CN110133272A

CN110133272A - Method for the excretion body in astroglia source to be enriched with or detected from biological fluid

Info

Publication number: CN110133272A
Application number: CN201810136747.8A
Authority: CN
Inventors: 章京; 于震维; 兰国钰
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2019-08-16
Also published as: WO2019153748A1

Abstract

The invention discloses a kind of methods of excretion body for being enriched with from biological fluid or detecting astroglia source.Wherein, the excretion body in astroglia source is enriched with method includes the following steps: (a) makes the biological fluid of the excretion body containing astroglia source contact anti-GLT1 antibody to form immune complex, wherein, biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And the excretion body in astroglia source (b) is enriched with using the method for solid phase or liquid phase by immune complex.The biomarker of the excretion body in detectable astroglia source, such as auxiliary diagnosis, antidiastole and monitoring central nervous system disease, Alzheimer disease, Parkinson's disease, multi-system atrophy, prion disease and nervous system neoplasm such as glioma etc. avoids passing through traumatic operation and obtains marker.

Description

For the excretion body in the enrichment from biological fluid or detection astroglia source Method

Technical field

The present invention relates to field of biomedicine technology, in particular to one kind for being enriched with or examining from biological fluid The method for surveying the excretion body in astroglia source.

Background technique

Such as auxiliary diagnosis, antidiastole and monitoring central nervous system disease, Alzheimer disease (AD), Parkinson The biomarker demand of sick (PD), multi-system atrophy (MSA) and prion disease are very urgent.In current biomarker, It shows preferable marker and is all based on what cerebrospinal fluid (CSF) was developed.CSF directly contacts brain and spinal cord, can be preferably The change of reaction center nervous system.But the acquisition of CSF is traumatic operation, and this severely limits cerebrospinal fluid biomarkers Conventional application.Therefore, periphery biomarker (such as in blood) research and development of central nervous system disease are particularly significant.So And due to the presence of blood-brain barrier, it is difficult to detect central nervous system (such as astroglia) source in the body fluid of periphery Ingredient.For example, in blood plasma only can be easy detection fraction human brain/CSF derived protein (Pan et al, J.Proteome Res.,13:4535-4545,2014).For these reasons, although taking time of decades and a large amount of Funds, there is presently no the periphery biomarkers established for AD, PD, MSA or prion disease.In addition, existing when using Technology measurement periphery biological fluid in the disease related proteins such as tau, A β, α-syn when, the protein in periphery source can be significant Influence the signal of central nervous system disease.

Therefore, how the biology for being able to reflect central nervous system disease variation is enriched with and marked from the biological fluid of periphery Marker is focus of the invention.

Summary of the invention

The present invention is intended to provide a kind of for the excretion body in astroglia source to be enriched with or detected from biological fluid Method, with solve it is existing for auxiliary diagnosis, antidiastole and monitor central nervous system disease biomarker obtain It takes and belongs to the technical issues of traumatic operation is without can be carried out conventional application.

To achieve the goals above, according to an aspect of the invention, there is provided it is a kind of for being enriched with from biological fluid The method of the excretion body in astroglia source.Method includes the following steps: (a) makes containing astroglia source The biological fluid of excretion body contacts anti-GLT1 antibody to form immune complex, wherein biological fluid be selected from blood, serum, One of blood plasma and saliva are a variety of；And star glue (b) is enriched with using the method for solid phase or liquid phase by immune complex The excretion body in cell plastid source.

Further, step (b) includes, by immune complex will the astroglia source in biological fluid it is outer It secretes body and is incorporated into solid phase；The excretion body of solid phase binding is separated from biological fluid to be enriched with the excretion in astroglia source Body.

Further, the anti-GLT1 antibody in step (a) is fixed in solid phase.

Further, this method further includes the steps that the excretion body (c) from solid phase elution of bound.

Further, anti-GLT1 antibody is by the antibody of label, and step (b) includes being enriched with star by flow cytometer The excretion body in shape spongiocyte source.

Further, anti-GLT1 antibody is by fluorescent marker, isotope, enzyme marker, chemiluminescent agent or quantum The antibody of point label.

According to another aspect of the present invention, a kind of anti-GLT1 antibody is provided in preparation for detecting in biological fluid Purposes in the preparation of the biomarker in the astroglia source of the nervous system disease.The purposes the following steps are included: (a) make the anti-GLT1 antibody of biological fluid contact of the excretion body containing astroglia source to form immune complex, In, biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And (b) determine astroglia The level of the biomarker of cell origin.

Further, biomarker is nucleic acid, protein, lipid or astrocyte source excretion body itself.

Further, step (b) includes: that (b1) passes through immune complex for the astroglia source in biological fluid Excretion body be incorporated into solid phase；(b2) the excretion body of solid phase binding is separated from biological fluid；And (b3) determines step (b2) The level of the biomarker in the astroglia source of the excretion body of isolated solid phase binding.

Further, when biomarker is astrocyte source excretion body itself, anti-GLT1 antibody is by label Antibody, step (b) include: by means of marking star cell origin excretion body in directly detection biological fluid；Preferably, anti-GLT1 Antibody is the antibody by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloid gold label；It is preferred that , star cell origin in biological fluid is directly detected by Particle Size Analyzer, gamma counter or small animal living body imager Concentration, particle diameter distribution and the Tissue distribution of excretion body；It is furthermore preferred that fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

Further, the nervous system disease includes that Parkinson's disease, Alzheimer disease, prion disease and nervous system are swollen Tumor；When the nervous system disease is Parkinson's disease, biomarker is the alpha-synapse nucleoprotein and star of alpha-synapse nucleoprotein, phosphorylation Shape cell origin excretion body itself；When the nervous system disease is Alzheimer disease, biomarker is Protein tau and phosphorylation Protein tau；When the nervous system disease is prion disease, biomarker is prion protein；When the nervous system disease is mind Through system tumor, the nervous system neoplasm includes glioma, and biomarker is the intracorporal biology of astrocyte source excretion The concentration and particle diameter distribution of marker level and astrocyte source excretion body, wherein the intracorporal life of astrocyte source excretion Substance markers object include alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, phosphorylation Protein tau and Abeta albumen.

According to a further aspect of the invention, it is refreshing for detecting in subject in preparation to provide a kind of anti-GLT1 antibody Purposes in preparation through systemic disease.The purposes is the following steps are included: (a) makes the excretion body containing astroglia source Biological fluid contact anti-GLT1 antibody to form immune complex, wherein biological fluid is selected from blood, serum, blood plasma, saliva One of liquid and urine are a variety of；And (b) determine the level of the biomarker in astroglia source, also, with The control level of subject from not the nervous system disease compares, outer from astroglia source in subject The level difference for secreting the biomarker of body significantly shows subject with the nervous system disease.

Further, step (b) includes: that (b1) passes through immune complex for the astroglia source in biological fluid Excretion body be incorporated into solid phase；(b2) the excretion body of solid phase binding is separated；And (b3) determines that step (b2) is isolated and consolidates The level of the biomarker in the astroglia source of the excretion body combined, also, there is no nervous system disease with coming from The control level of the subject of disease compares, the biomarker of the excretion body in subject from astroglia source Level difference significantly shows subject with the nervous system disease.

Further, the nervous system disease includes that Parkinson's disease, Alzheimer disease, prion disease and nervous system are swollen Tumor；When the nervous system disease is Parkinson's disease, biomarker is the alpha-synapse nucleoprotein and star of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of shape cell origin excretion body；When the nervous system disease is Alzheimer disease, biomarker is tau The Protein tau of albumen and phosphorylation；When the nervous system disease is prion disease, biomarker is prion protein；Work as nerve Systemic disease is nervous system neoplasm, and the nervous system neoplasm includes glioma, and biomarker is outside astrocyte source Secrete the concentration and particle diameter distribution of intracorporal biomarker level and astrocyte source excretion body, wherein astrocyte source The intracorporal biomarker of excretion includes alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, phosphorylation Protein tau and Abeta albumen.

According to a further aspect of the invention, it provides a kind of for detecting the star of the nervous system disease in biological fluid The device of the biomarker in shape spongiocyte source.The device includes: excretion body enrichment module, for making containing astroglia The biological fluid of the excretion body of cell origin contacts anti-GLT1 antibody to form immune complex, biological fluid be selected from blood, One of serum, blood plasma, saliva and urine are a variety of；And biomarker level determination module, for determining star glue The level of the biomarker in cell plastid source.

Further, excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source Anti- GLT1 antibody is contacted to form immune complex, by immune complex by the astroglia source in biological fluid Excretion body is incorporated into solid phase, and the excretion body of solid phase binding is separated from biological fluid；And biomarker level determines mould Block, the biology in the astroglia source of the excretion body for determining solid phase binding isolated in excretion body enrichment module The level of marker.

Further, anti-in excretion body enrichment module when biomarker is astrocyte source excretion body itself GLT1 antibody is by the antibody of label, and biomarker level determination module directly detects biological fluid culminant star by means of marking Shape cell origin excretion body；Preferably, anti-GLT1 antibody is by fluorescent marker, isotope, enzyme marker, chemiluminescence The antibody of agent, quantum dot or colloid gold label；Preferably, biomarker level determination module passes through Particle Size Analyzer, gamma Counter or small animal living body imager directly detect in biological fluid the concentration, particle diameter distribution of star cell origin excretion body and Tissue distribution；It is furthermore preferred that fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to a further aspect of the invention, it provides a kind of for detecting the dress of the nervous system disease in subject It sets.The device includes: excretion body enrichment module, for contacting the biological fluid of the excretion body containing astroglia source Anti- GLT1 antibody to form immune complex, biological fluid be selected from one of blood, serum, blood plasma, saliva and urine or It is a variety of；And comparison module, the level of the biomarker for determining astroglia source, also, do not have with coming from The control level of the subject of the nervous system disease compares, the life of the excretion body in subject from astroglia source The level difference of substance markers object significantly then exports the information that subject suffers from the nervous system disease.

Further, excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source Anti- GLT1 antibody is contacted to form immune complex, by immune complex by the astroglia source in biological fluid Excretion body is incorporated into solid phase, and the excretion body of solid phase binding is separated from biological fluid；And comparison module, for determining excretion The level of the biomarker in the astroglia source of the excretion body of the isolated solid phase binding of body enrichment module, and And compared with the control level of the subject from not the nervous system disease, astroglia is come from subject The level difference of the biomarker of the excretion body in source significantly then exports the information that subject suffers from the nervous system disease.

Further, anti-in excretion body enrichment module when biomarker is astrocyte source excretion body itself GLT1 antibody is by the antibody of label, and comparison module directly detects star cell origin excretion in biological fluid by means of marking Body, also, compared with the control level of the subject from not the nervous system disease；Preferably, anti-GLT1 antibody is to pass through Fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloid gold label antibody；Preferably, biology mark Note object level determination module is directly detected in biological fluid by Particle Size Analyzer, gamma counter or small animal living body imager Concentration, particle diameter distribution and the Tissue distribution of astrocyte source excretion body；It is furthermore preferred that fluorescent marker is fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to a further aspect of the invention, it provides a kind of for detecting the star of the nervous system disease in biological fluid The method of the biomarker in shape spongiocyte source.Method includes the following steps: (a) makes containing astroglia source The biological fluid of excretion body contact anti-GLT1 antibody to form immune complex, wherein biological fluid is selected from blood, blood Clearly, one of blood plasma, saliva and urine or a variety of；And (b) determine the water of the biomarker in astroglia source It is flat.

Further, when biomarker is astrocyte source excretion body itself, anti-GLT1 antibody is by label Antibody, step (b) include: by means of marking star cell origin excretion body in directly detection biological fluid；Preferably, anti-GLT1 Antibody is the antibody by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloid gold label；It is preferred that , biomarker level determination module is directly detected by Particle Size Analyzer, gamma counter or small animal living body imager Concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in biological fluid；It is furthermore preferred that fluorescent marker is glimmering Signal object Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800。

According to a further aspect of the invention, it provides a kind of for detecting the side of the nervous system disease in subject Method.Resist method includes the following steps: (a) makes the biological fluid of the excretion body containing astroglia source contact anti-GLT1 Body is to form immune complex, wherein biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or more Kind；And (b) determine the level of the biomarker in astroglia source, also, with come from no the nervous system disease The control level of subject compare, the water of the biomarker of the excretion body in subject from astroglia source Flat significant difference shows subject with the nervous system disease.

Further, step (b) includes: that (b) passes through immune complex for the astroglia source in biological fluid Excretion body be incorporated into solid phase；(c) the excretion body of solid phase binding is separated；And (d) determine the isolated solid phase knot of step (c) The level of the biomarker in the astroglia source of the excretion body of conjunction, also, with from not the nervous system disease The control level of subject compares, the level of the biomarker of the excretion body in subject from astroglia source Significant difference shows subject with the nervous system disease.

Further, when biomarker is astrocyte source excretion body itself, anti-GLT1 antibody is by label Antibody, step (b) include: directly to detect star cell origin excretion body in biological fluid by means of marking, and do not have with coming from There is the control level of the subject of the nervous system disease to compare, the excretion body in subject from astroglia source The level difference of biomarker significantly shows subject with the nervous system disease；Preferably, anti-GLT1 antibody is by glimmering Signal object, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloid gold label antibody；Preferably, pass through granularity Analyzer, gamma counter or small animal living body imager directly detect the dense of star cell origin excretion body in biological fluid Degree, particle diameter distribution and Tissue distribution；It is furthermore preferred that fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

According to a further aspect of the invention, a kind of excretion body biology mark of GLT1 as astroglia source is provided Remember the application of object.

Further, the application include: from biological fluid be enriched with astroglia source excretion body, preparation be used for The preparation of the biomarker in the astroglia source of the nervous system disease is detected in biological fluid, in biological fluid It detects the biomarker in the astroglia source of the nervous system disease and detects the nervous system disease in subject

Apply the technical scheme of the present invention enrichment astroglia source excretion body, can be used for detecting astroglia The biomarker of the excretion body of cell origin, auxiliary diagnosis, antidiastole and monitoring central nervous system disease such as, A Erci The silent disease in sea, Parkinson's disease, multi-system atrophy, prion disease and nervous system neoplasm such as glioma etc., avoid traumatic operation Marker is obtained, the pain of patient is alleviated, is suitble to routine clinical application.

Detailed description of the invention

The accompanying drawings constituting a part of this application is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:

Fig. 1 shows: being captured in A. embodiment 1 by anti-GLT1 capture or normal mouse IgG, Alix (general excretion body label Object) and GLT1 western blot；B. the extracellular vesica concentration of the blood plasma of GLT1 antibody capture and normal mouse in embodiment 1 The comparison of the extracellular vesica concentration of blood plasma of IgG antibody capture.

Fig. 2 shows: in A. embodiment 3 in clinical blood sample the excretion bulk concentration of middle GLT1 fluorescent marker assessment；B. The blood plasma excretion bulk concentration of GLT1 fluorescent marker is measured in embodiment 3 using Particle Size Analyzer (Nanosight300) and is divided Cloth.

Fig. 3 shows: for PD diagnosis and the ROC with the GLT1 fluorescent marker excretion bulk concentration of MSA antidiastole in embodiment 3 (receiver operating curves, Receiver OperatingCharacteristics) analysis.In entire queue, (46 MSA suffer from Person, 49 PD patients and 50 normal healthy controls) in, the antidiastole efficiency of multi-system atrophy and Parkinson's disease is observed, under curve Area (AUC)=0.6948, sensitivity=65.91%, specificity=78.57%；Observation Parkinson's disease and normal healthy controls are examined Disconnected efficiency, area under the curve=0.6750, sensitivity=66%, specificity=70.45%.

Fig. 4 shows: the testing result of α-syn in embodiment 5.

Specific embodiment

It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

Definition

" in conjunction with to " refers to be attracted to as used in this article is incorporated into mutual two kinds each other and specifically Molecule.In conjunction with pair example include but is not limited to antigen and the antibody for above-mentioned antigen, ligand and its receptor, complementary nucleic acid Chain, biotin and avidin, biotin and Streptavidin, phytolectin (agglutinin, lectin) and carbon aquation Close object.It is preferred combine to be biotin and Streptavidin, biotin and avidin, fluorescein and anti-fluorescein, Digoxigenin (digioxigenin)/anti-digoxigenin.

" the excretion body in astroglia source " refers to outer from astroglia as used in this article Secrete body.

" excretion body (exosome) " is the extracellular vesica of 30~150nm as used in this article, can be by numerous differences The cell of type is discharged, and executes different cell functions, including cell-cell communication, antigen presentation and protein and The transfer of nucleic acid.

" fixation " refers to that reagent is fixed to the surface of solids as used in this article.When reagent is fixed to solid table When face, it is non-to be covalently bonded in or be covalently bonded in surface.

" GLT1 " refers to the glutamate transporter 1 of astroglia expression as used in this article.

The inventors discovered that although mechanism not yet determines that the excretion body in astroglia source can cross over blood brain screen Hinder and enters in peripheral blood.

The pleasantly surprised discovery of the present inventor, " GLT1 " are unique, the disease that the excretion body in astroglia source carries The biomarker of specificity, can be used as the biomarker of the excretion body in astroglia source, can be accordingly in blood The excretion body in vivo detection astroglia source in liquid and other periphery body fluid, that astroglia is enriched with from biological fluid is thin The biology mark in astroglia source of the excretion body, preparation in born of the same parents source for detecting the nervous system disease in biological fluid Remember object preparation, in biological fluid detect the nervous system disease astroglia source biomarker and tested The nervous system disease is detected in person.

The inventors have discovered that for separating and marking in biological fluid such as blood, serum, blood plasma, saliva or urine The method of excretion body from astroglia.

The present inventor have also been found that can by the excretion body of the enrichment from astroglia in biological fluid Lai The central nervous system biomarker of detection and quantization astroglia source, and the above results can be used for detecting nerveous system Unite disease such as AD, PD, MSA and prion disease, can also be by the mark of these astroglia specificity based on periphery body fluid Will object is used for the antidiastole of related disease, monitors progression of disease and objectively evaluates the treatment effect of central nervous system disease Fruit.

Based on above-mentioned discovery, the present inventor is proposed for the star glue by measurement in enrichment from biological fluid Biomarker level in the excretion body in cell plastid source determines the substance (biomarker) in astroglia source Horizontal method.

The present invention relates to for from the biological fluid of subject mark astroglia source excretion body method, Method includes the following steps: (a) makes anti-GLT1 antibody and fluorescent dye form chemical coupling structure；(b) make containing star glue The anti-GLT1 antibody of the biological fluid contact conjugated fluorescent dyes of the excretion body in cell plastid source is to form immune complex；(c) Use excretion bulk concentration and the distribution that fluorescent dye is had in Nanosight analysis biological fluid.

The invention further relates to for from the biological fluid of subject enrichment astroglia source excretion body method, Method includes the following steps: (a) makes the biological fluid of the excretion body containing astroglia source contact anti-GLT1 antibody To form immune complex；(b) by immune complex, by the excretion body knot in the astroglia source in biological fluid Together in solid phase；And separate the excretion body of solid phase binding from biological fluid (c) to be enriched with the excretion in astroglia source Body.Being suitable for the invention biological fluid includes blood, serum, blood plasma, saliva and urine, wherein with blood, serum, blood plasma or Saliva is as priority option.

In order to specifically be enriched with the excretion body from the astroglia in biological fluid, the present invention uses immune parent The excretion body containing GLT1 is separated from the biological fluid of subject with catching method.GLT1 is to be originated from astroglia Marker on the surface of excretion body, astrocytes specifically express GLT1, and at other tracts (up to the present), Including being expressed without GLT1 in haemocyte and blood platelet, because without non-specifically capturing from non-astroglia source Excretion body.

For capturing, the anti-GLT1 antibody of the excretion body in astroglia source can be polyclonal antibody, monoclonal resists Body, single-chain antibody or antibody fragment such as Fab or F (ab') 2 segment containing GLT1 antigen binding domain.Anti- GLT1 antibody can be with thing First be fixed in solid phase, or when contact biological fluid in astroglia source excretion body with formed be immunized it is compound The excretion body that anti-GLT1 is combined is incorporated into solid phase after object, above method is added by means of that can capture the reagent of anti-GLT1 antibody With fixation.The method preferentially chosen is anti-GLT1 to be incorporated into solid phase in advance, then it is made to contact biological fluid.It is solid in solid phase Fixed method is related to covalent, hydrophobicity or electrostatic bond.For example, can be applied by being adsorbed onto the surface of solids or by being covalently bonded in The aminopropyl silane of cloth on a solid surface carrys out the first member of secure bond pair first (for example, Streptavidin, anti-fluorescence Element etc.).It may then pass through the combination of biotin-Streptavidin or fluorescein and anti-fluorescein (in conjunction with to), it will be with combining Pair the second member (for example, biotin, fluorescein etc.) label anti-GLT1 antibody be incorporated into the surface of solids.By means of solid phase And after specifically capturing the excretion body in astroglia source by GLT1 and anti-GLT1 antibody immune complex, from Biological fluid separates excretion body to be enriched with the excretion body in astroglia source.The excretion of solid phase binding can also directly be used Body can elute them from solid phase, for further using and measuring.Because step of the present invention is not related to ultracentrifugation, institute It further more tries out after optimization in routine clinical inspection with the inventive method.

It has been found by the present inventors that the measurement result of the excretion body in the astroglia source in biological fluid is available In detection the nervous system disease.Biomarker is also possible to protein, nucleic acid such as DNA or RNA, lipid or excretion body itself. For example, in the excretion body in astroglia source α-syn or phosphorylation α-syn (for example, serine 129- α-syn, The phosphorylation of α-syn at residue serine -129 or ps129) it is biomarker for PD.In astroglia source Excretion body in, tau or acidification tau, for example, p-tau¹⁸¹, it is the biomarker of AD.In the outer of astroglia source Secreting prion protein or the phosphorylation prion protein in body is prion disease such as creutzfeldt-jakob disease (Creutzfeldt-Jakob Disease biomarker).The biomarker as AD contained in the excretion body in astroglia source RNA include with gene UBAP2L, YBX1, SERF2, UBE2B, RPL10A, H3F3AP4, PPP2CA, NMD3, RNF7, RPLP0, SPARC、WTAP、HNRNPU、LINC00265、INMT、SLC35E2、CT60、SYNCRIP、RGS2、SMC6、ARSA、SPDYE7P、 SMIM17、TRAF3IP2-AS1、KCNC2、SLC24A1、HLCS、GOSR1、MN1、MGAT5、NBPF14、FBXO31、WDR52、 TBC1D2B、ZNF648、NBPF16、PAGR1、AQP2、PRKCI、SCN2B、DPYSL3、TMEM26、TSPAN11、ELL2、 FAM186A、CD59、THSD4、GOLGA6B、ARHGEF5、PKD1、BPTF、FLG、POM121L10P、NXPH3、H3F3A、 The associated RNA of SH3TC2, GGCX and GREB1.The biology mark as PD contained in the excretion body in astroglia source Note object RNA include with gene BLOC1S1, UBE2L3, RNF149, FAM89B, LCE6A, NT5DC2, PPP1CC, CCL5, HDLBP、HNRNPAB、NXN、SLC9A4、EIF2AK1、PAPOLA、TRIM50、SIX4、RAB3IP、VANGL2、DHRSX、 The associated RNA of FOXP4, SYNM, ZNF543, ATF6, LOC100132832 and BLOC1S1-RDH5.When being trapped in solid phase When, or after eluting from solid phase, protein-based biomarker can be measured in the excretion body of enrichment.For being exposed on Protein biomarker object on the surface of excretion body can measure it and crack without excretion body.For being included in excretion body Interior protein biomarker object can measure after the cracking of excretion body.Well known by persons skilled in the art can be passed through Where method measures protein biomarker object, such as immunoassays such as ELISA, Luminex and newest Quanterix be to use In the preferred method of measurement protein biomarker object.For nucleic acid biomarker object, before measuring nucleic acid biomarker object, Need to crack the excretion body of capture.Can by any method known to those skilled in the art, for example, DNA or rna probe, Or any of sequencing technologies detect nucleic acid.

The invention further relates to for detecting the nervous system disease in subject or for the method for antidiastole.Above-mentioned side Method is the following steps are included: (a) makes the biological fluid from subject contact anti-GLT1 antibody to form immune complex, wherein giving birth to Object liquid is blood, serum, blood plasma, saliva or urine；(b) by immune complex, by the astroglia in biological fluid The excretion body of cell origin is incorporated into solid phase；(c) thin to be enriched with astroglia from the excretion body of biological fluid separation solid phase binding The excretion body in born of the same parents source；(d) level for carrying out the biomarker of the excretion body in astroglia source of self enrichment is determined, and And it (is examined for identifying with from normal subjects (for diagnosing) or from the subject with another the nervous system disease It is disconnected) control level compare, the level error of the biomarker of the excretion body in subject from astroglia source It is different that significant there were significant differences shows subject with the nervous system disease (such as AD, PD or prion disease).In a kind of embodiment party In formula, biomarker is α-syn, phosphorylation α-syn and astrocyte origin marking object concentration itself, and the nervous system disease is PD.In another embodiment, biomarker is tau, phosphorylation tau or A beta substance, and the nervous system disease is AD.Another In a kind of embodiment, biomarker is prion protein, and the nervous system disease is prion disease.

The invention further relates to the method for the progress of the nervous system disease is monitored in subject.Above method packet It includes following steps: (a) obtaining biology from subject in different time points (for example, at time zero, 6 months, 1 year or 2 years) Humoral sample, wherein biological fluid is blood, serum, blood plasma, saliva or urine；(b) each sample is made to contact anti-GLT1 antibody To form immune complex；(c) by immune complex, make the excretion in the astroglia source in every kind of biological fluid Body is incorporated into solid phase；(d) the excretion body in astroglia source is enriched with from the excretion body of each sample separation solid phase binding； (e) level of biomarker is determined in the excretion body in the astroglia source of the enrichment from each sample, wherein Further significant difference shows that disease is progressive to level in the sample of later point.In one embodiment, Biomarker is α-syn or phosphorylation α-syn and the nervous system disease is PD.In another embodiment.Biology mark Remember that object is tau or phosphorylation tau or A beta substance and the nervous system disease is AD.In another embodiment, biomarker Object is prion protein or phosphorylation prion protein and the nervous system disease is prion disease.

The invention further relates to the method for the drug therapy of the nervous system disease is monitored in subject.Above-mentioned side Method the following steps are included: (a) obtains body fluid example before and after drug therapy, from subject, wherein biological fluid be blood, Serum, blood plasma, saliva or urine；(b) each sample is made to contact anti-GLT1 antibody to form immune complex；(c) by immune The excretion body in the astroglia source in every kind of biological fluid is incorporated into solid phase by compound；(d) from each sample point Excretion body from solid phase binding is to be enriched with the excretion body in astroglia source；(e) in the star of the enrichment from each sample The level of biomarker is determined in the excretion body in shape spongiocyte source, wherein the horizontal drop after the drug therapy in sample It is low to show that drug therapy is effective.In one embodiment, biomarker is α-syn or phosphorylation α-syn and mind It is PD through systemic disease.In another embodiment, biomarker is tau or phosphorylation tau or A beta substance and nerve Systemic disease is AD.In another embodiment, biomarker be prion protein or phosphorylation prion protein and The nervous system disease is prion disease.

Subject of the invention is mammalian subject such as people, horse and dog, and wherein people is preferred subject.

It is a kind of typically according to the present invention that embodiment there is provided one kind is thin for being enriched with astroglia from biological fluid The method of the excretion body in born of the same parents source.Method includes the following steps: (a) makes the life of the excretion body containing astroglia source Object liquid contacts anti-GLT1 antibody to form immune complex, wherein biological fluid be selected from blood, serum, blood plasma, saliva and One of urine is a variety of；And astroglia (b) is enriched with using the method for solid phase or liquid phase by immune complex The excretion body in source.

It can separate or pass through by solid phase by the excretion body that immune complex is enriched with astroglia source Liquid phase separation, according to the present invention a kind of typical embodiment, step (b) include, by the immune complex by organism The excretion body in the astroglia source in liquid is incorporated into solid phase；The excretion body of solid phase binding is separated from biological fluid with richness Collect the excretion body in astroglia source.Preferably, the anti-GLT1 antibody in step (a) is fixed in solid phase, in this way Facilitate subsequent operation.In order to further be enriched with excretion body, this method can also include the excretion body from solid phase elution of bound Step (d).Another typical embodiment according to the present invention, anti-GLT1 antibody are by the antibody of label, step (b) packet It includes, the excretion body in astroglia source is enriched with by flow cytometer；Preferably, anti-GLT1 antibody is by fluorescent marker Object, isotope, enzyme marker, chemiluminescent agent or quantum dot-labeled antibody.

It is a kind of typically according to the present invention that embodiment there is provided a kind of anti-GLT1 antibody to prepare in biological fluid Purposes in the preparation of the biomarker in the astroglia source of middle detection the nervous system disease.The purposes includes following Step: so that the biological fluid of the excretion body containing astroglia source is contacted anti-GLT1 antibody with formed be immunized it is compound Object, wherein biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And (b) determine star The level of the biomarker in spongiocyte source.Wherein, it is thin to can be nucleic acid, protein, lipid or star for biomarker Born of the same parents source excretion body itself, naturally it is also possible to be that other in the excretion body in astroglia source can be used as biomarker The substance of object.

A kind of typical embodiment according to the present invention, step (b) include: that (b1) passes through immune complex for biological fluid In the excretion body in astroglia source be incorporated into solid phase；(b2) the excretion body of solid phase binding is separated from biological fluid； And (b3) determines the biomarker in the astroglia source of the excretion body of the isolated solid phase binding of step (b2) Level.

In another exemplary embodiment of the invention, when biomarker is astrocyte source excretion body itself, Anti- GLT1 antibody is by the antibody of label, and step (b) includes: that star in biological fluid is directly detected by means of fluorescent marker is thin Born of the same parents source excretion body；The label of anti-GLT1 antibody includes but is not limited to fluorescent marker, isotope, enzyme marker, chemiluminescence Agent, quantum dot or colloid gold label；Preferably, by corresponding instrument, such as Particle Size Analyzer, gamma counter or small animal living body Imager etc. directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in biological fluid；More preferably , fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

The nervous system disease of the invention includes all diseases relevant to nervous system, including but not limited to Parkinson Disease, Alzheimer disease, prion disease and nervous system neoplasm；According to the difference of the nervous system disease type, can choose not Same biomarker, for example, biomarker is alpha-synapse nucleoprotein, phosphorylation when the nervous system disease is Parkinson's disease Alpha-synapse nucleoprotein and astrocyte source excretion body concentration and particle diameter distribution；When the nervous system disease is Alzheimer Disease, biomarker are the Protein taus of Protein tau and phosphorylation；When the nervous system disease is prion disease, biomarker is Prion protein；When the nervous system disease is nervous system neoplasm, the nervous system neoplasm includes glioma, biomarker It is the concentration and particle diameter distribution of astrocyte source excretion intracorporal biomarker level and astrocyte source excretion body, In, the astrocyte source intracorporal biomarker of excretion includes but is not limited to alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomerization Body, PS129, Protein tau, the Protein tau of phosphorylation and Abeta albumen.

It is a kind of typically according to the present invention that embodiment there is provided a kind of anti-GLT1 antibody to prepare in subject Detect the purposes in the preparation of the nervous system disease.The purposes is the following steps are included: (a) makes containing astroglia source The biological fluid of excretion body contacts anti-GLT1 antibody to form immune complex, wherein biological fluid be selected from blood, serum, One of blood plasma, saliva and urine are a variety of；And (b) determine the level of the biomarker in astroglia source, Also, compared with the control level of the subject from not the nervous system disease, astroglia is come from subject The level difference of the biomarker of the excretion body in source significantly shows subject with the nervous system disease.Wherein, biology mark Note object can be nucleic acid, protein, lipid or astrocyte source excretion body itself, naturally it is also possible to be astroglia Other in the excretion body in source can be used as the substance of biomarker.

A kind of typical embodiment according to the present invention, step (b) include: that (b1) passes through immune complex for biological fluid In the excretion body in astroglia source be incorporated into solid phase；(b2) the excretion body of solid phase binding is separated；And (b3) is determined The level of the biomarker in the astroglia source of the excretion body of the isolated solid phase binding of step (b2), wherein Compared with the control level of the subject from not the nervous system disease, from astroglia source in subject The level difference of the biomarker of excretion body significantly shows subject with the nervous system disease.

In another exemplary embodiment of the invention, anti-GLT1 antibody is the antibody by fluorescent marker, step (b) It include: that star cell origin excretion body in biological fluid is directly detected by means of fluorescent marker；Preferably, by corresponding instrument, Such as Particle Size Analyzer, gamma counter or small animal living body imager directly detect star cell origin excretion in biological fluid Concentration, particle diameter distribution and the Tissue distribution of body；It is furthermore preferred that fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

It is a kind of according to the present invention that typically embodiment there is provided one kind for detecting nervous system disease in biological fluid The device of the biomarker in the astroglia source of disease.The device includes: excretion body enrichment module, for making containing star The biological fluid of the excretion body in shape spongiocyte source contacts anti-GLT1 antibody to form immune complex, and biological fluid is to be selected from One of blood, serum, blood plasma, saliva and urine are a variety of；And biomarker level determination module, for determining star The level of the biomarker in shape spongiocyte source.Wherein, biomarker can be nucleic acid, protein, lipid or star Cell origin excretion body itself, naturally it is also possible to be that other in the excretion body in astroglia source can be used as biological mark Remember the substance of object.

A kind of typical embodiment according to the present invention, excretion body enrichment module, for making containing astroglia The anti-GLT1 antibody of biological fluid contact of the excretion body in source, will be in biological fluid by immune complex to form immune complex The excretion body in astroglia source be incorporated into solid phase, and the excretion body of solid phase binding is separated from biological fluid；And Biomarker level determination module, the star of the excretion body for determining solid phase binding isolated in excretion body enrichment module The level of the biomarker in shape spongiocyte source.

In another exemplary embodiment of the invention, when biomarker is astrocyte source excretion body itself, Anti- GLT1 antibody in excretion body enrichment module is by the antibody of label, and the label of anti-GLT1 antibody includes but is not limited to fluorescence Marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloid gold label；Preferably, fluorescent marker is fluorescence Marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800；Biomarker level determination module directly detects star cell origin excretion body in biological fluid by means of marking； Preferably, biomarker level determination module is by corresponding instrument, such as Particle Size Analyzer, gamma counter or small animal living body Imager etc. directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in biological fluid.

It is a kind of according to the present invention that typically embodiment there is provided one kind for detecting the nervous system disease in subject Device.The device includes: excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source Anti- GLT1 antibody is contacted to form immune complex, biological fluid is one in blood, serum, blood plasma, saliva and urine Kind is a variety of；And comparison module, the level of the biomarker for determining astroglia source, also, with come from There is no the control level of the subject of the nervous system disease to compare, from the excretion body in astroglia source in subject Biomarker level difference significantly then export subject suffer from the nervous system disease information.Wherein, biomarker It can be nucleic acid, protein, lipid or astrocyte source excretion body itself, naturally it is also possible to be astroglia source Other in excretion body can be used as the substance of biomarker.

A kind of typical embodiment according to the present invention, excretion body enrichment module, for making containing astroglia The anti-GLT1 antibody of biological fluid contact of the excretion body in source, will be in biological fluid by immune complex to form immune complex The excretion body in astroglia source be incorporated into solid phase, and the excretion body of solid phase binding is separated from biological fluid；And Comparison module, the astroglia source of the excretion body for determining the isolated solid phase binding of excretion body enrichment module The level of biomarker, also, compared with the control level of the subject from not the nervous system disease, in subject The level difference of the biomarker of excretion body from astroglia source significantly then exports subject with nerveous system The information for disease of uniting.

In another exemplary embodiment of the invention, when biomarker is astrocyte source excretion body itself, Anti- GLT1 antibody in excretion body enrichment module is by the antibody of label, and the label of anti-GLT1 antibody includes but is not limited to fluorescence Marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or colloid gold label；Preferably, fluorescent marker is fluorescence Marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800；Comparison module directly detects star cell origin excretion body in biological fluid by means of marking, also, does not have with coming from There is the control level of the subject of the nervous system disease to compare；Preferably, comparison module passes through corresponding instrument, such as grain size analysis Instrument, gamma counter or small animal living body imager etc. directly detect the concentration of star cell origin excretion body in biological fluid, Particle diameter distribution and Tissue distribution, also, compared with the control level of the subject from not the nervous system disease.

It is a kind of according to the present invention that typically embodiment there is provided one kind for detecting nervous system disease in biological fluid The method of the biomarker in the astroglia source of disease.Method includes the following steps: (a) make it is thin containing astroglia The biological fluid of the excretion body in born of the same parents source contacts anti-GLT1 antibody to form immune complex, wherein biological fluid is selected from blood One of liquid, serum, blood plasma, saliva and urine are a variety of；And (b) determine the biomarker in astroglia source Level.Wherein, biomarker can be nucleic acid, protein, lipid or astrocyte source excretion body itself, certainly may be used Be astroglia source excretion body in other can be used as the substance of biomarker.

In another exemplary embodiment of the invention, when biomarker is astrocyte source excretion body itself, Anti- GLT1 antibody is by the antibody of label, and step (b) includes: by means of marking astrocyte in directly detection biological fluid Source excretion body；The label of anti-GLT1 antibody includes but is not limited to fluorescent marker, isotope, enzyme marker, chemiluminescent agent, amount Son point or colloid gold label；Preferably, it is imaged by corresponding instrument, such as Particle Size Analyzer, gamma counter or small animal living body Instrument etc. directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in biological fluid；It is furthermore preferred that glimmering Signal object be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

It is a kind of according to the present invention that typically embodiment there is provided one kind for detecting the nervous system disease in subject Method.Resist method includes the following steps: (a) contacts the biological fluid of the excretion body containing astroglia source GLT1 antibody is to form immune complex, wherein biological fluid is selected from one of blood, serum, blood plasma, saliva and urine Or it is a variety of；And (b) determine the level of the biomarker in astroglia source, also, with come from no nervous system The control level of the subject of disease compares, the biomarker of the excretion body in subject from astroglia source Level difference significantly show subject with the nervous system disease.Wherein, biomarker can be nucleic acid, protein, rouge Class or astrocyte source excretion body itself, naturally it is also possible to be that other in the excretion body in astroglia source can be made For the substance of biomarker.

A kind of typical embodiment according to the present invention, step (b) include: that (b) passes through immune complex for biological fluid In the excretion body in astroglia source be incorporated into solid phase；(c) the excretion body of solid phase binding is separated；And (d) determine step Suddenly the level of the biomarker in the astroglia source of the excretion body of (c) isolated solid phase binding, also, with Compare from the control level of the subject of not the nervous system disease, the excretion from astroglia source in subject The level difference of the biomarker of body significantly shows subject with the nervous system disease.

In another exemplary embodiment of the invention, when biomarker is astrocyte source excretion body itself, Anti- GLT1 antibody is by the antibody of label, and step (b) includes: by means of marking astrocyte in directly detection biological fluid Source excretion body；The label of anti-GLT1 antibody includes but is not limited to fluorescent marker, isotope, enzyme marker, chemiluminescent agent, amount Son point or colloid gold label；Preferably, it is imaged by corresponding instrument, such as Particle Size Analyzer, gamma counter or small animal living body Instrument etc. directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in biological fluid；It is furthermore preferred that glimmering Signal object be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800, and compared with the control level of the subject from not the nervous system disease, in subject The level difference of the biomarker of excretion body from astroglia source significantly shows subject with nervous system Disease.

Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.These embodiments are intended only to illustrate the present invention And it is not necessarily to be construed as restrictive.

Embodiment 1

The separation of excretion body

It is from the agreement of Tauro et al (Methods 56:293-304,2012) and coated using antibody according to modification Superparamagnetic microballon separates excretion body from mouse or human plasma.

It briefly describes: by the anti-GLT1 antibody (ab178401, Abcam, Cambridge, MA, USA) of 10 μ g or normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA) is coated on one group of (1mg) M-270 (as negative control) On epoxy pearl, wherein utilizing antibody coupling kit (Life Technologies, Grand according to the explanation of manufacturer Island, NY, USA).At 37 DEG C after quick-thawing (in 2 minutes), plasma sample (> 300 μ are centrifuged at 2,000 X g L it) 15 minutes, is then centrifuged at 12,000 X g 30 minutes, is then diluted with phosphate buffered saline (PBS) (PBS) (pH7.4) 1:3 Supernatant.At 4 DEG C, by means of mildly rotating, the coated pearl of one group of antibody and 900 μ L diluting plasmas about 24 hours are incubated.Then Pearl is washed four times with 0.1% bovine serum albumin(BSA) (BSA) of 1mL/PBS (pH7.4) and is transferred to new pipe.

By means of the 0.1%BSA/PBS (pH7.4) and fixed buffer (4% paraformaldehyde/5% glutaraldehyde) of 60 μ L 1:1 mixture elutes excretion body from pearl, is used for Electron microscopy.Alternatively, at room temperature, by means of gently shaking, passing through Add 10% protease inhibitor cocktail in the 1%Triton X-100 of 110 μ L in 0.1%BSA/PBS (pH7.4) (P2714, Sigma-Aldrich, St Louis, MO, USA；In the H of 10ml₂Prepared in O) medium temperature grow cultured pearls 1 hour it is outer to crack Body is secreted, for Luminex measurement and other analyses, the result is shown in Figure 1, wherein A is shown through anti-GLT1 capture or normal small Mouse IgG capture, the western blot of Alix (general excretion body marker) and GLT1；B shows the blood plasma of GLT1 antibody capture The comparison of extracellular vesica concentration and the extracellular vesica concentration of blood plasma of normal mouse IgG antibody capture, this result illustrate anti-GLT1 Antibody can specifically be enriched with the excretion body from the astroglia in biological fluid.

The excretion body in clinical blood sample is extracted in batches, and PD and control sample are assigned to each batch.It will converge Collect and each batch is added from two reference plasma samples of 30 normal healthy controls to help to eliminate batch variation.

Embodiment 2

The characterization of the excretion body of anti-GLT1 capture from human plasma

Luminex measurement: by means of established Luminex experimental program (Brain 133:713-726,2010), examination With excretion system spare unit (extracting from 300 μ L blood plasma) Lai Dingliang α-syn, oligomer a-syn, PS129, tau, the A β of 100 μ L Protein concentration.

Western blot analysis: following standard protocols, carries out western blot.With Laemmli sample buffer To dissolve excretion body sample (~10 μ g protein) and divide on 1D PAGE gel before being transferred to polyvinylidene fluoride film From.By means of following primary antibody: mouse anti human GLT1 (Abcam, 1:500) and mouse anti human Alix (Cat#ABC40, Millipore, Billerica, MA, USA；1:1000) detect the protein on above-mentioned film.

Embodiment 3

The assessment of blood plasma GLT1 fluorescent marker excretion body in clinical sample

Collect clinical queue (46 MSA patients, 49 PD patients and 50 ages and the matched normal healthy controls of gender) Plasma sample and the concentration that the excretion body for containing GLT1 in blood plasma is measured using Nanosight measurement, are as a result shown in Fig. 2.Fig. 2 Middle A is to mark multi-system atrophy (MSA), Parkinson's disease (PD), normal healthy controls (CT) clinical using the GLT1 antibody of fluorescent marker Star cell origin excretion body in queue blood plasma, and excretion bulk concentration is calculated by Nanosight, the results showed that, compared to strong Health control, it is significantly higher (p < 0.01) in the excretion bulk concentration that blood plasma contains GLT1 fluorescent marker in PD patient.B in Fig. 2 For the astrocyte source excretion body size distribution for observing GLT1 antibody label in clinical queue blood plasma by Nanosight.*, As a result p < 0.01 is shown, PD with compare, the excretion body distribution situation of GLT1 fluorescent marker is not significantly different.

The potentiality of PD diagnosis are used in the excretion bulk concentration containing GLT1 fluorescent marker for further evaluation, at 46 ROC (receiver operating curves) are generated in MSA patient, 49 PD patients and 50 normal healthy controls, to be characterized in difference PD and be good for Their sensibility (sensitivity and specificity) in health control subject.Fig. 3 is the star marked using GLT1 antibody in blood plasma Cell origin excretion bulk concentration carries out multi-system atrophy (MSA), Parkinson's disease (PD) and normal healthy controls (CT) diagnosis and examines with identification Disconnected receiver operating curves analyze (Receiver Operating Characteristic Analysis, ROC analysis).The antidiastole efficiency of multi-system atrophy and Parkinson's disease is observed, finds the excretion body of blood plasma GLT1 fluorescent marker The PD diagnosis efficiency of concentration is moderate (AUC=0.6750, sensibility=66.00%, specificity=70.45%), the mirror of PD and MSA Other diagnosis efficiency is moderate (AUC=0.6948, sensibility=65.91%, specificity=78.57%) (Fig. 3).

Embodiment 4

In people's saliva detect GLT1 according to by Devic et al (Brain.134 (Pt 7): el78.doi: 10.1093brain/awr015.Epub2011Feb 24) method of description collects people's saliva from normal subjects.? Between 9~11AM of the morning, saliva sample is collected from healthy individuals.At 4 DEG C and 15,000g Centrifuge A sample 15 minutes to remove not Soluble substance and fragment.Supernatant is added in 10% (v/v) protease inhibitor cocktail immediately, followed by 20% (v/v) Precipitating proteins 1 hour on ice TCA.Centrifugal mixture 15 minutes under 4 DEG C and 15,000g.Remove supernatant and with ice-cold Acetone washing sediment twice.By means of BCA protein determination kit (Thermo Scientific Pierce) and use BSA assesses protein concentration as standard, then by sample storage at -20 DEG C until analysis.Foundation is retouched in embodiment 1 The agreement stated, by means of the pearl of anti-GLT1 antibody coating or pearl (negative control) Lai Wenyu supernatant of normal mouse IgG coating.According to According to the agreement described in example 2, the excretion body of capture is cracked, is used for Western blot analysis.By means of mouse anti human Alix carrys out detection membrane.Alix (a kind of common excretion body marker) is obvious detectable in the excretion body captured by anti-GLT1 , but in the excretion body captured by normal mouse IgG (negative control) it is undetectable to.Except being respectively provided with 50kDa and 25kDa Appropriate molecular weight human IgG known heavy chain and light chain other than, there is no other non-specific bands.

Embodiment 5

According to the method described in embodiment 1, GLT1- excretion body and normal mouse are enriched with from the blood plasma of subject IgG negative control.By means of established Luminex agreement (Brain133:713-726,2010), the excretion system of 100 μ L Spare unit (extracting from 300 μ L blood plasma) are used to quantify α-syn, as a result as shown in Figure 4.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims

1. a kind of method for being enriched with the excretion body in astroglia source from biological fluid, which is characterized in that including Following steps:

(a) biological fluid of the excretion body containing astroglia source is made to contact anti-GLT1 antibody with formed be immunized it is compound Object, wherein the biological fluid is to be selected from one of blood, serum, blood plasma and saliva or a variety of；And

(b) excretion in the astroglia source is enriched with using the method for solid phase or liquid phase by the immune complex Body.

2., will be biological by the immune complex the method according to claim 1, wherein step (b) includes The excretion body in the astroglia source in body fluid is incorporated into solid phase；From biological fluid separate solid phase binding excretion body with It is enriched with the excretion body in astroglia source.

3. according to the method described in claim 2, it is characterized in that, the anti-GLT1 antibody in step (a) is fixed on In the solid phase.

4. according to the method described in claim 2, it is characterized in that, the method further includes from the solid phase elution of bound The excretion body the step of (c).

5. the method according to claim 1, wherein the anti-GLT1 antibody is by the antibody of label, step It (b) include that the excretion body in the astroglia source is enriched with by flow cytometer.

6. according to the method described in claim 5, it is characterized in that, the anti-GLT1 antibody is by fluorescent marker, same to position Element, enzyme marker, chemiluminescent agent or quantum dot-labeled antibody.

7. a kind of anti-GLT1 antibody is in preparation for detecting the astroglia source of the nervous system disease in biological fluid Biomarker preparation in purposes, which comprises the following steps:

(a) biological fluid of the excretion body containing astroglia source is made to contact anti-GLT1 antibody with formed be immunized it is compound Object, wherein the biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；And

(b) level of the biomarker in the astroglia source is determined.

8. purposes according to claim 7, which is characterized in that the biomarker is nucleic acid, protein, lipid or star Shape cell origin excretion body itself.

9. purposes according to claim 8, which is characterized in that step (b) includes:

(b1) the excretion body in the astroglia source in the biological fluid is incorporated by the immune complex solid Phase；

(b2) the excretion body of the solid phase binding is separated from the biological fluid；And

(b3) life in the astroglia source of the excretion body of the isolated solid phase binding of step (b2) is determined The level of substance markers object.

10. purposes according to claim 8, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody are by the antibody of label, and step (b) includes: by means of described in the label directly detection Star cell origin excretion body in biological fluid；

Preferably, the anti-GLT1 antibody be by fluorescent marker, isotope, enzyme marker, chemiluminescent agent, quantum dot or The antibody of colloid gold label；

Preferably, it is directly detected in the biological fluid by Particle Size Analyzer, gamma counter or small animal living body imager Concentration, particle diameter distribution and the Tissue distribution of astrocyte source excretion body；

It is furthermore preferred that the fluorescent marker be fluorescent marker Qdot525, Qdot545, Qdot565, Qdot585, Qdot605, Qdot625, Qdot655, Qdot705 or Qdot800.

11. purposes according to claim 8, which is characterized in that the nervous system disease includes Parkinson's disease, A Erci The silent disease in sea, prion disease and nervous system neoplasm；

When the nervous system disease is Parkinson's disease, the biomarker is the alpha-synapse of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of nucleoprotein, astrocyte source excretion body；

When the nervous system disease is Alzheimer disease, the biomarker is the tau egg of Protein tau and phosphorylation It is white；

When the nervous system disease is prion disease, the biomarker is prion protein；

When the nervous system disease is nervous system neoplasm, the nervous system neoplasm includes glioma, the biomarker Object is the concentration and particle diameter distribution of astrocyte source excretion intracorporal biomarker level and astrocyte source excretion body, Wherein, the intracorporal biomarker of astrocyte source excretion include alpha-synapse nucleoprotein, alpha-synapse nucleoprotein oligomer, PS129, Protein tau, the Protein tau of phosphorylation and Abeta albumen.

12. a kind of anti-GLT1 antibody is in preparation for the use in the preparation of detection the nervous system disease or tumour in subject On the way, which comprises the following steps:

(b) determine the level of the biomarker in the astroglia source, and with come from no nervous system disease The control level of the subject of disease compares, described in the excretion body from the astroglia source in the subject The level difference of biomarker significantly shows the subject with the nervous system disease.

13. purposes according to claim 12, which is characterized in that the biomarker be nucleic acid, protein, lipid or Astrocyte source excretion body itself.

14. purposes according to claim 13, which is characterized in that step (b) includes:

(b2) the excretion body of the solid phase binding is separated；And

(b3) life in the astroglia source of the excretion body of the isolated solid phase binding of step (b2) is determined The level of substance markers object, and compared with the control level of the subject from no the nervous system disease, described tested The level difference of the biomarker of excretion body from the astroglia source in person significantly show it is described by Examination person suffers from the nervous system disease.

15. purposes according to claim 13, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody are by the antibody of label, and step (b) includes: by means of described in the label directly detection Star cell origin excretion body in biological fluid；

16. purposes according to claim 13, which is characterized in that the nervous system disease includes Parkinson's disease, A Er Ci Haimo disease, prion disease and nervous system neoplasm；

When the nervous system disease is Parkinson's disease, the biomarker is the alpha-synapse of alpha-synapse nucleoprotein, phosphorylation The concentration and particle diameter distribution of nucleoprotein and astrocyte source excretion body；

When the nervous system disease is Alzheimer disease, the biomarker is the tau egg of Protein tau and phosphorylation It is white；When the nervous system disease is prion disease, the biomarker is prion protein；

17. a kind of for detecting the dress of the biomarker in the astroglia source of the nervous system disease in biological fluid It sets characterized by comprising

Excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source contact anti-GLT1 antibody To form immune complex, the biological fluid is to be selected from one of blood, serum, blood plasma, saliva and urine or a variety of；With And

Biomarker level determination module, the level of the biomarker for determining the astroglia source.

18. device according to claim 17, which is characterized in that the biomarker be nucleic acid, protein, lipid or Astrocyte source excretion body itself.

19. device according to claim 18, which is characterized in that

The excretion body enrichment module, for making the biological fluid of the excretion body containing astroglia source contact anti-GLT1 Antibody, will be outside the astroglia source in the biological fluid by the immune complex to form immune complex It secretes body and is incorporated into solid phase, and separate the excretion body of the solid phase binding from the biological fluid；And

The biomarker level determination module, for determining the solid phase isolated in the excretion body enrichment module In conjunction with excretion body the astroglia source biomarker level.

20. device according to claim 18, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody in the excretion body enrichment module are by the antibody of label, and the biomarker is horizontal really Cover half block directly detects star cell origin excretion body in the biological fluid by means of the label；

Preferably, the biomarker level determination module by Particle Size Analyzer, gamma counter or small animal living body at As instrument directly detects concentration, particle diameter distribution and the Tissue distribution of star cell origin excretion body in the biological fluid；

21. device according to claim 18, which is characterized in that the nervous system disease includes Parkinson's disease, A Er Ci Haimo disease, prion disease and nervous system neoplasm；

22. a kind of for detecting the device of the nervous system disease in subject characterized by comprising

Comparison module, the level of the biomarker for determining the astroglia source, also, with come from no institute The control level for stating the subject of the nervous system disease compares, from the astroglia source in the subject The level difference of the biomarker of excretion body significantly then exports the letter that the subject suffers from the nervous system disease Breath.

23. device according to claim 22, which is characterized in that the biomarker be nucleic acid, protein, lipid or Astrocyte source excretion body itself.

24. device according to claim 23, which is characterized in that

The comparison module, the institute of the excretion body for determining the isolated solid phase binding of the excretion body enrichment module State the level of the biomarker in astroglia source, also, with the subject from no the nervous system disease Control level compare, the biomarker of the excretion body from the astroglia source in the subject Level difference significantly then export the subject suffer from the nervous system disease information.

25. device according to claim 23, which is characterized in that when the biomarker is astrocyte source excretion Body itself, the anti-GLT1 antibody in the excretion body enrichment module are by the antibody of label, and comparison module is by means of the mark Note directly detects star cell origin excretion body in the biological fluid, also, with from no the nervous system disease The control level of subject compares；

26. device according to claim 23, which is characterized in that the nervous system disease includes Parkinson's disease, A Er Ci Haimo disease, prion disease and nervous system neoplasm；

Application of the 27.GLT1 as the excretion body biomarker in astroglia source.

28. application according to claim 27, which is characterized in that the application includes: to be enriched with star from biological fluid Astroglia source of the excretion body, preparation in spongiocyte source for detecting the nervous system disease in biological fluid The preparation of biomarker, in biological fluid detect the nervous system disease astroglia source biomarker and The nervous system disease is detected in subject.