CN108776215A - Systemic sclerosis diagnoses and the biomarker of PAH predictions - Google Patents
Systemic sclerosis diagnoses and the biomarker of PAH predictions Download PDFInfo
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Abstract
The invention discloses the adam protein 4 with 1 type thrombospondin motif, i.e. ADAMTSL4 or its segment are being prepared for the purposes in the reagent of pulmonary hypertension possibility in forecasting system sclerosis.The present invention filters out 113 SSc related auto-antibodies target antigens using high-density protein chip technology.Target antigen structure, which is corresponded to, using 113 SSc related auto-antibodies prepares the clinical enlarged sample verification of SSc related auto-antibodies target antigen protein chips progress.As a result show that positive rate of the anti-ADAMTSL4 antibody in SSc merges PAH patient is 14.9%, the positive rate 2.88% being significantly higher than in SSc nonjoinder PAH patients can be used for the possibility for predicting that PAH occurs in SSc.
Description
Technical field
The invention belongs to field of biological detection, and in particular to systemic sclerosis diagnoses and the biomarker of PAH predictions
And application thereof.
Background technology
Systemic sclerosis (Systemic sclerosis, SSc), also referred to as chorionitis (scleroderma), are one
Kind is using limitation or diffusivity pachyderma and fibrosis as the Autoimmune diseases of main feature.The main feature of this disease
For inherent immunity and adaptive immunity dysfunction and microvascular lesions, the excessive production of extracellular matrix and collagen is finally resulted in
Raw and accumulation.Although being clinically usually hardened with the pachyderma of progressive and fiber turning to main feature, except skin by
Tired outer, fibrosis lesion can also involve multiple internal organs (lung, heart, kidney and alimentary canal etc.), and these changes are only
The deciding factor of clinical prognosis.Wherein interstitial lung lesion (Interstitial lung Disease, ILD) and pulmonary artery are high
Pressure (pulmonary hypertension, PAH) be not only most serious and prognosis it is worst complication and patient it is main dead
Die reason.Newest epidemic data shows that about 50% will appear ILD in SSc patient, and about 15% will appear PAH.Other are simultaneously
Hair symptom further includes Esophagus Function obstacle, and renal crises, finger tip ulcer, first pleat Microcirculation abnormality and dermal pathology show skin corium
Collagenous fibres increase fusion etc..In addition, SSc is as a kind of autoimmune disease, often with the generation of autoantibody, wherein
Include anti-topoisomerase I antibody (Anti-topoisomerase I) with the most important autoantibody of diagnostic significance, also referred to as
For Anti-Scl-70, anti-centromere antibody (Anticentromere, ACA) and anti-rna plymerase iii antibody (Anti-
RNApolymerase III)。
Research at present thinks that SSc is to lead to morbidity by environmental factor in the patient with genetic predisposition, and be
Triggered with the multifocal process of cytokine mediated chronic Cascaded amplification by immunocyte by a kind of, this process mainly with
Blood vessel pattern, inflammation, self-immunprocess and fibrosis are main feature.For example, vascular endothelial cell is pierced by the external world
After sharp or abnormal activation, the Endothelin of cell surface and a variety of receptors combine and cause transforming growth factor β
(transforming growth factor β, TGF-β) mediate and II type bone morphogenetic protein receptors (Bone
Morphogenetic protein receptor type II, BMPR2) the Smad signal paths transduction that mediates is abnormal, afterwards
Influence the differentiation of the secretion and T cell of B cell autoantibody；T cell abnormal differentiation, interleukins (Interleukin, IL)
The change of the cytokine levels such as IL-1, IL-6, TGF-β leads to regulatory T cells (Regulatorycells, Tregs)
Treg/Th17 cell Imbalances make nuclear Factor-Kappa B (Nuclear factor- κ B, NF- κ B) downstream etc. promote inflammation
The signal path effect of effect weakens；Abnormal adjust of TGF-β signal path can break its promotion inflammation and promote fibrosis
Balance between double effect passes through Connective Tissue Growth Factor (connective tissue growthfactor, CTGF)
Downstream transmitting promotes fibroblast to the signal of myofibroblast transdifferentiation, so as to cause the big volume production of extracellular matrix
Raw and collagen deposition.The further pathogenesis of SSc is still not known at present, needs constantly to further investigate to supplement current institute
It was found that possible approaches.
In Asia, the incidence of SSc is 7.2-10.9/1000,00 people, illness rate 38-56.3/1000,00 people, women
Morbidity crowd is significantly more than male.Also difference is larger for sex ratio, and women is common, and incidence is about 4-12 times of male.Exist more
Middle age morbidity, children are relatively rare.In addition, SSc patient often merges various autoimmune disease, there are about 26% SSc patients
Exist simultaneously one or more autoimmune diseases.According to the degree of skin involvement, it is by SSc points often clinically：Topical type
SSc (lcSSc), pachyderma are confined to the distal end of elbow or knee, and internal organs involvement is less；Diffuse type (dcSSc), skin
Range of being involved is wider, is rapidly developed in early stage and severe complication.2013, American society of rheumatism/Europe rheumatism connection
Alliance (American College of Rheumatology/EuropeanLeague Against Rheumatism, ACR/
EULAR newest SSc classification diagnosis standard) has been issued, importance of autoantibody during SSc is diagnosed is highlighted.
SSc autoantibodies marker used at present is not very ideal.Anti-Scl-70 is SSc Clinical practices
Autoantibody, but positive rate only 30%-40% or so of the antibody in SSc.The early lesion of SSc diseases typically belongs to
Clinical treatment reversible refunding or part reversible refunding, therapeutic effect are preferable；And later stage or whole latter stage, to belong to clinical treatment irreversible
In the refunding, rear bad, the death rate is high, and consequence is serious.Therefore, the early diagnosis of SSc diseases, early treatment are particularly important.Research
Show that histopathology biopsy, biochemical analysis and the autoantibodies inspection used is relied on to be only capable of finding in time simultaneously at present
The diagnosis of diagnosis of partial SSc patient, such disease still seem very imperfect.Since autoantibody can be in autoimmune
Disease Clinical symptom occurs first 10 years, or even appears in patient's body earlier, therefore, thoroughly to accomplish the early stage on practical significance
Diagnosis and early stage effectively treatment, as a whole with autoantibody on the front burner in pathogenesis, further system comprehensively
The serum antibody overall picture of system research SSc diseases, it is grinding for current SSc diseases to find for the relevant marker of such medical diagnosis on disease
Study carefully emphasis and clinical problem anxious to be resolved.
Invention content
To solve the above-mentioned problems, the present invention provides ADAMTSL4 or its segment and is preparing for forecasting system hardening
Purposes in disease in the reagent of pulmonary hypertension possibility.
Wherein, described diagnose includes：Measure the biology for being obtained from and the patient that systemic sclerosis merges pulmonary hypertension being presented
To the level of the reactive antibody of ADAMTSL4 or its segment in sample；Optionally,
With the level of antibody in the contrasting data biological sample, wherein relative to the contrasting data, the sample
The possibility for suffering from systemic scleroderma is shown to the detectably raising that ADAMTSL4 is reactive antibody in product.
Wherein, the biological sample is blood serum sample.
Wherein, the level of ADAMTSL4 antibody is measured by following steps, including：
A. the biological sample from patient is made to be contacted with ADAMTSL4 or its segment；
B. existing in the biological sample to form antibody-protein complex between antibody and ADAMTSL4 or its segment；
C. it washs to remove any unbonded antibody；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. it washs to remove any unbonded labeled detection antibody；With
F. the marker of the detection antibody is converted to detectable signal；The presence of wherein detectable signal shows described
There are anti-ADAMTSL4 antibody in patient.
Wherein, the ADAMTSL4 or its segment are deposited or are fixed on solid support.
Wherein, the support is the form of latex pearl, porous flat plate or film item.
Wherein, the detection antibody is by being covalently attached to enzyme, the marker with fluorescent chemicals or metal or having
The marker of chemiluminescence compound marks.
The present invention also provides a kind of diagnostic reagents for detecting and/or quantifying biological sample moderate resistance ADAMTSL4 antibody
Box, including：A kind of solid support, wherein the ADAMTSL4 or its segment deposition are fixed on solid support, institute
Biomarker of the anti-ADAMTSL4 antibody stated as pulmonary hypertension possibility in forecasting system sclerosis.
Wherein, the diagnostic kit further includes labeled and is reactive inspection to the antibody for carrying out biological sample
Survey antibody.
Wherein, the support is the form of latex pearl, porous flat plate or film item.
The present invention filters out 113 SSc related auto-antibodies target antigens using high-density protein chip technology.Using 113
A SSc related auto-antibodies correspond to target antigen structure and prepare the clinical expansion of SSc related auto-antibodies target antigen protein chips progress
Sample is verified.As a result it shows that positive rate of the anti-ADAMTSL4 antibody in SSc merges PAH patient is 14.9%, is significantly higher than SSc
Positive rate 2.88% in nonjoinder patients with pulmonary hypertension can be used for the possibility for predicting that PAH occurs in SSc.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
440 SSc patients that the present invention is included in from January, 2013 in January, 2018 BJ Union Hospital's outpatient service and
Patient SSc that inpatient and the outer institutes of sector fund project collect (includes the SSc trouble of the complication such as merging ILD, PAH
Person).Wherein male patient 38, female patient 402, the range of age 8-87 Sui, average age (48.35 ± 13.43) year.Enter
Group standard：The diagnosis of all patients meets the SSc classification diagnosis standards of the publications of ACR/EULAR in 2013.All SSc patients remove
Other outer autoimmune diseases (such as rheumatoid arthritis, systemic loupus erythematosus, Sjogren syndrome).Disease control group
190, including rheumatoid arthritis 42, inclusion criteria：It is closed using the rheumatoid of rheumatology association of U.S. publication in 1987
Save inflammation diagnostic criteria；Systemic loupus erythematosus 44, inclusion criteria：The system issued using rheumatology association of the U.S. in 1997
Property lupus erythematosus diagnosis standard；The diagnosis of Sjogren syndrome 44, all patients meets diagnosis of xerosis mark in 2002
It is accurate；Dermatomyositis 30, polymyositis 30, the diagnosis of all patients meet the diagnostic criteria of Bohan/Peter in 1975.
Other chronic diseases compare 40, and Normal group 130 derives from BJ Union Hospital's medical center.Strictly press protein
Group learns research and requires to leave and take sample, acquires peripheric venous blood, is dispensed after detaching serum in 2 hours, -80 DEG C freeze it is spare.
Embodiment 1
Using 40 SSc patients of high-throughput protein chip technology pair comprising 21065 kinds of nonredundancy recombination human source protein,
30 autoimmunity disease control patients and 20 normal controls carry out the screening of SSc related auto-antibodies target antigens.It is true through analyzing
Surely 113 SSc related auto-antibodies target antigens are filtered out.In order to confirm the sensibility of these SSc related auto-antibodies and special
Property and clinical value, then using 113 SSc related auto-antibodies corresponds to target antigen structure prepare SSc correlation itself resist
400 SSc patients of body target antigen protein chip pair, 160 from exempt from disease compare patient (including rheumatoid arthritis patients 36,
Patients with SLE 37, Patients with Sjogren Syndrome 37, patient with dermatomyositis 25 and patients with polymyositis 25
Example), 40 other chronic diseases compare patient (endocrine system disease 7, disease in the urological system 8, disease of cardiovascular system
8, disease of digestive system 7, respiratory disease 8 and cancer 2) and 100 serum of normal control carry out clinic expansions
Sample is verified.
It is corresponding with a small amount of SSc serum screening by hybridization candidate's SSc patient's autoantibody first with high throughput protein chip
Target antigen；Then it analyzes and determines itself related target antigen of SSc, SSc is prepared certainly using candidate SSc itself target antigens screened
Body antigen protein chip, and verification and the screening operation of a large amount of clinical serum samples are used it for, to identify SSc specificity certainly
The sensibility and specificity of body antigen.
1 high-density protein cDNA microarray SSc related auto-antibodies
The equal band of all albumen n ends for being included on high-density protein chip is useful for the GST labels of the protein purification.First
Use rabbit-anti GST monoclonal antibodies and chip protein hybridization proofing chip quality.Then choose 40 parts of SSc patients serums, 30
Serum of Patients With Autoimmune Diseases control serum and 20 normal healthy controls serum and 90 high-density protein chip hybridizations, pass through signal acquisition
And candidate's SSc correlation autoantigens are identified in data analysis.
2 high-density protein chip qualities detect
Utilize the quantity and parallel protein of effective protein spots on anti-GST antibody and protein-chip hybridization verification chip
The correlation of signal strength between point, assesses the detection quality of high-density protein chip, and concrete operation step is as follows：
(1) the high-density protein chip preserved is taken out from -80 DEG C, first air-isolation restores to room temperature, is subsequently dipped to contain 3%
In the 5ml PBST chip confining liquids of BSA, 1 hour (h) is closed at room temperature；
(2) take 0.5 μ l rabbit-anti GST monoclonal antibodies according to 1：4000 dilution proportion is in the PBST of lml3%BSA, shake
Mixing, 10000rpm*l0min centrifugations are swung, supernatant is the primary antibody diluted.Chip in confining liquid after taking out, if albumen
Chip surface has bubble, then uses 1ml sample loading guns to draw and be incubated sealing liquid in box and rinsed from top, with blotting paper by extra envelope
It closes liquid to blot as possible from side, be placed in wet box.Then it draws the primary antibody that 150 μ l have diluted to be added on protein chip, slowly add
Coverslip avoids generating bubble, is incubated at room temperature lh；
(3) protein chip being incubated is placed in 10ml PBST, carefully removes coverslip, chip is put into and is washed in box
It is rinsed 3 times with the PBST 40rpm of room temperature, each 10min：
(4) the horse anti-rabbit IgG antibody for taking 0.5 μ l cy5 labels, according to l：2000 dilution proportion is in 3%BSA PBST
In, mixing, 10000rpm, 10min centrifugation are shaken, supernatant is the secondary antibody diluted.Protein chip is taken out in box from washing, if
ProteinChip surface has bubble, then uses lml；Sample loading gun is drawn liquid in incubation box and is rinsed from top, is inhaled from side with blotting paper
Dry extra washing lotion, is placed in wet box.The secondary antibody that 150 μ l have diluted is added on protein chip, is slowly capped slide, avoids generating
Bubble, room temperature, which is protected from light, is incubated secondary antibody lh；
(5) protein chip being incubated is placed in PBST, carefully removes coverslip, protein chip is put into wash in box and is used
PBST 40rpm are protected from light rinsing 3 times, each 10min；
(6) and then with room temperature distilled water (double distilled water, ddH20) 40rpm is protected from light rinsing 3 times, often
Secondary 10min；
(7) take out protein chip, be placed in 50ml centrifuge tubes, there is the one of probe to face outwardly on protein chip, 1000rpm from
Heart 2min dries ddH remaining on protein chip20；
(8) GenePiX 4000B chip scanners scan protein chip, and identical scanning is all arranged after the completion of hybridization every time
Parameter scanning chip.The parameter setting of scanner is as follows：Photomultiplier (PMT) is set as 800, and laser energy (Power) is set
It is set to 100%, the pixel of scanning is 5um.
3 high-density protein chip scanning data are extracted
High-density protein chip scanning is carried out using GenePix 4000B chip scanners, obtains tiff format gray-scale map
As after, according to the operation manual of 6.0 softwares of GenePix Pro, according to high-density protein chip loading dot matrix parameter, by dividing
Analysis software is automatically performed each protein site signal pixels segmentation extraction, while manually checking one by one, and manual setting is avoided impurity, drawn
The human factor of protein signal point position caused by trace etc. and size offset, checks the letter intensity of all protein sites on chip, most
Complete the extraction of all proteantigen point signal strengths on every high-density protein chip afterwards, and generate data file it is standby subsequently into
The analysis of one step uses.
4 high-density protein chips detect quality evaluation
Hybridize the gray level image after high-density protein chip using anti-GST antibody, it is strong to complete each protein site signal of chip
After degree acquisition, when the signal-to-noise ratio (Signal to Noise Ratio, SNR) of two parallel points of probe on chip is simultaneously greater than 2
Just think that the protein site can be detected on chip.Then the ratio for the protein spots that can be detected on computing chip and each albumen
The correlation of signal strength between two parallel points.
5 high-density protein chips detect autoantibodies
Serum specimen for the detection of high-density protein chip includes SSc patient 40, exempts from disease control patient 30 certainly, just
Often control 20.Concrete operation step is as follows：
(1) after the high-density protein chip for preserving -80 DEG C takes out, air-isolation is balanced to room temperature, is immersed 5ml and is contained 3%
In the protein chip confining liquid of BSA PBST, room temperature closes 1 hour (h)；
(2) take l μ l serum specimens according to 1：1000 dilution proportion shakes mixing in lml 3%BSA PBST,
10000rpm*l0min is centrifuged, and supernatant is the serum diluted.By protein chip after being taken out in confining liquid, if albumen core
There is bubble on piece surface, then uses lml sample loading guns to draw and be incubated sealing liquid in box and rinsed from top, with blotting paper by extra closing
Liquid blots as possible from side, is placed in wet box.Then it draws the serum that 150 μ l have diluted to be added on protein chip, pipette tips should not
Chip is encountered in order to avoid destroying the proteantigen on chip, is slowly capped slide closing, is avoided generating bubble, is incubated at room temperature 1h；
(3) protein chip being incubated is placed in 10ml PBST washing lotions, carefully removes coverslip, chip, which is put into, washes box
It is middle to be rinsed 3 times with the PBST 40rpm of room temperature, each 10min；
(4) goat anti-human IgG antibodies for taking Alexa 647 to mark, according to 1：1000 dilution proportion is in 3%BSA PBST
In, mixing, 10000rpm*10min centrifugations are shaken, supernatant is the secondary antibody diluted.Protein chip is taken out in box from washing, if
Chip surface has bubble, then uses 1ml sample loading guns to draw and be incubated liquid in box and rinsed from top, it is extra to be blotted from side with blotting paper
Washing lotion, be placed in wet box.The 150 μ l fluorescence secondary antibodies diluted are added on chip, slide closing is slowly capped, avoids generating
Bubble, room temperature, which is protected from light, is incubated secondary antibody 1h；
(5) protein chip being incubated is placed in PBST washing lotions, carefully removes coverslip, chip is put into wash in box and use
PBST 40rpm are protected from light rinsing 3 times, each 10min；
(6) and then in room temperature distilled water (double distilled water, ddH20) 40rpm is protected from light rinsing albumen
Chip 3 times, each 10min；
(7) protein chip is taken out, is placed in 50ml centrifuge tubes, have antigen protein one is put on chip and is faced outwardly, 1000rpm
2min is centrifuged, ddH remaining on chip is dried20；
(8) GenePix 4000B chip scanners scan protein chip, and identical scanning is all arranged after the completion of hybridization every time
Parameter scanning chip.
The determination of 6SSc related auto-antibodies target antigens
By 6.0 software collections of GenePix Pro to every high-density protein chip on all protein sites signal strength
Information imports in Excel tables.With being carried on the back around the foreground signal intensity (F635median) of each protein site divided by the protein site
Signal value of the scape signal strength (B635median) as the protein site.That is Iij=F635median/B635median (Iij generations
The signal value of protein i points in table block j).The signal value of proteantigen protein site more levels off to 1, illustrates in serum
Corresponding autoantibody concentrations are lower, which is less susceptible to be detected.Signal value is higher to illustrate this in serum specimen itself
The ability of the combination target antigen protein of antibody is stronger.Using the average value of different two signal strengths of the same albumen as this
Signal strength of the protein site on the chip.When the average value>When 2, it is believed that the protein site is the positive.Then every part of serum is counted
With the information of each proteantigen immune response positive rate on high-density protein chip.Using high-throughput chip biological information analysis
In technology common genetic chip significance analysis (SAM) method to SSc patient groups, exempt from certainly disease control group and Normal group into
Row data analysis, with Q values<35% enters to be selected as the candidate related autoantigens of SSc.
6SSc related auto-antibodies target antigen protein chips detect autoantibodies
(1) standing 30min (avoids condensing after taking out the SSc related auto-antibodies target antigen protein chips of -80 DEG C of preservations
Water), after air-isolation restores room temperature, packaging is cut off, protein chip is carefully taken out, against fluorescent lamp, according to each on chip
The position of Block places fence in the front of chip and is firmly compacted post, and then faces up and is placed into chip hybridization wet box
In；
(2) small side of 50 microlitres of PBST (0.1%Tween20, the v/v) buffer solution in each fence containing 3%BSA is added
In battle array, closed 1 hour at 37 DEG C.
(3) protein chip is positioned in 50ml centrifuge tubes, there is the one of proteantigen to face outwardly on chip, 1000rpm
2min is centrifuged to remove the liquid in each small square formation；
(4) take 1u1 serum according to 1：1000 dilution proportion shakes mixing in lml 3%BSA PBST,
10000rpm*10min is centrifuged, and supernatant is the serum diluted.
(5) blood that 50ul has diluted is separately added into SSc related auto-antibodies target antigens protein chip each small square formation
Clearly, it avoids generating bubble, is incubated at room temperature 1h；
(6) protein chip is soaked in 10 milliliters of PBST washing lotions to be placed on decolorization swinging table and slowly shakes rinsing 3 times, 40
Rev/min, each 10min.
(7) protein chip is taken out in box from washing, chip is positioned in 50ml centrifuge tubes, there is proteantigen on chip
One faces outwardly, and 1000rpm centrifuges 2min, removes the liquid in each small square formation；
(8) goat anti-human IgG antibodies for taking Alexa 647 to mark, according to 1：1000 dilution proportion is in 3%BSA PBST
In, mixing, 10000rpm*10min centrifugations are shaken, supernatant is the secondary antibody diluted.
(9) 50 microlitre 1 is added in protein chip each small square formation:1000 diluted A1exa647 mark Goat anti-Human
IgG antibody, 37 DEG C are protected from light incubation 1 hour.
(10) after carefully 12 hole fences being pasted onto on protein chip being torn, chip is soaked in 10 milliliters of PBST washing lotions
In, be placed on decolorization swinging table and slowly shake, be protected from light rinsing 3 times, 40 revs/min, each 10min；
(11) and then with 10ml ddH20 is protected from light rinsing protein chip 3 times, each 10min.Protein chip is positioned over
There is the one of protein probe to face outwardly in 50ml centrifuge tubes, on chip, 1000rpm centrifuges 2min to remove remaining liquid on chip
Body；
(12) protein chip scanning is carried out with chip scanner.The setting of parameter is as follows：The a length of 635nm of excitation light wave, light
Electric multiplier detectors (PMT) are set as 650, and power supply (Power) is set as 90, and scanning element is set as 5 μm.
4. result：
113 SSc related auto-antibodies target antigens are filtered out through analysis determination, details are shown in Table 1.
Table 1 prepares the albumen of SSc related auto-antibodies target antigen protein chips
Testing result on 2 SSc related auto-antibodies target antigen chips of table
Then, anti-to itself of SSc merging related complications (such as ILD, PAH) and SSc nonjoinder related complication patients
Body positive rate has carried out analysis and has compared, it is found that merge PAH groups in SSc has significant difference compared with SSc nonjoinder PAH groups
Point, it is as a result as follows：
Subgroup analysis result on 3 SSc related auto-antibodies target antigen chips of table
Table 1 is to determine to filter out 113 SSc related auto-antibodies target antigens through analysis, and table 2 is 113 SSc correlations itself
Positive rate difference of the antibody target antigen in SSc experimental groups and control group, table 3 are that 113 SSc related auto-antibodies target antigens exist
SSc merges the positive rate difference in PAH groups and SSc nonjoinder PAH groups.Result of study shows that anti-ADAMTSL4 antibody is closed in SSc
And the positive rate in PAH patient is 14.9%, the 2.88% (P of positive rate being significantly higher than in SSc nonjoinder patients with pulmonary hypertension
=3.46x 10-5), result difference has conspicuousness, can be used for the possibility for predicting that PAH occurs in SSc.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (10)
1. the adam protein 4 with 1 type thrombospondin motif, i.e. ADAMTSL4 or its segment are being made
It is ready for use on the purposes in the reagent of pulmonary hypertension possibility in forecasting system sclerosis.
2. purposes as described in claim 1, which is characterized in that the diagnosis includes：It measures to be obtained from and systemic sclerosis is presented
Merge the level of the reactive antibody in the biological sample of the patient of pulmonary hypertension to ADAMTSL4 or its segment；Optionally
Ground,
With the level of antibody in the contrasting data biological sample, wherein relative to the contrasting data, in the sample
The possibility for suffering from systemic sclerosis merging pulmonary hypertension is shown to the detectably raising that ADAMTSL4 is reactive antibody
Property.
3. purposes as claimed in claim 1 or 2, wherein the biological sample is blood serum sample.
4. purposes as claimed in claim 1 or 2, wherein the level of ADAMTSL4 antibody is measured by following steps, is wrapped
It includes：
A. the biological sample from patient is made to be contacted with ADAMTSL4 or its segment；
B. existing in the biological sample to form antibody-protein complex between antibody and ADAMTSL4 or its segment；
C. it washs to remove any unbonded antibody；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. it washs to remove any unbonded labeled detection antibody；With
F. the marker of the detection antibody is converted to detectable signal；The presence of wherein detectable signal shows the patient
In there are anti-ADAMTSL4 antibody.
5. purposes as claimed in claim 4, wherein the ADAMTSL4 or its segment deposition are fixed on solid support
On.
6. purposes as claimed in claim 5, wherein the support is the form of latex pearl, porous flat plate or film item.
7. purposes as claimed in claim 4, wherein the detection antibody is by being covalently attached to enzyme, having fluorescent chemicals
Metal marker or marker with chemiluminescence compound mark.
8. a kind of diagnostic kit for detecting and/or quantifying biological sample moderate resistance ADAMTSL4 antibody, including：A kind of solid phase
Support, wherein the ADAMTSL4 or its segment deposition are fixed on solid support, and the anti-ADAMTSL4 is anti-
Biomarker of the body as pulmonary hypertension possibility in forecasting system sclerosis.
9. diagnostic kit as claimed in claim 8, which is characterized in that further include labeled and to carrying out biological sample
Antibody be reactive detection antibody.
10. diagnostic kit as claimed in claim 8, which is characterized in that the support be latex pearl, porous flat plate or
The form of film item.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070224600A1 (en) * | 2006-03-24 | 2007-09-27 | Bruce Wang | Methods for diagnosing and treating kidney and colorectal cancer |
| CN106810606A (en) * | 2015-11-27 | 2017-06-09 | 西藏自治区人民医院 | A kind of preparation and application of Apolipoprotein C-III antigen polypeptide and its polyclonal antibody |
-
2018
- 2018-06-04 CN CN201810564668.7A patent/CN108776215B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070224600A1 (en) * | 2006-03-24 | 2007-09-27 | Bruce Wang | Methods for diagnosing and treating kidney and colorectal cancer |
| CN106810606A (en) * | 2015-11-27 | 2017-06-09 | 西藏自治区人民医院 | A kind of preparation and application of Apolipoprotein C-III antigen polypeptide and its polyclonal antibody |
Non-Patent Citations (1)
| Title |
|---|
| LUIS A. R. GABRIEL ET AL.: "ADAMTSL4, a Secreted Glycoprotein Widely Distributed in the Eye, Binds Fibrillin-1 Microfibrils and Accelerates Microfibril Biogenesis", 《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》 * |
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