolic proteins LAMP1 1.2 2.0 PSEN1 3.3 5.6 BACE
3.5 2.2 AchE 3.2 3.5 Secretary proteins IL6 17.3 2.4 TNFa 0.5 1.0
CX3CL1 0.6 1.6 ENK 3.6 3.7 Membrane proteins OR 3.4 2.4 DRD2 1.4
1.8 KOR 2.5 2.5 TREM2 2.1 2.2 Control PBS SNAP25 1.1 0 CD81 1.2
2844 O SNAP25 0.4 227 O Cytosolic proteins LAMP1 0.3 250 O PSEN1
1.3 334 O BACE 1.1 267 O AchE 1.1 381 O Secretary proteins IL6 4.3
440 O TNFa 0.5 704 O CX3CL1 0.5 988 O ENK 1.0 1257 O Membrane
proteins OR 1.1 543 O DRD2 0.5 437 O KOR 0.7 563 O TREM2 0.9 638
O
[0175] These results showed that methods of the present invention
are useful for detecting cytosolic proteins, secretary proteins,
and membrane proteins and receptors on the surface of exosomes.
These results showed that the methods and compositions of the
present invention are useful for identifying, detecting, and
measuring biomarkers on exosomes, wherein the exosomes are not
lysed or permeabilized. These results further suggested that the
methods and compositions of the present invention would be useful
for diagnosing neurological disease in a subject. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
Example 12: Quantification Standards for Multi-Target ELISA
[0176] ELISA assays require quantification standards to determine
levels of target molecules in a test sample. The exemplary assays
of the present invention utilize two antibodies against different
target molecules. Accordingly, purified or recombinant proteins are
not applicable as a standard for such ELISA assays. Thus, various
plasma samples were screened, and one particular plasma sample was
selected, for having high values of exosomes including NDE, ADE,
and ODE positive exosomes. This plasma sample was subsequently used
as a quantification standard. As shown in FIG. 11, this plasma
sample performed well as a standard for multiple ELISAs of the
present invention. A standard curve was generated for each target
by using four parameter logistic regression analysis.
Example 13: Differential Diagnosis of Alzheimer's Disease and Mild
Cognitive Impairment in Humans
[0177] The methods and compositions of the present invention were
used for differential diagnosis of Alzheimer's disease and mild
cognitive impairment in biological samples from humans as follows.
Anti-SNAP25 antibodies were immobilized on an ELISA plate. Eight
samples of EDTA plasma from subjects having Alzheimer's disease
(AD, n=4) or mild cognitive impairment (MCI, n=4) and eight age and
gender-matched controls were applied to ELISA wells, and antibodies
against 16 cytosolic proteins, secretary proteins, and membrane
proteins and receptors were used as detection antibodies (FIG.
12).
[0178] As shown in FIG. 12, duplicate variation for all 16
biomarkers was small. FIG. 13 shows the standard curves for all 16
biomarkers. Quantification of target surface protein biomarkers is
shown in FIG. 14. As shown in FIG. 14, AchE, LAMP1, and CCL2 levels
were significantly higher in MCI compared to control sample levels.
These results showed that the methods and compositions of the
present invention could be used to diagnose MCI and for the
differential diagnosis of Alzheimer's disease and MCI.
[0179] To account for variability in CD81 levels in the plasma
samples, the remaining 15 exosomal biomarker levels were normalized
by CD81 levels. As shown in FIG. 15, AchE and LAMP1 levels were
significantly greater in plasma samples from subjects with MCI and
AD+MCI compared to control samples. Additionally, NGRN, GYS, DR6,
HSP, IL12b, and BACE levels were significantly different between
AD, MCI, AD+MCI and control samples. These results showed that the
methods and compositions of the present invention could be used to
diagnose AD, MCI, and for differential diagnosis of AD and MCI.
[0180] In another series of experiments, the target biomarker
levels in the plasma samples assayed above were normalized to SYT
levels. Since neural-derived exosomes were captured by
SNAP25-immobilized plates, the SYT normalization results in a
second normalization of neuron markers. As shown in FIG. 16,
AchE/SYT, LAMP1/SYT, REST/SYT, CCL2/SYT, HSP/SYT, and IL12b/SYT
levels were significantly different between AD, MCI, AD+MCI, and
control samples. These results showed that the methods and
compositions of the present invention could be used to diagnose AD,
MCI, and for differential diagnosis of AD and MCI. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
Example 14: Enumeration of Brain-Derived Exosomes in Human Subjects
with Parkinsonism-Plus Syndromes
[0181] The methods and compositions of the present invention were
used to enumerate the plasma levels of neuron-derived,
astrocyte-derived, and oligodendrocyte-derived exosomes (NDEs, ADEs
and ODEs, respectively) obtained from patients with Parkinson's
Disease (PD), multiple system atrophy (MSA), progressive
supranuclear palsy (PSP) and control subjects as follows. A total
of 52 subjects, including 15 patients with PD (ages 46-79,
mean.+-.SD of 64.7.+-.10.8, 9 male and 6 female), 15 patients with
MSA (ages 44-74, mean.+-.SD of 63.3.+-.8.16, 10 male and 5 female),
7 patients with PSP (ages 58-81, mean.+-.SD of 71.6.+-.9.8, 2 male
and 5 female), and 15 disease controls (ages 47-77, mean.+-.SD of
64.7.+-.8.5, 10 male and 5 female) were included in this study, and
these groups were age-matched.
[0182] Plasma samples were taken through venous puncture, and a
total of 8 mL of blood was collected in EDTA-containing tubes.
After collection, plasma was separated by centrifugation for 15 min
at 2,000 g and distributed in polypropylene vials, then stored at
-80.degree. C. until analysis.
[0183] Various concentrations of antibodies were suspended in
1.times. coating buffer (BioLegend, San Diego, Calif.), and 50
.mu.L was applied to each well of a white flat bottom strip
microplate (Sigma-Aldrich, St. Louise, Mo.). After 1-hour
incubation at room temperature, each well was washed with 1.times.
wash buffer (BioLegend), then 75 .mu.L of blocker casein (Thermo
Fisher Scientific, Waltham, Mass.), supplemented with 1% blocker
bovine serum albumin (BSA) (Thermo Fisher) was added, and
incubation was continued for an another 1 hour. After washing each
well, each plate was used immediately for enzyme-linked
immunosorbent assay (ELISA).
[0184] Forty .mu.L each of standards, controls, and plasma samples
were applied to each well of ELISA plates, and incubated in a
refrigerator overnight. Next day, after each well was washed, 40
.mu.L of biotinylated detection antibodies suspended in phosphate
buffered saline, pH 7.4 (PBS) supplemented with 0.1% tween-20
(Sigma-Aldrich), 1% blocker BSA (Thermo Fisher), 8 .mu.g/mL mouse
IgG (Equitech-Bio, Kerrville, Tex.) was added, and incubated at
room temperature for 1 hour. After incubation, each well was
washed, then 40 .mu.L of streptavidin-horseradish peroxidase
(SA-HRP) (Thermo Fisher) suspended in PBS supplemented with 0.1%
Tween-20, 1% blocker BSA (Thermo Fisher), and 5% blocker casein was
applied. After 30 min incubation, each well was washed, then 50
.mu.L of substrate (SuperSignal, Thermo) was applied. After 4-min
incubation, chemiluminescent signals (relative light units, RLU)
was determined in a luminometer (ANSH Labs, Webster, Tex.).
[0185] Since our sandwich ELISA used a combination of 2 different
target antibodies, such as anti-CD81 and anti-SNAP25
(synaptosomal-associated protein 25), purified or recombinant
protein was not applicable as a quantification standard. Thus, we
first screened various plasma samples obtained from commercial
sources (Innovative Research, Novi, Mich., and EquiTech Enterprise,
Kerrville, Tex.), and found plasma samples with high concentrations
of all of NDE, ADE, and ODE. By assigning 100 units/mL to this
plasma, dilution study was carried out in each ELISA to obtain RLU
in each dilution. Then using 4 parameter logistic analysis, RLU of
each sample was converted to units/mL.
[0186] In order to find antibodies against NDE, ADE, and ODE in
plasma, various dilutions of plasma were applied to anti-CD81
monoclonal antibody (clone JS-81, BD Biosciences, Sparks,
Md.)-immobilized ELISA plates to capture whole exosomes as well as
control mouse IgG (Santa Cruz Biotechnology, Dallas,
Tex.)-immobilized ELISA plate to be used as a negative control.
After extensive washing step to remove any non-bound materials from
each well, biotinylated antibodies against various targets were
applied, and ELISA was carried out as described above. For
biotinylation, we used EZ-Link Sulfo-NHS-LC-Biotin (Thermo,
Rockford, USA). From this, we found that monoclonal antibody
against SNAP25 (clone B-8, Santa Cruz Biotechnology), polyclonal
antibodies against excitatory amino acid transporter 1 (EAAT1)
(=glutamate aspartate transporter (GLAST)) and
oligodendrocyte-myelin glycoprotein (OMG) (Bioss Antibodies,
Woburn, Mass.), showed very strong signals on CD81-immobilized
plates, not on control mouse IgG-immobilized plates. After
discovery of appropriate antibodies, anti-SNAP25, anti-EAAT1, and
anti-OMG were immobilized onto ELISA plates, then probed with
biotinylated anti-CD81 (Clone 1.3.3.22, LS Bio, Seattle, Wash.). We
called NDE for SNAP25.sup.+, CD81.sup.+ ELISA results, ADE for
EAAT1.sup.+, CD81.sup.+ ELISA results, and ODE for OMG.sup.+,
CD81.sup.+ ELISA results, respectively (FIG. 17).
[0187] Logarithmic transformations of measured values of BDE were
used as appropriate to a particular analysis to reduce excessive
skewing and outlier influence in analyses for group differences and
regressions between parameters. The mean differences in BDE between
diagnostic groups and the regressions between the levels of BDE and
clinical parameters were assessed by t-test (Excel). Because the
number of samples was limited, additional non-parametric
Mann-Whitney U test was performed (Prism, GraphPad, La Jolla,
Calif.). We also derived receiver operating characteristics (ROC)
curves for the diagnosis of PD using the levels of BDE as the
predictor, and estimated the area under the curve (AUC; AUC=0.5
indicates no discrimination and AUC=1 would indicate a perfect
diagnostic test) to evaluate a diagnostic ability of each predictor
(the plasma levels of NDE, ADE and ODE). The level of significance
was set at p<0.05.
[0188] ELISA specificity was determined as follows. ELISA plates
were immobilized with either target antibodies (anti-SNAP25,
anti-EAAT1, and anti-OMG) or control IgG, then serial dilutions of
plasma was applied. As shown in FIGS. 18A-18C, ELISA readings (RLU)
increased in proportion to the volume of plasma on target
antibodies-immobilized ELISA plates (filled circles; .cndot.), not
control IgG-immobilized plates (open triangles; A). We also tested
ELISA without biotin antibodies to assess non-specific binding of
SA-HRP, however, no signal was detected. Since biotin antibodies
were mixed with 10.times. volume of control mouse IgG, non-specific
IgG binding was eliminated in the assay.
[0189] Next, the performance of the ELISA assays was determined.
Intra-assay precision was determined by ELISAs carried out in
duplicate using 52 different subjects. As shown in FIGS. 19A-19C,
duplicate variation was very small in all of the three ELISAs
(r.sup.2=0.9644, 0.9541, and 0.9791 and slope=1.08, 0.99, and 1.01
for NDE, ADE, and ODE, respectively). Inter-assay precision was
determined using 3 control plasma samples run 9 times. As shown in
FIGS. 20A-20C, CV % of high and medium range plasma was <=20%,
although very low range plasma (<10 units/mL for NDE, <1
units/mL for ADE and ODE) was 35-63%. The CV % was acceptable
because we constructed ELISA plates in each experiment.
[0190] In this ELISA, quantification of target plasma samples is
based on the dilution curve of standard plasma. This calculation is
applicable when the dilution curve between sample and standard are
identical. To validate this hypothesis, dilutional linearity was
determined by high, medium, and low range plasma samples. As shown
in FIGS. 21A-21C, all 3 plasma showed linear dilution in NDE, ADE,
and ODE, with almost identical slopes, even though the quantity of
these 3 plasma varied widely around 1,000 folds. As shown in FIGS.
21A-21C, linear detection range was 0.1-5 uL plasma (NDE), 0.01-5
uL plasma (ADE and ODE), respectively.
[0191] Limit of detection (LOD) of NDE, ADE, and ODE was 0.519, 0,
and 0.062 units/mL, respectively.
[0192] Enumeration of plasma BDE and comparison between patients
and controls was determined as follows. Plasma levels of NDE, ADE
and ODE were significantly higher in PD samples than in control
samples, respectively (FIGS. 22A-22C; p=0.003 (t-test), p=0.003 (U
test) for NDE, p=0.028 (t-test), p=0.021 (U test) for ADE, and
p=0.016 (t-test), p=0.008 (U test) for ODE). When compared between
PD and MSA groups, plasma levels of NDE were significantly higher
in patients with PD than those with MSA (FIGS. 22A-22C; p=0.028
(t-test), p=0.023 (U test)), whereas no significant difference was
found in ADE and ODE. Patients with PSP also showed significantly
higher levels of plasma NDE, and ADE compared with the controls
(FIGS. 22A-22B; p=0.038 (t-test), p=0.041 (U test) for NDE, and
p=0.012 (t-test), p=0.025 (U test) for ADE).
[0193] FIGS. 23A-23C shows the ROC curves for the classification of
patients with PD and controls based on the levels of plasma NDE,
ADE, and ODE. The AUC of the ROC curve for plasma NDE, ADE and ODE
were 0.89, 0.83, and 0.88, respectively, indicating that plasma
levels of NDE, ADE, and ODE will be applicable to diagnostic tests
for PD with substantial accuracy.
[0194] The correlation between BDE and clinical severity of the
subjects with PD and MSA was determined as follows. We investigated
the correlation between the levels of plasma BDE and scores of
clinical severity in the patients with PD. As shown in FIGS.
24A-24F, plasma levels of all BDE (NDE, ADE and ODE) were
significantly higher in the patients with advanced PD, namely in
both groups with mRS grade 4 (n=4) (p=0.003 (t-test), p=0.008 (U
test) for NDE, p=0.001 (t-test), p=0.006 (U test) for ADE, and
p<0.001 (t-test), p=0.006 (U test) for ODE, and those with Hoehn
and Yahr (HY) stage 4 (n=5) (p=0.001 (t-test), p=0.003 (U test) for
NDE, p=0.001 (t-test), p=0.003 (U test) for ADE, and p<0.001
(t-test), p=0.003 (U test) for ODE, compared with the control
group. Furthermore, plasma levels of NDE were significantly higher
even in the mild PD patients, who were in mRS grade 1-2 (n=5)
(p=0.001 (t-test), p=0.002 (U test)) or in HY stage 1-2 (n=4)
(p=0.003 (t-test), p=0.004 (U test)), compared with the controls.
Less remarkably, but nevertheless significantly, plasma levels of
ODE were also higher even in the mild PD patients with mRS grade
1-2 (p=0.002 (t-test), p=0.005 (U test)) or in HY stage 1-2
(p=0.011 (U test)) compared with the controls. Moreover, plasma
levels of ADE were also higher even in the mild PD patients with
mRS grade 1-2 (p=0.023 (U test)) compared with the controls. These
results indicate that the plasma levels of NDE and ODE could be
useful as a diagnostic biomarker for PD patients especially in
early disease stages. Although the levels of ODE were fluctuated
during early disease courses of mRS and Yahr score 1-3, the levels
of ODE were significantly higher in mRS and Yahr score 4 than score
1-3 (p=0.002 (t-test), p=0.011 (U test) for mRS, and p=0.004
(t-test), p=0.0024 (U test) in Yahr). This suggests that ODE may be
a potential surrogate biomarker of the monitoring of disease
progression of PD.
[0195] We further investigated the correlations between the levels
of BDE or the ratios between them (ADE/NDE, ODE/NDE, and ODE/ADE)
and various clinical parameters in the PD group (see Table 4). The
ratio of ADE/NDE showed statistically significant high r.sup.2
values for disease duration of the patients (r.sup.2=0.51, n=15,
p=0.0028), grades of mRS (r.sup.2=0.47, n=15, p=0.0048), and UPDRS
part III scores (r.sup.2=0.52, n=14, p=0.0036), as well as ODE/NDE
for UPDRS part III scores (r.sup.2=0.51, n=14, p=0.0041) (FIGS.
25A-25D). These results also suggest that the plasma levels of BDE
could possibly be useful for monitoring severity of patients with
PD.
TABLE-US-00004 TABLE 4 Correlation between BDE and PD severity. * =
r.sup.2 values. Quantity of BDE Ratio NDE ADE ODE A/N O/N O/A
Severity .sup. 0.00 * 0.21 0.19 0.38 0.31 0.03 Dis.duration(M) 0.02
0.33 0.22 0.51 0.30 0.00 mRS 0.00 0.20 0.19 0.47 0.39 0.04
mRS/duration 0.01 0.12 0.11 0.33 0.27 0.03 Yahr grade 0.00 0.21
0.19 0.38 0.31 0.03 Yahr/duration 0.02 0.08 0.09 0.29 0.24 0.03
UPDRS part3(motor) 0.03 0.35 0.36 0.52 0.51 0.12 UPDRS/duration
0.01 0.07 0.04 0.23 0.11 0.01 DM 0.16 0.12 0.17 0.04 0.10 0.16 MMSE
0.01 0.03 0.02 0.03 0.02 0.01 * r.sup.2 values
[0196] Next, we investigated the correlations between the levels of
BDE and various clinical parameters in the MSA group. As shown in
FIG. 26A-26D, plasma levels of ODE did not correlate with the
grades of mRS or disease duration in the patients with MSA as a
whole, or not with ICARS scores in MSA-C, but had a significant
correlation with UPDRS part III scores (r.sup.2=0.57, n=6,
p=0.048).
[0197] These results showed that plasma levels of both NDE and ODE
are significantly increased when compared to controls, even in the
early stages of PD and that the levels of ODE increased according
to the progression of disease severity in PD. These results further
showed that plasma levels of BDE could be diagnostic and
severity-level biomarkers for PD and related diseases. These
results showed that the methods and compositions of the present
invention are useful for identifying, detecting, and measuring
biomarkers on exosomes, wherein the exosomes are not lysed or
permeabilized. These results further suggested that the methods and
compositions of the present invention would be useful for
diagnosing neurological disease in a subject. These results further
suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
Example 15: Detection of Pathological Form of .alpha.-Synuclein on
the Surface of Neuron-, Astrocyte-, and Oligodendrocyte-Derived
Exosomes
[0198] The detection of pathological form of .alpha.-synuclein on
the surface of neuron-, astrocyte-, and oligodendrocyte-derived
exosomes was performed as followed. Forty uL of EDTA plasma samples
(1/4 dilution in PBS) were added into anti-SNAP25-, EAAT1-. And
OMG-immobilized immobilized ELISA plate to capture neuron-(NDE),
astrocyte-(ADE), and oligodendrocyte-(ODE) derived exosomes. After
each well was washed, biotinylated antibodies against aggregated
forms (pathological forms) of .alpha.-synuclein (Syn-F1, Syn-02
from BioLegend) were applied, and incubation was continued for
another hour, followed by ordinary ELISA procedures (HRP reaction,
SuperSignal, measurement of chemiluminescence signals). As shown in
FIGS. 27A-27F, pathological .alpha.-synuclein was detected in
patients with PD, MSA, and PSP. Statistical significance was based
on Mann-Whitney U test.
[0199] These results showed that the methods and compositions of
the present invention are useful for identifying, detecting, and
measuring biomarkers on exosomes, wherein the exosomes are not
lysed or permeabilized. These results further suggested that the
methods and compositions of the present invention would be useful
for diagnosing neurological disease in a subject. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
Example 16: Detection of Dopamine Receptor D2 (DRD2) on the Surface
of Neuron-Derived Exosomes
[0200] The detection of dopamine receptor D2 (DRD2) on the surface
of neuron-derived exosomes was performed as followed. Anti-SNAP25
(neuron marker), anti-DRD2, and control mouse IgG were immobilized
on an ELISA plate. Next, human EDTA plasma or buffer control
(phosphate buffered saline, PBS) was applied to the ELISA plate,
followed by a reaction with biotinylated anti-SNAP25. As shown in
FIG. 28A, plasma was positive on both anti-SNAP25 and anti-DRD2
antibody ELISA plate, whereas PBS was not.
[0201] In another series of experiments, anti-SNAP25 and control
mouse IgG were immobilized on an ELISA plate, human EDTA plasma or
PBS was applied to the ELISA plate, followed by a reaction with
biotinylated antibodies against SNAP25, CD81 (exosome common
marker), DRD2, as well as PBS control. As shown in FIG. 28B, plasma
was positive in anti-SNAP25, CD81, and DRD2 probes on anti-SNAP25
antibody ELISA plate.
[0202] A plasma dilution study was performed as follows.
Anti-SNAP25 was immobilized on an ELISA plate and serial dilutions
of human EDTA plasma samples (3 different donors) were applied to
the ELISA plate, followed by the reaction with biotinylated
anti-DRD2 and anti-CD81. As shown in FIGS. 29A and 29B, plasma
dilution slope was almost identical among 3 donors. Therefore, the
ratio of DRD2/CD81 was consistent (see FIG. 29C).
[0203] In another series of experiments, DRD2 was detected in
neuron-derived exosomes in plasma samples from humans with
neurological disease. Standard plasma was assigned to 100 units/mL,
then using the dilution curve, ELISA reading (DRD2 probes on SNAP25
plate, and CD81 probes on SNAP25 plate (NDE)) of each sample was
converted to units/mL (U/mL). Plasma samples from 15 each of
control, Parkinson's disease (PD), multiple system atrophy (MSA),
and 7 patients with progressive supranuclear palsy (PSP) were
analyzed. PD and MSA patients were further categorized by the
severity of disease (mRS score). As shown in FIGS. 30A-30G, the
levels of DRD2 in PD were significantly lower than control,
although the levels of NDE were significantly higher in PD than
control. Thus, the ratio (DRD2/NDE) showed more significant
differences between PD samples and control samples. Moreover, the
decrease in DRD2 was shown in the mild cases of PD (mRS score 1+2),
indicating that DRD2 may be used as a marker for the early
screening of PD patients as well as the monitoring of PD
progression. ROC curve and area under the curve (AUC) for DRD2
levels between PD and control samples are shown in FIG. 31.
[0204] These results showed that the methods and compositions of
the present invention are useful for identifying, detecting, and
measuring biomarkers on vesicles, wherein the vesicles are not
lysed or permeabilized. These results further suggested that the
methods and compositions of the present invention would be useful
for diagnosing a neurological disorder in a subject. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
Example 17: Detection of DAT-Positive Extracellular Vesicles
[0205] DAT-positive extracellular vesicles were captured on ELISA
plates and analyzed as follows. Plasma samples obtained from three
different human control donors were diluted in PBS. Sample were
applied to a pair of enzyme linked immunosorbent assay (ELISA)
wells, one well was immobilized with anti-DAT mouse monoclonal
antibody (FIG. 32, .cndot..tangle-solidup..box-solid.) and the
other well was immobilized with control mouse IgG (FIG. 32,
.smallcircle..DELTA..quadrature.). After DAT.sup.+ extracellular
vesicles (EV) were captured on the ELISA wells, unbound materials
were removed by extensive washing, then probed with labeled
anti-CD81 to quantify DAT.sup.+-CD81.sup.+ double positive signals
(=DAT.sup.+ EV) (FIG. 32A), as well as antibodies against monomer
(FIG. 32B), oligomer .alpha.-synuclein (SNCA) (FIG. 32C), and
tyrosine hydroxylase (TH) (FIG. 32D) to quantify SNCA and TH on the
surface of DAT.sup.+ EV.
[0206] As shown in FIG. 32, the plasma samples on DAT.sup.+ ELISA
wells all showed dose-dependent signal changes, whereas those of
control IgG ELISA wells stayed very low, 1/10 to 1/100 lower than
those of DAT.sup.+ ELISA wells. More importantly, the slopes of
three plasma samples were very similar, indicating that the
dilution curve of one particular plasma can be used as a universal
quantification standard.
[0207] These results showed that the methods and compositions of
the present invention are useful for capturing and detecting
DAT-positive extracellular vesicles. These results further showed
that the methods and compositions of the present invention are
useful for identifying, detecting, and measuring biomarkers on
vesicles, wherein the vesicles are not lysed or permeabilized.
These results further suggested that the methods and compositions
of the present invention would be useful for diagnosing a
neurological disease or disorder in a subject.
Example 18: Detection of SNAP25-Positive, EAAT1-Positive, and
OMG-Positive Extracellular Vesicles
[0208] SNAP25-positive, EAAT1-positive, and OMG-positive
extracellular vesicles were captured on ELISA plates and analyzed
as follows. Plasma samples obtained from control donors were
applied to ELISA plates where antibodies against SNAP25, EAAT1, and
OMG were previously immobilized. After captured extracellular
vesicles (EV) were washed extensively, EVs were eluted by
incubation with a pH 12.5 elution buffer for 5 minutes, then
immediately neutralized. These samples were applied to Nanoparticle
Tracking Analysis (NanoSight).
[0209] As shown in FIG. 33, EV was detected in all samples with the
size range from 100-400 nm. Moreover, EV size of SNAP25 was smaller
than those of EAAT1 and OMG.
[0210] These results showed that the methods and compositions of
the present invention are useful for capturing and detecting
SNAP25-positive, EAAT1-positive, and OMG-positive extracellular
vesicles. These results further showed that the methods and
compositions of the present invention are useful for identifying,
detecting, and measuring biomarkers on vesicles, wherein the
vesicles are not lysed or permeabilized. These results further
suggested that the methods and compositions of the present
invention would be useful for diagnosing a neurological disease or
disorder in a subject.
Example 19: Detection of Biomarkers on the Surface of Exosomes
[0211] Multiple membrane biomarkers were detected on the surface of
exosomes as follows. Various antibodies were suspended in a coating
buffer and applied to an ELISA plate. After an hour of incubation,
each well was washed once, then blocking solution (0.5% BSA in
Blocker casein) was added, and incubation was continued for an
additional hour. After washing each well twice, plasma or buffer
control (phosphate buffered saline, PBS) was added, then incubation
was continued in a refrigerator overnight. After washing each well
twice, biotinylated anti-EAAT1, anti-SNAP25, anti-OMG, or
anti-CD11b was added and incubation was continued for an hour.
After washing each well twice, streptavidin-horseradish peroxidase
was added, and incubation was continued for another 30 minutes.
After washing each well three times, SuperSignal substrate was
added and chemiluminescent signals were measured in a
luminometer.
[0212] As shown in FIG. 35, multiple biomarkers were detected on
the surface of SNAP25+, EAAT1+, OMG+, and CD11b+ exosomes.
[0213] As shown in FIG. 34, all three control plasma showed
particle ranging from 50-400 nm in size. These results demonstrated
that the particles captured according to the methods described in
Example 17 above were extracellular vesicles.
[0214] These results showed that methods of the present invention
are useful for detecting biomarkers on the surface of exosomes.
These results showed that the methods and compositions of the
present invention are useful for identifying, detecting, and
measuring biomarkers on exosomes, wherein the exosomes are not
lysed or permeabilized. These results further suggested that the
methods and compositions of the present invention would be useful
for diagnosing neurological disease in a subject. These results
further suggested that the methods and compositions of the present
invention would be useful for diagnosing a disease or disorder.
[0215] Various modifications of the invention, in addition to those
shown and described herein, will become apparent to those skilled
in the art from the foregoing description. Such modifications are
intended to fall within the scope of the appended claims.
[0216] All references cited herein are hereby incorporated by
reference herein in their entirety.
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