CN110320368A - Diagnostic kit based on TREM2 and its application on diagnosis of Parkinson disease product - Google Patents
Diagnostic kit based on TREM2 and its application on diagnosis of Parkinson disease product Download PDFInfo
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- CN110320368A CN110320368A CN201910462078.8A CN201910462078A CN110320368A CN 110320368 A CN110320368 A CN 110320368A CN 201910462078 A CN201910462078 A CN 201910462078A CN 110320368 A CN110320368 A CN 110320368A
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- trem2
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- diagnostic kit
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
Abstract
The invention discloses a kind of diagnostic kit and its applications on preparation Parkinson's disease clinical diagnosis product.The diagnostic kit includes: specific antibody and one or more kinds of detection reagents；The specific antibody has the ability in conjunction with TREM2 protein-specific, and the detection reagent is used to cooperate with the specific antibody, the TREM2 gene expression dose of test sample.The biomarker that the diagnostic kit is early diagnosed using TREM2 albumen as PD.It is compared with the diagnostic method of traditional Parkinson's disease, have the advantages that timeliness, specificity and sensitivity, can know disease risks in time in disease early stage, and for risk height, corresponding prevention and treatment measure is taken, the therapeutic effect for improving patient is conducive to.
Description
Technical field
Face the present invention relates to technical field of biological more particularly to a kind of diagnostic kit and its in preparation Parkinson's disease
Application on bed diagnostic products.
Background technique
Parkinson's disease (PD) is a kind of central nervous system degenerative disease, and clinical manifestation mainly includes static tremor, fortune
Move slow, myotonia and posture gait disorder.The pathological key feature of PD includes central nervous system (CNS) nigrostriatum
The loss of dopaminergic neuron (DA), microglia abnormal activation and neuron alpha-synapse nucleoprotein (α-
Synuclein deposition).
Currently, the goldstandard of diagnosis of Parkinson disease need to rely on biopsy of brain, and traditional clinical diagnosis relies primarily on medical history, disease
The comprehensive assessment of shape, sign and iconography.There is many shortcomings for traditional clinical diagnosis assessment mode.
Firstly, by the clinical diagnosing system of tradition PD, when patient shows symptom, sign, nigrostriatum area
DA neuron is largely lost, and has missed early intervention and has protected the opportunity of neuron.Secondly, traditional clinical diagnostic system
There are misdiagnosis rate height, make a definite diagnosis the disadvantages of needing the time long.
In order to overcome the defect of conventional diagnostic mode, it can choose using suitable biological marker and realize the morning of PD
Phase diagnosis, the accuracy for improving PD diagnosis and susceptibility.
Wherein, alpha-synapse nucleoprotein is the popular molecule of current candidate PD diagnostic marker.Alpha-synapse nucleoprotein can be in PD
Abnormal deposition in Intestinal Mucosal Injury in Patients Undergoing, digestion body of gland and skin.But in PD early diagnosis, cerebrospinal fluid alpha-synapse nucleoprotein is as diagnosis
The accuracy of marker and susceptibility are undesirable, and individual difference range is big.
In addition, there are diversified forms for alpha-synapse nucleoprotein, including there are monomer, the oligomer polymerizeing in various degree such as dimerization
Body, tripolymer, tetramer etc..Too many existence form increases the difficulty of alpha-synapse nucleoprotein clinical application.
Therefore, people's early clinical diagnosis that there is an urgent need to provide ideal biomarker to assist completing PD.
Summary of the invention
Place in view of above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a kind of diagnostic kit and its in pa
Application on the gloomy sick diagnostic products of gold, it is intended to solve the problems, such as that PD is early diagnosed ineffective in the prior art.
In order to achieve the above object, first aspect of the embodiment of the present invention provides a kind of diagnostic kit.The diagnostic kit
It include: specific antibody and one or more kinds of detection reagents；The specific antibody has and TREM2 protein-specific knot
The ability of conjunction, the detection reagent are used to cooperate with the specific antibody, the TREM2 gene expression dose of test sample.
Optionally, the specific antibody is selected from monoclonal antibody and/or polyclonal antibody.
Optionally, the sample is selected from the cerebrospinal fluid and blood of people.
Optionally, the diagnostic kit is immunity detection reagent；By the detection reagent and the specific antibody,
Water-soluble TREM2 albumen by the method for immune detection, in sample described in quantitative detection.
Optionally, the TREM2 albumen is the expression product of TREM2 gene, including soluble people TREM2 albumen and
The functional equivalent of people's TREM2 albumen.
Optionally, the TREM2 albumen is selected from one or more of following protein:
(1) protein of the composition of the amino acid sequence as shown in SEQ ID No.1；
(2) derived from the amino acid sequence as shown in SEQ ID No.1, and have and ammonia shown in SEQ ID No.1
The protein of base acid sequence function having the same；
(3) there is the amino acid sequence of 80% or more homology to constitute with amino acid sequence shown in SEQ ID No.1
Protein.
Optionally, derived from the amino acid sequence as shown in SEQ ID No.1, and have and SEQ ID No.1
Shown in amino acid sequence function having the same protein, specifically include:
By amino acid sequence shown in SEQ ID No.1 by the substitution of one or more amino acid and/or missing and/
Or it adds and derivative protein.
Optionally, the amino acid quantity of the substitution and/or deletion and/or addition is 1-50.
Optionally, there is the amino acid sequence of 90%-95% or more homology with amino acid sequence shown in SEQ ID No.1
Arrange the protein constituted.
Second aspect of the embodiment of the present invention provides a kind of diagnostic kit as described above and examines in preparation Parkinson's disease clinic
Application on stopping pregnancy product.
The biomarker that detection kit provided in an embodiment of the present invention is early diagnosed using TREM2 albumen as PD.Its
Compared with the diagnostic method of traditional Parkinson's disease, have the advantages that timeliness, specificity and sensitivity, it can be in disease early stage
Disease risks are known in time, and for risk height, take corresponding prevention and treatment measure, are conducive to the treatment for improving patient
Effect.
Detailed description of the invention
One or more embodiments are illustrated by the picture in corresponding attached drawing, these exemplary theorys
The bright restriction not constituted to embodiment, the element in attached drawing with same reference numbers label are expressed as similar element, remove
Non- to have special statement, composition does not limit the figure in attached drawing
Fig. 1 is the plasma soluble TREM2 albumen of the control group of the embodiment of the present invention 1 and the correlativity of synapse nucleoprotein
Schematic diagram；
Fig. 2, which is that the cerebrospinal fluid soluble T REM2 albumen of the control group of the embodiment of the present invention 1 is related to synapse nucleoprotein, to close
It is schematic diagram；
Fig. 3 is that the cerebrospinal fluid soluble T REM2 albumen of the embodiment of the present invention 1 and the correlativity of synapse nucleoprotein level show
It is intended to；
Fig. 4 is that the plasma soluble TREM2 albumen of the embodiment of the present invention 1 and the correlativity of synapse nucleoprotein level are illustrated
Figure；
Fig. 5 is that the cerebrospinal fluid soluble T REM2 level of the embodiment of the present invention 1 identifies the ROC curve of PD and normal aging people；
Fig. 6 be the embodiment of the present invention 1 cerebrospinal fluid soluble T REM2 level identify PD and normal aging people sensitivity and
Accuracy schematic diagram.
The amino acid sequence of people's TREM2 albumen are as follows: MEPLRLLILLFVTELSGAHNTTVFQGVAGQSLQVSCPYDSMK
HWGRRKAWCRQLGEKGPCQRVVSTHNLWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEA
DTLRKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEHSISRSLLEGEIPFPPTSILLLLACIFLIKILAASALW
AAAWHGQKPGTHPPSELDCGHDPGYQLQTLPGLRDT(SEQ ID No.1)
Specific embodiment
To make the purpose of the present invention, technical solution and effect clearer, clear and definite, right as follows in conjunction with drawings and embodiments
The present invention is further described.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not used to
Limit the present invention.
Unless otherwise stated, there are Science and Technology noun used in this specification those skilled in the art to lead to
The meaning understood.Also, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue culture, micro- life
Object, immunology relational language and laboratory operation step are widely used term and conventional steps in corresponding field.
The numerical value and its numberical range disclosed in the embodiment of the present invention is approximation, and and non-determined value.In error or
It may include all values in error range in the case that experiment condition allows.The numerical value model provided in the embodiment of the present invention
Enclose the temperature or other parameters enumerated in relative quantity and the other methods embodiment for indicating component in the mixture
Range.
In the present specification, " diagnosis Parkinson's disease (PD) " or " assessment Parkinson's disease " both includes whether judging subject
It suffered from Parkinson's disease, also included the risk that judges subject and whether there is with Parkinson's disease.From the state change of disease
Divide, may include disease alleviation and disease complete healing situations such as.
As documented by background technique, alpha-synapse nucleoprotein is considered as the morbid substance of PD.Alpha-synapse nucleoprotein is certainly
Body polymerizing power is strong, can form oligomer more stronger than alpha-synapse nucleoprotein monomer toxicity and fiber.Therefore, judge a kind of base
Whether the correlativity between Parkinson's disease can be dashed forward by detection gene or its expression product and α-for cause or its expression product
The correlation between nucleoprotein is touched indirectly to determine.
In order to realize that diagnosis or assessment Parkinson's disease, applicant's creativeness provide a kind of diagnostic kit.The diagnosis
Kit includes: specific antibody and one or more kinds of detection reagents.
Wherein, the specific antibody has an ability with TREM2 protein-specific ining conjunction with, the detection reagent for
The specific antibody cooperation, the TREM2 gene expression dose of test sample.
TREM2 is the specific receptor of microglia, can swallow brain Abnormal Eggss with assistant regulating and controlling microglia
The function of white (including abnormal alpha-synapse nucleoprotein).Applicant passes through it is experimentally confirmed that it has between abnormal alpha-synapse nucleoprotein
There is certain correlation, can be used as the molecular marker of PD early diagnosis and there is no one that alpha-synapse nucleoprotein is faced
A little actual application problems.
In the present specification, term " antibody " refers to usually (each pair of to have one " light " (L) by two pairs of identical polypeptide chains
Chain and " weight " (H) chain) composition immunoglobulin molecules.Antibody light chain can be classified as κ and lambda light chain.Heavy chain can be classified as
μ, δ, γ, α or ε, and the isotype of antibody is defined as IgM, IgD, IgG, IgA and IgE respectively.
In light chain and heavy chain, variable region is connected with constant region by area " J " of about 12 or more amino acid, heavy chain
It also include area " D " of about 3 or more amino acid.Each heavy chain is by heavy chain variable region (VH) and heavy chain constant region (CH) group
At.
Heavy chain constant region is made of 3 structural domains (CH1, CH2 and CH3).Each light chain is by light chain variable region (VL) and light chain
Constant region (CL) composition.Constant region of light chain is made of a domain C L.The constant region of antibody can mediated immunity globulin and place
Main tissue or the factor, the first component of various cells (for example, effector cell) and classical complement system including immune system
(C1q) combination.
The area VH and VL can be also subdivided into denatured region (referred to as complementary determining region (CDR)), be interspersed with
The more conservative region for being known as framework region (FR).Each VH and VL are by the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3,
3 CDR and 4 FR composition that FR4 is arranged from amino terminal to carboxyl terminal.The variable region (VH and VL) of each heavy chain/light chain pair
It is respectively formed paratope.The distribution of amino acid to each region or structural domain follows Kabat Sequences of
Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.
(1987and 1991)) or Chothia&Lesk (1987) J.Mol.Biol.196:901-917；Chothia et al. (1989)
The definition of Na ture 342:878-883.
In the present specification, the specific antibody is not limited by any specific method for generating antibody.Such as it recombinates
Antibody, monoclonal antibody and polyclonal antibody.The specific antibody can also be the antibody of not isotype, for example, IgG (for example,
IgG1, IgG2, IgG3 or IgG4 hypotype), IgA1, IgA2, IgD, IgE or IgM antibody.
In the present embodiment, the specific binding capacity of specific antibody is from " antigen-binding portion thereof ".The antigen knot
It closes part and refers to one or more parts of full length antibody, same antigen that the part keeps binding antibody to be combined (for example,
TREM2 ability and complete antibody) competes the specific binding to antigen.
Specifically antigen-binding portion thereof can be generated by recombinant DNA technology or by the enzymatic or chemical disruption of complete antibody.
In some embodiments, antigen-binding portion thereof includes Fab Fab', F (ab') 2, Fd, Fv, dAb and complementary determining region (CDR) piece
Section, single-chain antibody (for example, scFv), chimeric antibody, double antibody (diabody) and such polypeptide are more it includes being enough to assign
At least part of the antibody of peptide specific antigen binding capacity
It should be noted that the specific antibody or its segment are only required to retain and TREM2 specific binding
Ability can (i.e. antigen-binding portion thereof).Those skilled in the art can select to use any conjunction according to the needs of actual conditions
Suitable method or technological means prepares the specific antibody or its segment.
Based on invention thought disclosed by the embodiments of the present invention, those skilled in the art can apply the diagnostic kit, special
The expression of anisotropic quantitative detection TREM2 albumen or TREM2 gene suffers from Parkinson's disease to auxiliary diagnosis PD, assessment
Risk.
In the preferred embodiment, the TREM2 albumen in testee's cerebrospinal fluid and peripheral blood, and root can be detected simultaneously
Whether normally help to judge the state and α-of CNS No microglial according to cerebrospinal fluid TREM2 level and peripheral blood TREM2 level
The case where synapse nucleoprotein, auxiliary diagnosis testee suffer from the risk of PD.
The extracellular soluble fragments (Soluble TREM2, sTREM2) of TREM2 can be detected in cerebrospinal fluid and peripheral blood
It measures.STREM2 level and central nervous system abnormal protein level in cerebrospinal fluid is closely related, and peripheral blood
The change of mononuclear macrophage in the horizontal more reflection peripheral-systems of sTREM2.
In other words, cerebrospinal fluid and blood of the sample of the diagnostic kit from patient to be measured.Diagnostic kit can be with
The TREM2 albumen in cerebrospinal fluid and blood is detected simultaneously using same or different method.
Compared with the mode of the TREM2 protein content or gene expression dose of individually detection peripheral blood or cerebrospinal fluid, individually
The influence of when detection often due to periphery mononuclear macrophage can not correct response central nervous system situation, can not accurate evaluation
Encephalic alpha-synapse nucleoprotein and intracranial lesion.And the mode for detecting cerebrospinal fluid and blood sample simultaneously can obtain encephalic alpha-synapse
Nucleoprotein and the more accurate assessment result of intracranial lesion.
It should be noted that diagnostic kit provided in an embodiment of the present invention specifically can be based on any suitable principle simultaneously
Quantitative detection to TREM2 albumen is realized using any suitable way.It is any to be based on carrying out TREM2 albumen specific inspection
It surveys, to carry out the kit or relevant detection device of PD early diagnosis, the combination of device or its reagent belongs to the present invention
One of way of realization of disclosed diagnostic kit.
In some embodiments, the diagnostic kit can be immunity detection reagent.The detection reagent and described
Specific antibody, the water-soluble TREM2 albumen by the method for enzyme linked immunosorbent detection, in sample described in quantitative detection.
In other words, it can wrap containing plurality of reagents compatible with enzyme linked immunological, cooperated with complete in diagnostic kit
The detection of pairs of TREM2.Certainly, during enzyme linked immunological, specifically used detection reagent is for those skilled in the art institute
It is well known, it can select to use suitable reagent according to the needs of actual conditions.
In some embodiments, the TREM2 albumen is the expression product of TREM2 gene, containing related to Parkinson's disease
Functional domain, such as may include the functional equivalent of soluble people TREM2 albumen and people's TREM2 albumen.
The functional equivalent is that have identical or approximation function polypeptide, and including but not limited to people TREM2 albumen is conservative
Property variant protein matter or its active fragment or its reactive derivative, allelic variant, natural mutation, induced mutants,
It can be with the encoded protein of DNA of the DNA hybridization of people TREM2 etc. under high or low stringent condition.
Specifically, the TREM2 albumen is selected from one or more of following three kinds of protein:
(1) protein of the composition of the amino acid sequence as shown in SEQ ID No.1.
(2) derived from the amino acid sequence as shown in SEQ ID No.1, and have and ammonia shown in SEQ ID No.1
The protein of base acid sequence function having the same.
In some embodiments, can be will be shown in SEQ ID No.1 for the derivative protein with the same function
The derivative protein of substitution and/or deletion and/or addition that amino acid sequence passes through one or more amino acid.
Typically, the amino acid quantity of the substitution and/or deletion and/or addition is 1-50.It is preferred that can control
System is between 1-30.More preferably selected from 1-20.In the preferred embodiment, control is in 1-10 amino acid.
(3) there is the amino acid sequence of 80% or more homology to constitute with amino acid sequence shown in SEQ ID No.1
Protein.
Homology can be referred to as sequence identity again, show the close degree between two amino acid sequences.Compared with
In good embodiment, it can be with the amino with the above homology of amino acid sequence 90%-95% shown in SEQ ID No.1
Acid sequence.In further embodiments, specifically can choose be have with amino acid sequence 96% shown in SEQ ID No.1,
97%, the polypeptide that the amino acid sequence of 98%, 99% homology is constituted.
It should be noted that the modification of one or more amino acid in specific protein will not influence the function of protein
Energy.Those skilled in the art is appreciated that the amino acid for changing single amino acids or small percentage or to the individual of amino acid sequence
Addition, missing, insertion, replacement are conservative modifications.
The change of protein generates the protein with identity function, provides the Conservative substitution tables of intimate amino acid
It is well known in the art.
For example, the fusion protein of TREM2 albumen is by one amino acid of addition or more amino acid modification
Protein.In embodiments of the present invention, for the peptide or protein with TREM2 protein fusion, there is no limit as long as gained
Fusion protein retain TREM2 albumen biological activity.
In further embodiments, TREM2 albumen provided in an embodiment of the present invention further includes to shown in SEQ ID No.1
The non-conservative modification of amino acid sequence, as long as the protein by modification still is able to retain the biological activity of TREM2 albumen
?.Typically, in such modification protein the amino acid number that is mutated can be 10 perhaps less such as 6 or
Less, such as 3 or less.
The embodiment of the invention provides carry out TREM2 gene difference table by the total protein in cerebrospinal fluid and peripheral blood
The invention thinking reached, discloses the correlation of TREM2 protein level with Parkinson's disease.It will be understood by those skilled in the art that brain
Cell expression TREM2 in tissue, which can be fallen in cerebrospinal fluid, to be recycled, i.e. solubility TREM2 albumen, therefore in cerebrospinal fluid
Soluble T REM2 level can react the expression of TREM2 gene in brain tissue.
The diagnostic kit provided through the embodiment of the present invention as a result, can be to the soluble T REM2 of cerebrospinal fluid and peripheral blood
The expression of gene is detected, and is further used for judging whether subject suffers from Parkinson's disease.
Diagnostic kit provided in an embodiment of the present invention can detect subject's cerebrospinal fluid and peripheral blood T REM2's simultaneously
Protein level can be used as a kind of PD clinical diagnosis product and use, for judging whether subject suffers from Parkinson's disease or sentence
Disconnected subject whether there is the risk with Parkinson's disease, so that clinician be instructed to provide prevention scheme to subject or control
Treatment scheme.
Below in conjunction with specific example, the diagnostic kit of the present invention is described in detail embodiment offer and its in PD early diagnosis
On using effect.It should be noted that the experiment side of actual conditions is not specified in a particular embodiment in order to state simplicity
Method, usually according to normal condition, for example (,) Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring
HarborLaboratoryPress, 1989) condition described in, or according to item proposed by reagent or device fabrication manufacturer
Part executes.
Embodiment 1:
1) collection of peripheral blood and CSF sample:
As experimental group PD patient come from the First Affiliated Hospital of Guangzhou Medical University, totally 50.Age Jie 57-66
Year, all cases are diagnosed as PD, UK think-tank clinical criteria of the diagnostic criteria referring to Parkinson's disease.
Control group totally 40, be selected from the First Affiliated Hospital of Guangzhou Medical University's routine physical examination crowd, it is all enter examination person arrange
Except diseases such as blood lipid metabolism, nervous system chronic degenerative diseases, the age is situated between 55-68 years old.
All equal signeds of research object provide peripheral blood and brain ridge to the informed consent form of the detection project
Liquid is used for the detection of gene.
2) detection kit of building measurement cerebrospinal fluid and blood soluble T RME2 albumen:
21) it (is used using the anti-TRME2 antibody of monoclonal of two different extracellular parts for people TRME2 albumen
Anti-human TRME2 monoclonal antibody uses the anti-human TRME2 monoclonal antibody of Biotinylated mouse anti-as detection as capture antibody
Body) quantitative determination soluble T RME2 content.
22) antibody is captured in 96 hole detection plates, and with the ultimate density of 2.0 μ g/ml, the room temperature (RT) in PBS buffer solution is wrapped
It buries overnight.
23) after being cleaned 3 times with Wash Buffer (0.05%Tween20in PBS, PH=7.2), under the conditions of 25 DEG C,
It will test plate closing 1h with Reagent Diluents (1%BSA in PBS).
3) detection kit of building measurement synapse nucleoprotein:
31) (anti-human synapse nucleoprotein is used with the anti-TRME2 antibody of monoclonal of two kinds of differences to people's synapse nucleoprotein albumen
Monoclonal antibody uses the anti-human synapse nucleoprotein monoclonal antibody of Biotinylated mouse as detection antibody as capture antibody)
Quantitative determine the content of synapse nucleoprotein.
32) with the ultimate density of 4.0 μ g/ml, the room temperature in PBS buffer solution embedded capture antibody in 96 hole detection plates
Night.
33) after being cleaned 3 times with Wash Buffer (0.05%Tween20in PBS, PH=7.2), under the conditions of 25 DEG C,
It will test plate closing 1h with Reagent Diluents (1%BSA in PBS).
4) in serum and cerebrospinal fluid soluble T RME2 measurement (in all tests using soluble human TRME2-FC and
People a-synuclein-FC is as standard items):
41) take whole blood or 250 μ l (or 0.25g) of cerebrospinal fluid into RNase-Free Filter column, 13000rpm is centrifuged 2 points
Clock collects supernatant.
42) the 100 fresh rewarmings of μ l good CSF, serum and standard items room temperature are added in coating board under test and are being incubated for 2h, use
It is added after Wash Buffer cleaning to the detection antibody (final concentration: 35.0ng/ of the final concentration of soluble T RME2 albumen of 100 μ l
Ml) and a-synuclein detection antibody (final concentration: 25.0ng/ml) is incubated at room temperature 2h.
43) after being cleaned with Wash Buffer, the Streptavidin of 100 μ l horseradish peroxidase-labeleds is added in room
It is protected from light under temperature and is incubated for 20min.
44) after being cleaned with Wash Buffer, 100 μ l Substrate Solution (R&D Catalog# are added
DY999 it) is protected from light at room temperature and is incubated for 20min.
45) stop the development of color by 50 μ l 2N sulfuric acid (R&D Catalog#DY994) of addition.
46) the OD value in every hole is measured in 450 nanometers, while being corrected using the OD value of 540 nanometers measurement.
5) statistical method:
Each experiment is repeated 3 times, and data are indicated in a manner of mean+SD, is counted using SPSS21.0
Software is for statistical analysis.Difference between the two is examined using t, it is believed that has statistical significance as P < 0.05.
6) experimental result:
As shown in Figure 1, control group blood soluble T REM2 and synapse nucleoprotein level have raising compared with normal population
Trend, no significant difference (P < 0.05).
As shown in Fig. 2, the level of control group cerebrospinal fluid soluble T REM2 synapse nucleoprotein is significant compared with normal population
It increases, difference is statistically significant (P < 0.05).
As shown in figure 3, cerebrospinal fluid soluble T REM2 protein content and synapse nucleoprotein level are positively correlated, difference has statistics
It learns meaning (P < 0.05).
As shown in figure 4, plasma soluble TREM2 and synapse nucleoprotein level are positively correlated, no significant difference (P <
0.05)。
As shown in figure 5, cerebrospinal fluid soluble T REM2 level identifies the ROC curve (AUC=of PD and normal aging people
0.92)。
As shown in fig. 6, the soluble T REM2 of cerebrospinal fluid is used as sensitivity and accuracy schematic diagram when PD is diagnosed.
It is shown by the above experimental result, compared with normal population, the soluble T REM2 water of Parkinsonian's cerebrospinal fluid
Flat to increase, soluble T REM2 level shows without significant relation by solvable in measurement subject's cerebrospinal fluid and blood in peripheral blood
The level of property TREM2 helps to judge the risk that subject suffers from Parkinson's disease.Thus, it is possible to using TREM2 as assessment PD disease
The molecular marker of disease.
The embodiment of the invention also provides a kind of diagnostic kit and its applications.Diagnostic kit simultaneous quantitative detection is outer
All blood and cerebrospinal fluid TREM2 albumen, subject is assessed by the expression of TREM2 gene and suffers from Parkinson's disease risk, is mentioned
The clinical diagnosis and treatment of high Parkinson's disease is horizontal.
It, can according to the technique and scheme of the present invention and this hair it is understood that for those of ordinary skills
Bright design is subject to equivalent substitution or change, and all these changes or replacement all should belong to the guarantor of appended claims of the invention
Protect range.
Sequence table
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Met Glu Pro Leu Arg Leu Leu Ile Leu Leu Phe Val Thr Glu Leu Ser
1 5 10 15
Gly Ala His Asn Thr Thr Val Phe Gln Gly Val Ala Gly Gln Ser Leu
20 25 30
Gln Val Ser Cys Pro Tyr Asp Ser Met Lys His Trp Gly Arg Arg Lys
35 40 45
Ala Trp Cys Arg Gln Leu Gly Glu Lys Gly Pro Cys Gln Arg Val Val
50 55 60
Ser Thr His Asn Leu Trp Leu Leu Ser Phe Leu Arg Arg Trp Asn Gly
65 70 75 80
Ser Thr Ala Ile Thr Asp Asp Thr Leu Gly Gly Thr Leu Thr Ile Thr
85 90 95
Leu Arg Asn Leu Gln Pro His Asp Ala Gly Leu Tyr Gln Cys Gln Ser
100 105 110
Leu His Gly Ser Glu Ala Asp Thr Leu Arg Lys Val Leu Val Glu Val
115 120 125
Leu Ala Asp Pro Leu Asp His Arg Asp Ala Gly Asp Leu Trp Phe Pro
130 135 140
Gly Glu Ser Glu Ser Phe Glu Asp Ala His Val Glu His Ser Ile Ser
145 150 155 160
Arg Ser Leu Leu Glu Gly Glu Ile Pro Phe Pro Pro Thr Ser Ile Leu
165 170 175
Leu Leu Leu Ala Cys Ile Phe Leu Ile Lys Ile Leu Ala Ala Ser Ala
180 185 190
Leu Trp Ala Ala Ala Trp His Gly Gln Lys Pro Gly Thr His Pro Pro
195 200 205
Ser Glu Leu Asp Cys Gly His Asp Pro Gly Tyr Gln Leu Gln Thr Leu
210 215 220
Pro Gly Leu Arg Asp Thr
225 230
Claims (10)
1. a kind of diagnostic kit characterized by comprising specific antibody and one or more kinds of detection reagents；It is described
Specific antibody has the ability in conjunction with TREM2 protein-specific, and the detection reagent with the specific antibody for matching
It closes, the TREM2 gene expression dose of test sample.
2. diagnostic kit according to claim 1, which is characterized in that the specific antibody is selected from monoclonal antibody
And/or polyclonal antibody.
3. diagnostic kit according to claim 1, which is characterized in that the sample is selected from the cerebrospinal fluid and blood of people.
4. diagnostic kit according to claim 1, which is characterized in that the diagnostic kit is immunologic function test reagent
Box；The detection reagent and the specific antibody, the water solubility by the method for immune detection, in sample described in quantitative detection
TREM2 albumen.
5. diagnostic kit according to claim 1, which is characterized in that the TREM2 albumen is the expression of TREM2 gene
Product, the functional equivalent including soluble people TREM2 albumen and people's TREM2 albumen.
6. diagnostic kit according to claim 1, which is characterized in that the TREM2 albumen is in following protein
One or more:
(1) protein of the composition of the amino acid sequence as shown in SEQ ID No.1；
(2) derived from the amino acid sequence as shown in SEQ ID No.1, and have and amino acid shown in SEQ ID No.1
The protein of sequence function having the same；
(3) albumen that there is the amino acid sequence of 80% or more homology to constitute with amino acid sequence shown in SEQ ID No.1
Matter.
7. diagnostic kit according to claim 6, which is characterized in that the amino acid as shown in SEQ ID No.1
Derived from sequence, and there is the protein with amino acid sequence function having the same shown in SEQ ID No.1, it is specific to wrap
It includes:
Amino acid sequence shown in SEQ ID No.1 by the substitution of one or more amino acid and/or missing and/or is added
Add and derivative protein.
8. diagnostic kit according to claim 7, which is characterized in that the ammonia of the substitution and/or deletion and/or addition
Base acid quantity is 1-50.
9. diagnostic kit according to claim 6, which is characterized in that with amino acid sequence shown in SEQ ID No.1
The protein that amino acid sequence with 90%-95% or more homology is constituted.
10. a kind of if the described in any item diagnostic kits of claim 1-9 are on preparation Parkinson's disease clinical diagnosis product
Using.
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| CN116041481A (en) * | 2022-09-13 | 2023-05-02 | 北京湃德智健科技有限公司 | Antigen polypeptide for detecting TREM2 autoantibody and application thereof |
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| CN114010658A (en) * | 2021-11-25 | 2022-02-08 | 浙江大学 | TREM2hiApplication of macrophage in preparation of medicine for treating cardiac dysfunction |
| CN114010658B (en) * | 2021-11-25 | 2024-01-02 | 浙江大学 | TREM2 hi Application of macrophage in preparing medicine for treating cardiac dysfunction |
| CN116041481A (en) * | 2022-09-13 | 2023-05-02 | 北京湃德智健科技有限公司 | Antigen polypeptide for detecting TREM2 autoantibody and application thereof |
| CN116041481B (en) * | 2022-09-13 | 2024-01-19 | 北京湃德智健科技有限公司 | Antigen polypeptide for detecting TREM2 autoantibody and application thereof |
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