WO2018209215A1

WO2018209215A1 - Method for enriching oligodendrocyte-derived exosomes

Info

Publication number: WO2018209215A1
Application number: PCT/US2018/032290
Authority: WO
Inventors: Jing Zhang
Original assignee: Jing Zhang
Priority date: 2017-05-11
Filing date: 2018-05-11
Publication date: 2018-11-15

Abstract

The present invention relates to a method for enriching or isolating CNS-derived exosomes from a biological fluid such as blood, serum, plasma, or saliva by using an anti-CNPase antibody. The present invention further relates to a method for detecting a CNS disease by determining the biomarker level or by determining the particle number or size in the oligodendrocyte-derived exosomes enriched or isolated from the biological fluid. The method is useful in detecting, differentiating or monitoring CNS diseases, especially for those centered on oligodendroglia such as multiple system atrophy, multiple sclerosis, and oligodendroglioma.

Description

The present invention relates to a method for enriching or isolating oligodendrocyte- derived exosonies from a biological fluid such as blood, serum, plasma, or saliva by using an anti-CNPase antibody. The present invention further relates to a method for detecting a CNS disease by measuring the biomarker level in the oligodendrocyte-derived exosonies enriched or isolated from the peripheral biological fluids.

BACKGROUND OF THE INVENTION

Objective biomarkers are critically needed for assisting with diagnosis, differential diagnosis and or monitoring disease progression of CNS disorders, e.g., Alzheimer's disease (AD), Parkinson's disease (PD), stroke, infections, multiple system atrophy (MSA), multiple sclerosis (MS) and deadly glial neoplasm, including astrocytoma and oligodendroglioma. Of those CNS diseases, MSA, MS and oligodendroglioma are the disorders with

oligodendrocytes as one of major targets or initiating cell types. To date, the best performing markers are all based on cerebrospinal fluid (CSF) that is in direct contact with the brain and spinal cord or based on diseased brain tissue directly, which is particularly true for oligodendroglioma. However, obtaining CSF is an invasive procedure that severely limits its routine application; brain biopsy (routinely performed for oligodendroglioma) requires even more invasive procedures. Consequently, peripheral biomarkers (e.g. in blood) of CNS diseases are desperately needed. The challenge, however, is that not all CNS-derived materials can be detected in peripheral body fluids because of tightly regulated various barriers such as blood-brain-barrier (BBB). For instance, only a small fraction of proteins seen in human brain/CSF can be readily detected in plasma (Pan et al, J. Proteome Res., 13 :4535-4545, 2014). In addition, many CNS-derived materials, e.g. amyloid β (Αβ) and α- synuclein (α—syn), are also produced by other organ systems. This is indeed one of the fundamental underlying reasons for the fact that, despite decades of research and millions of dollars spent, no peripheral biomarkers have been established for major CNS diseases discussed above.

Research shows that oligodendrocytes secrete microvesicles, which include exosomes, that regulate morphological differentiation of oligodendrocyte and myelin formation and function. However, the overall role of these microvesicles or exosonies in i MSA, oligodendroglioma or demyelinating diseases is unclear. At this point, there is no prior art available to address specifically the CNS-derived microvesicles or exosomes in blood when coming to MSA, oligodendroglioma or demyelination.

Therefore, there exists a need to provide a method to specifically enrich the CNS- derived biomarkers in peripheral body fluids.

BRIEF DESCRIPTION OF THE DRAWING FIG. 1 shows evidence of CNPase (+) exosomes in human blood. Panel A

demonstrates that CNPase antibody pulldown with positive CNPase band, along with typical common exosome markers (Alix and CD9). Brain tissue is used for positive control. Panel B, as measured by NanoSight shows that the si ze of these CNPase (+) microvesicles i s in the range of typical exosomes. Panel C confirms the specificity of CNPase pulldown (vs. IgG pulldown), whereas Panel D further makes the point of specificity by showing that the signals were substantially reduced by pre-removing exosomes before enrichment was performed.

FIG. 2 shows the evaluation of exosome concentration and distribution in clinical plasma samples. The distribution and size of CNPase (+) exosomes (A) and total exosome a- syn concentration (B) were measured by Nanosight 300 qdot immuno-labeling assay and Luminex assays, respectively, in plasma (after immunocapture) from patients and controls. (C) The ratio (of CNPase containing exosomal a-syn/exosome numbers) was calculated. Data shown are mean ± S.D. *p < 0.05; **p < 0.01

FIG. 3 shows that CNPase (+) exosomes increased substantially in patients with oligodendroglioma (lane 4) compared to healthy controls (lane 2) and those with astrocytoma (lane 3). Negative control (IgG pulldown exosomes) is shown in lane 1 . CNPase is shown as 46 Kd band.

FIG. 4 shows CNPase (+) exosomes are increased substantially in patients with multiple sclerosis (MS) compared to healthy controls (CT). CD 63 and Alix are common exosomal markers, whereas CNPase is unique to the exosomes secreted by oligodendrocytes.

DETAILED DESCRIPTION OF THE INVENTION

Definition

"A binding pair," as used herein, refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but not limited to, an antigen and an antibody against the antigen, a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectin and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and anti- fluorescein, digioxigenin/anti-digioxigenin.

"CNP or CNPase", 2',3'-cyclic-nucleotide 3 '-phosphodiesterase, is an enzyme that catalyzes the hydrolysis of phosphodiester bonds. CNPase is a myelin-associated enzyme that makes up 4% of total CNS myelin protein in oligodendrocytes. CNPase is expressed exclusively by oligodendrocytes in the CNS. CNPase has a molecular weight about 46 K daltons.

"Central nervous system", CNS, comprises the brain and spinal cord,

"CNS-derived exosomes," as used herein, refer to exosomes containing materials

(such as protein and nucleic acids) derived from the CNS, i.e., brain and/or spinal cord.

"Exosomes," as used herein, are 40-150nm extracellular vesicles that are released from a multitude of ceil types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of lipids, proteins as well as nucleic acids, e.g. niRNA and miRNA.

"Immobilized," as used herein, refers to reagents being fixed to a solid surface. When a reagent is immobilized to a solid surface, it is either be non-covalently bound or covalentl y bound to the surface.

"Oligodendrocytes" are one of the major classes of the cells existing in the central nervous system (CNS). The main function of oligodendrocytes is to form myelin around neuronal axons and to provide support and insulation to axons in the central nervous system of some vertebrates. Oligodendrocytes are found only in the central nervous system.

'Oligodendrocyte-derived exosomes," as used herein, refer to exosomes containing materials (such as lipids, proteins and nucleic acids) derived directly from the

oligodendrocytes.

The present invention provides a tool for identifying and enriching microvesicles (including exosomes) derived specifically from oligodendrocytes as opposed to microvesicles from other cell types throughout the body. This technology has the potential to elucidate the molecular mechanisms of oligodendrocyte-derived microvesicles and may serve as a platform for research on neurodegenerative disorders, neuro-oncology and demyelinating diseases.

The inventor has discovered that oligodendrocytes-derived exosomes may cross multiple layers of the blood-brain barrier (BBB), by the mechanisms yet to be defined. The inventor has discovered that oligodendrocytes-derived exosomes, which carry unique, disease-specific biomarkers, can be detected in vivo in blood and other peripheral body fluids.

The inventor has discovered a method for isolating and enriching exosomes derived from the oligodendrocytes in a biological fluid such as blood, serum, plasma, saliva, or urine. The inventor has also discovered that oligodendrocytes-derived neurological biomarkers can be detected and/or quantitated from the enriched exosomes derived from oligodendrocytes in a biological fluid, and the results are useful for detecting a CNS disease especially for those centered on oligodendrogiia, e.g. MSA, MS, and oligodendroglioma. These peripheral body fluids based but CNS specific markers may be used for diagnosis, differential diagnosis, monitoring disease progression, and objectively assessing treatment effects of CNS diseases.

The present invention is directed to a method for enriching or isolating

oligodendrocyte-derived exosomes from a biological fluid of a subject. The inventor has discovered that CNPase is a surface marker on oligodendrocyte-derived exosomes, and that anti -CNPase antibody is effective to enrich or isolate oligodendrocyte-derived exosomes from a biological fluid, by immunoaffinity capturing of CNPase expressed on the surface of oligodendrocytes. The inventor has demonstrated that antibodies against other proteins such as PLP (proteolipid protein or lipophiiin), opalin (TmemlO or HTMP10), MAG (myelin- associated glycoprotein), MOG (myelin oligodendrocyte glycoprotein), 04 (oligodendrocyte marker 04), MBP (myelin basic protein), and else (Oligo 2), all of which are reported to be expressed by oligodendrocytes, are not effective for immunoaffinity capturing (see Example 3). The method comprises contacting a biological fluid from a subject with an anti-CNPase antibody to bind the antibody to CNPase-containing oligodendrocyte-derived exosomes, wherein the biological fluid is blood, serum, plasma, or saliva. In one embodiment, the method further comprises separating the anti-CNPase bound exosomes from the biological fluid to enrich the oligodendrocyte-derived exosomes.

In one embodiment, the anti-CNPase antibody is immobilized on the solid phase, and the exosomes are bound to the soli d phase through the immunocomplex of anti-CNPase antibody and CNPase, The method may further comprise a step of eluting the bound exosomes from the solid phase.

The biological fluids suitable for this invention include blood, serum, plasma, saliva, and urine. A preferred biological fluid is blood, serum, plasma, or saliva.

The present invention uses an immunoaffinity capturing protocol to isolate CNPase- containing exosomes from a biological fluid of a subject, CNPase is a marker on the surface of exosomes derived from oligodendrocyte and is exclusively expressed in oligodendrocytes. Anti-CNPase antibody is specific to CNPases, and thus, it does not capture exosomes derived from immune cells, or other organ systems non-specifically, or exosomes without the surface marker of CNPase.

The anti -CNPase antibody used to capture the CNS-derived exosomes can be a polyclonal antibody, a monoclonal antibody, single chain antibody, or an antibody fragment containing the CNPase antigen binding domain such as Fab or F(ab')₂ fragment.

The anti-CNPases antibody can be immobilized on a solid phase, or it can be in a liquid phase when contacting oligodendrocyte-derived exosomes in a biological fluid to form an immunocomplex in situ, and then the anti-CNPase bound exosomes are bound to a solid phase immobilized with reagents that can capture anti-CNPase.

In one embodiment, the anti-CNPase is bound to a solid phase when contacting a biological fluid. Methods to immobilize reagents to the solid phase are common in immunochemistry and involve formation of covalent, hydrophobic or electrostatic bonds between the solid phase and reagent. Anti-CNPase can be directly immobilized on a solid phase. Alternatively, anti-CNPase can be indirectly immobilized on a solid phase through a binding pair. For example, a first member of a binding pair (e.g., streptavidin, anti- fluorescein, etc.) can be first immobilized either by adsorption to a solid surface or by covalently binding to aminopropyisilane coated on a solid surface. Then anti-CNPase that is labeled with a second member of a binding pair (e.g., biotin, fluorescein, etc.) can be bound to the solid surface through the binding of biotin-streptavidin or fluorescein and anti- fluorescein (a binding pair).

After the oligodendrocyte-derived exosomes are specifically captured by a solid phase through the immunocomplex of CNPase and anti-CNPase, the exosomes are separated from the biological fluid to enrich the oligodendrocyte-derived exosomes. The solid phase bound exosomes can be used directly or they can be eluted from the solid phase for further use and/or measurement.

The inventor has discovered that measurements of biomarkers contained in oligodendrocyte-derived exosomes, or determine the particle number and/or size of the anti- CNPase antibody-bound exosomes, in a biological fluid are useful in detecting CNS diseases, including but not limited to multiple system atrophy (MSA), multiple sclerosis (MS) and oligodendroglioma. Of those CNS diseases, MSA, MS and oligodendroglioma are the disorders with oligodendrocytes as one of major targets or initiating cell types. The biomarkers can be protein, nucleic acids such as DNA or RNA, or lipids. For example, a-syn or phosphorylated a-syn (e.g., serine 129-a-syn, the phosphorylation of a-syn at residue serine- 129 or ps l 29) in oligodendrocyte-derived exosomes are biomarkers for MSA and Parkinson's disease (PD). Other proteins and nucleic acids, such as inflammatory effectors, e.g. TNFa and interleuki s for multiple sclerosis and IDH for oligodendroglioma, contained in oligodendrocyte-derived exosomes also have tremendous advantages over the same proteins or nucleic acids in free blood (serum or plasma) as these free proteins and nucleic acids can also be produced by other body systems, with little or no relevance to CNS diseases. To this end, a pilot study demonstrated that these exosomes contain IDH protein (one of most important markers for predicting the outcome of gliomas, both

oligodendroglioma and astrocytoma).

Protein biomarkers can be measured in anti-CNPase bound exosomes while captured on a solid phase, or after eluted from the solid phase, or in situ. For a protein biomarker that is exposed on the surface of exosomes, it can be measured without lysis of the exosomes. For a protein biomarker that is contained within the exosomes, it can be measured after lysis of the exosomes. Protein biomarkers can be measured by any method known to a person skilled in the art. Immunoassays such as ELISA, Luminex, and more recently Quanterix are preferred methods for measuring protein biomarkers.

For nucleic acid biomarkers, the anti-CNPase bound exosomes need to be lysed before the nucleic acid biomarkers are measured. Nucleic acids can be detected by any method known to a person skilled in the art, e.g., DNA or RNA probe, or any known sequencing techniques.

The present invention is directed to a method for detecting a CNS disease such as MSA, MS and oligodendroglioma. In one embodiment, the method comprises: (a) contacting a biological fluid from a subject with an anti-CNPase antibody to bind the antibody to CNPase-containing oligodendrocyte-derived exosomes, and (b) determining the level of a CNS-derived biomarker from the anti-CNPase antibody-bound exosomes, wherein an elevated level of a biomarker from the anti-CNPase antibody -bound exosomes in the subject comparing with a control level from a subject without a CNS disease indicates that the subject has the CNS disease.

In another embodiment, a method for detecting a CNS disease such as MS A, MS and oligodendroglioma comprises the steps of: (a) contacting a biological fluid from a subject with an anti-CNPase antibody to bind the antibody to CNPase-containing oligodendrocyte- derived exosomes, and (b) determining the particle number and/or size of the anti-CNPase antibody-bound exosomes, wherei n a different particle number or si ze of the anti-CNPase antibody-bound exosomes in the subject comparing with a control particle number or size from a control subject without a CNS disease indicates that the subject has the CNS disease. In one aspect, the CNS disease is MSA, and the particle number of the anti-CNPase antibody- bound exosomes in the subject suffering from multiple system atrophy is lower (reduced) than that of a control subject. In another aspect, the CNS disease is MS or oligodendroglioma, and the particle number of the anti-CNPase antibody-bound exosomes in the subject suffering from MS or oligodendroglioma is higher (increased) than that of a control subject or more CNPase expressed by the same number of CNPase (+) exosomes in the MS or

oligodendroglioma subject than that of a control subject (see FIGs. 3 and 4).

The inventor has also demonstrated that the particle number of anti-CNPase captured oligodendrocyte-derived exosomes decreased in MSA patients in comparison with that in PD patients, which can be used for differential diagnosis between MSA and PD. The ratios of exosomal a-syn to CNPase+ exosome number are similar in MSA and PD patients. Similarly, the particle number (or the expression content of biomarkers) is likely to be helpful in differentiating patients with oligodendroglioma vs astrocytoma (Fig 3) or multiple sclerosis over controls.

The present invention is further directed to a method for monitoring the progression of a CNS disease in a subject. The method comprises the steps of; (a) obtaining biological fluid samples at different time points (e.g., at time zero, 6 months, 1 year, or 2 years) from a subject, (b) contacting each sample with an anti-CNPase antibody to bind the antibody to CNPase-containing oligodendrocyte-derived exosomes, (c) determining the level of a biornarker in the enriched CNS-derived exosomes from each sample, wherein an altered level in the sample of a later time point indicates that the disease is progressive.

For detecting or monitoring a CNS di sease, the anti-CNPases antibody can be immobilized on a solid phase, or it can be in a liquid phase when contacting oligodendrocyte- derived exosomes in a biological fluid to form an immunocomplex in situ. For in situ.

separation is not necessary, because immunocomplex can be detected by immunofluorescent signals directly when a flow cytometry type of methods is used. The subject of the present invention is a mammal subject such as a human, horse, and dog; with human being the preferred subject.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

1 ) Coll ecti on of human pi asma:

Patients with various diseases or healthy controls (age and sex matched) from TianTan Hospital were included in the study. The inclusion and exclusion criteria and sample collection were following PPMI protocols (www . pmi-info. org).

2) Exosomes containing 2, 3 -cyclic nucleotide-3-phosphodiesterase (CNP or CNPase), a common mature oligodendrocyte marker, were isolated from human plasma with a well- established immunocapture protocol published previously. Briefly, 10 g of anti-CNP antibodies (clone mAbcarn 44289, abeam, Cambridge, MA, USA) or normal mouse IgGs (Santa Cruz Biotechnology, Dallas, TX, USA) as negative controls were coated onto one set (1 mg) of M-270 epoxy beads using a Dynabeads® Antibody Coupling Kit (life Technologies, grand Island, NY, USA) according to the manufacturer's instructions.

After thawing quickly (within 2 min) at 37 °C, plasma samplers (-300 μΐ) were centrifuged at 2,000 ^y-g for 15 min followed by 12,000 ^<-g for 30 min, and then the supernatant was diluted 1 :3 with phosphate buffered saline (PBS) (pH7.4). One set of antibody-coated beads and 900 μΐ of diluted plasma were mixed and incubated for -24 h at 4 °C with gentle rotation. The beads were then washed four times and transferred into a new tube. Exosomes were eluted from the beads for western blot or Luminex

measurements (see below).

3) Exosome-poor plasma samples (Fig 1, Panel D) were prepared by removing exosomes after a 2-step ultracentrifugation (1 0,000 <> for 3 h at 4 °C ^χ 2).

Example 2, Fluorescence aissd scatter nanoparticle tracking analysis (NTA)

(Fig 1, Panel B and Fig 2, Panel A). CNPase and IgG antibodies (Cat# ab44289, abeam; cs-2025, Santa Cruz) were coupled to Qdot605 following the manufacture's instruction (SiteClick™ Qdot® 605 Antibody Labeling Kit). PBS and IgG were used as negative control. Human plasma (~120ul) was centrifuged by 2,000xg 15min followed by 12,000xg 30min. Carefully transfer the supernatant to a new tube. 19μ1 plasma and Ιμΐ Qdot605 labeled antibody were mixed and incubated on ice for 1 hour. Then the sample was diluted 1 :20 with filtered PBS in order to optimize the number of particles in each fraction. Serum free media were conducted for 4 h to collect cell release EVs, Collected culture media were then centrifuged by 3,000xg 5min to eliminate ceil debits. Label the EVs using Qdot605 coupled CNPase antibody or normal IgG with 1 : 1.000 dilution for 1 h. NTA measurements were performed with a NanoSight 300 ( anoSight, Amesbury, United Kingdom), equipped with a 405nm violet laser and a 430nm filter. For scatter measurement, no filter was chosen when recording.

Example 3, Western blot with or without immunoprecipitation

ExQsomes were isolated from human plasma with antibody-coated beads according to the protocols of Example 1, except different antibodies as shown below in Table 1 were used for capturing exosomes. To perform the western experiments, exosome samples (-10 μg proteins) were solubilized with laemmli sample buffer and separated on a SDS-page gel before transferring to a polyvinylidene difluoride membrane. Western blotting was performed following a standard protocol, after immunoprecipitation of potential oligodendroglial markers using 300-500 μΐ human plasma (of healthy controls).

Table 1

Antibody against: Company Cat. Dilution Clone

Proteolipid protein (PLP) abeam Ab 183493 1 1000 EPR12675(2)(B)

Proteolipid protein (PLP) Sigma WH0005354 1 1000 NP 000524

Opalin SantaCruz Sc-376128 1 100 E-8

Myelin-associated SantaCruz sc- 166921 1 100 E-l

glycoprotein (MAG)

Myelin-associated SantaCruz sc- 166849 1 100 A- 11

glycoprotein (MAG)

Myelin-associ ated SantaCruz sc- 166780 1 100 G-l 1

glycoprotein (MAG)

Myelin oligodendrocyte abeam ab32760 1 1000 ab32759 glycoprotein (MOG)

04 NovusBioiogieals MAB 1326 1 1000 # 04

Myelin basic protein abeam Ab40390 1 1000 A-3

(MBP)

2', 3 '-cyclic nucieotide-3^!- abeam ab44289 1 1000 44289

phosphodi esterase

(CNPase)

Oligodendrocyte abeam ab 109186 1 100 EPR2673 transcription factor

(01ig2)

However, except anti-CNPase, none of these antibodies in Table 1 pulldowns demonstrated a particular band(s) like CNPase antibody shown in FIG. 1. The results show that only anti- CNPase is effective in binding, capturing, or enrich oligodendrocyte-derived exosomes from plasma.

The membrane was also probed with the other primary antibodies: mouse anti-human CNPase (ab44289, abeam, 1 : 1000) and rabbit anti -human Alix (Cat# ABC40, Millipore, Biilerica, MA, USA; 1 :500). Antibodies against a-syn (BD, 6 0787) were used in WB and immunofluorescent (IF) studies. Horseradish peroxidase (HRP)-conjugated secondary antibodies used in WB and Alex a Fluor, 488, 594 conjugated secondary antibodies used in IF were purchased from Thermo Fisher Scientific. Example 4, Luminex assays

Exosome preparations of 100 μΐ (extracted from 300 μΐ of plasma) were used to quantify a-syn with an established Luminex protocol, along with total a-syn concentrations in plasma as previously described in our own publication that has been cited extensively (Hong et al, Brain. 2010 Mar; 133(Pt 3):713-26. doi : 10, 1093/brain/awq008, Epub 2010 Feb 15). Hemoglobin concentrations as an index of the blood contamination in plasma were measured using an ELISA kit (the method is also described in great details in the same Hong's publication).

Example 5, CNPase (+) exosomes in patients with oligodendroglioma (Fig 3).

a. Patients with WHO grade II oligodendroglioma and WHO grade II astrocytoma, with pathology as well molecular validation, and age-matched controls are those enrolled at SanBo Brain Hospital affiliated with Capital University in China, with an average age of 40+ 7, 43+ 5, and 46 + 1 1, respectively. Male/female ratios are 10/10 for all three groups. b. Blood samples were collected using the same protocol described in Example 1.

c. CNPase (+) exosomes were isolated, with Western blot performed, as described for

Example I .

Example 6, CNPase (+) exosomes in patients with multiple sclerosis (Fig 4} a. Patients with multiple sclerosis and age-matched controls are those enrolled at XuanWu Hospital affiliated with Capital University in China, with an average age of 38+ 6 and 40 ± 9, respectively. Male/female ratios are 10/5 vs. 11/4 for MS patients and controls, respectively,

b. Blood samples were collected using the same protocol described in Example 1. c. CNPase (+) exosomes were isolated, with Western blot performed, as described for Example 1.

Example 7. General characterization of CNPase (+) exosomes

Detailed methods including materials used are provided in the above Examples, FIG. 1 shows the evidence of CNPase (+) exosomes in human blood. Panel A demonstrates that CNPase antibody pulldown with positive CNPase band, along with typical common exosome markers (Alix and CD9). Brain tissue is used for positive control. Panel B, as measured by NanoSight shows that the size of these CNPase (+) microvesicles is in the range of typical exosomes (40-15 nM). Panel C confirms the specificity of CNPase pulldown, i.e. CNPase pulldown showing a much higher signal than IgG (non-specific immunoprecipitation control) showing much reduced signal when a-syn is measured by Luminex technology. Panel D further makes the point of specificity by showing that the a-syn signals were substantially reduced (bl ack bar) by pre-removing exosomes (black bar) before enrichment was performed.

Example 8, Evaluation of exosonie concentration and distribution in clinieal plasma samples.

Detailed methods including materials used are provided in the above Examples.

FIG. 2 shows the evaluation of exosome concentration and distribution in clinical plasma samples. The distribution and size of CNPase (+) exosomes (A) and total exosome a-syn concentration (B) were measured by Nanosight 300 qdot immuno-labeling assay and

Luminex assays, respectively, in plasma (after immunocapture) from patients and controls. (C) The ratio (of CNPase containing exosomal a-syn/exosome numbers) was calculated. Data shown are mean ± S.D. *p < 0.05; **p < 0.01

Example 9. CNPase (+) exosomes in oligodendroglioma

Detailed methods including materials used are provided in the above Examples.

FIG. 3 shows that CNPase (+) exosomes are increased substantially in patients with oligodendroglioma compared to healthy controls and those with astrocytoma.

Oligodendroglioma is a type of gliomas that have a much better prognosis and responsiveness to several chemotherapies than other gliomas, especially astrocytoma. Oligodendroglioma group (lane 4) is a pool of 20 patients compared with equal number of healthy controls (lane 2) and those with astrocytoma (lane 3). Negative control (IgG pulldown) is shown in lane 1. Isolation method of exosomes is identical to those described for FIG. 1. CD 63 and Alix are common exosome markers, whereas CNPase is unique to the exosomes secreted by oligodendrocytes.

Example 10. CNPase (+) exosomes in multiple sclerosis

FIG. 4 shows that CNPase (+) exosomes were increased substantially in patients with multiple sclerosis (MS) compared to healthy controls. MS is a de-myelin disease that has a predilection to women and certain geographic areas of populations. The sample of MS group was made of a pool of 15 patients with MS compared with equal number of healthy controls. Isolation method of exosomes was identical to those described for FIG. 1. CD 63 and Alix are common exosomal markers, whereas CNPase is unique to the exosomes secreted by oligodendrocyte. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims.

Claims

WHAT IS CLAIMED IS:

1. A method for enriching oligodendrocyte-derived exosomes from a biological fluid, comprising:

(a) contacting a biological fluid from a subject with an anti-CNPase antibody to bind the antibody to CNPase-containing oligodendrocyte-derived exosomes, wherein the biological fluid is blood, serum, plasma, or saliva: and

(b) separating the anti-CNPase bound exosomes from the biological fluid to enrich the oligodendrocyte-derived exosomes.

2 The method of Claim 1, wherein the anti-CNPase antibody in step (a) is immobilized on the solid phase.

3. In the method of Claim 2, further comprising a step (c) of eluting the bound exosomes from the solid phase.

4. A method for detecting a CNS disease in a subject, comprising:

(a) contacting a biological fluid from a subject with an anti-CNPase antibody to bind the antibody to CNPase-containing oligodendrocyte-derived exosomes, and

(b) determining the particle number or size of the anti-CNPase antibody-bound exosomes,

wherein the CNS disease is multiple system atrophy, multiple sclerosis, or oligodendroglioma, and a different particle number or size of the anti-CNPase antibody- bound exosomes in the subject comparing with a control particle number or size from a control subject without a CNS disease indicates that the subject has the CNS disease.

5. The method of Claim 4, wherein the anti-CNPase antibody in step (a) is immobilized on the solid phase.

6. The method according to Claim 4, wherein the CNS disease is multiple system atrophy, and the particle number of the anti-CNPase antibody-bound exosomes in the subject is lower than that of a control subject.

7, The method according to Claim 4, wherein the CNS disease is multiple sclerosis or oligodendroglioma, and the particle number or the CNPase expression of the anti-CNPase antibody -bound exosomes in the subject is higher than that of a control subject

8. A method for detecting a CNS disease in a subject, comprising:

(a) contacting a biological fluid from a subject with an anti-CNPase antibody to bind the antibody to CNPase-containing oiigodendrocyte-derived exosomes, and

(b) determining the level of a CNS-derived biomarker from the anti-CNPase antibody-bound exosomes, wherein the CNS disease is multiple system atrophy, multiple sclerosis, or oligodendroglioma, and an altered level of a biomarker from the anti-CNPase antibody-bound exosomes in the subject comparing with a control level from a subject without a CNS disease indicates that the subject has the CNS disease.

9. The method according to Claim 8, wherein the CNS disease is MSA, and the biomarker is a-syn, oligomer] c a-syn, or phosphorylated a-syn.

10. The method according to Claim 8, wherein the CNS disease is MS, and the biomarker is an inflammatory effector.

11. The method according to Claim 8, wherein the CNS disease is oligodendroglioma, and the biomarker is Π3Η.