CN108872591A - The biomarker and application thereof of juvenile dermatomyositis detection - Google Patents
The biomarker and application thereof of juvenile dermatomyositis detection Download PDFInfo
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Abstract
The invention discloses LIM Zinc finger domain albumen 1, i.e. LIMS1 or its segment are preparing the purposes in the reagent for diagnosing juvenile dermatomyositis (JDM).The present invention carries out the screening of polymyositis/dermatomyositis (PM/DM) related auto-antibodies target antigen using high-density protein chip technology, determines 91 PM/DM related auto-antibodies.Target antigen building preparation PM/DM related auto-antibodies target antigen chip is corresponded to using 91 PM/DM related auto-antibodies, carries out clinical enlarged sample verifying.Result of study show anti-LIMS1 (LIM Zinc finger domain albumen 1) antibody JDM group positive rate be 31.4%, be higher than 9.0% (P=2.4 × 10 of disease and healthy control group positive rate‑4), meanwhile, higher than 8.6% (P=3.3 × 10 of positive rate of APM/ADM group‑4), result difference all has conspicuousness.Show that anti-LIMS1 antibody can be used as diagnosis and the detection marker of juvenile dermatomyositis.
Description
Technical field
The invention belongs to field of biological detection, and in particular to the biomarker and application thereof of juvenile dermatomyositis detection.
Background technique
Idiopathic inflammatory myopathies (idiopathic inflammatory myopathies, IIMs) are with proximal end symmetry
The different substantiality disease that myasthenia and multiple organ involvement are characterized, mainly includes polymyositis (polymyositis, PM), musculus cutaneus
Inflammation (dermatomyositis, DM), immune-mediated gangrenosum acne myopathy (immune-mediated necrotizing
Myopathy, IMNM), sporadic inclusion body myositis (sporadic inclusion body myositis, sIBM) and childhood
The clinical subtypes such as inflammatory myopathies (juvenile idiopathic myositis, JIM).PM/DM is a kind of striated muscle non-ization
The autoimmune disease of purulence inflammatory myopathy.Its clinical characters is that muscle enzymes increase, and electromyogram changes in muscle-derived, with limb
There is inflammation in the muscular tissues such as band flesh, cervical muscle and pharynx flesh, denaturation changes, and lead to symmetry myasthenia and a degree of amyotrophia,
Finally involving multiple systems and organ leads to death.This disease can occur at any age, be in bimodal pattern, at children 5-14 years old
Respectively there is a peak with adult 45-60 years old.In JIM hypotype with juvenile dermatomyositis (juvenile dermatomyositis,
JDM) most commonly seen.Growing up, PM/DM (APM/ADM) is common with female patient, and male to female ratio is about 1：2.
Autoantibody refers to the antibody for autologous tissue, organ, cell and cell component, is human body fluid immune system
The externally invasion of defence pathogen and the internal important weapon for keeping body growth balance.Autoantibodies marker is certainly
It is generated in the pathogenic process of body immunological disease, and often there is typical clinical manifestations and any laboratory or iconography in disease
Occurred before even more than 10 years detection abnormal preceding several years.Evidence suggests the morbidity of PM/DM is related with autoimmunity：(1) pathology
There is T cell, macrophage, Dendritic Cells, B cell and thick liquid cell in the musculature of Microscopic examination showed patient；(2) blood
There is the autoantibody for nucleus cytosolic fraction in clear detection discovery PM/DM patient.Disease is examined according to autoantibody
Autoantibody related with myositis, is divided into two classes by disconnected value：Myositis-specific antibodies (myositis-specific
Autoantibodies, MSAs) and myositis correlation antibody (myositis-associated autoantibodies,
MAAs).Report that MSAs has anti-ARS antibody (anti-Jo-1, PL-7, PL-12, EJ, OJ, KS, YRS, Zo antibody), anti-Mi-2 at present
Antibody, anti-MDA5 antibody, anti-TIF1 antibody, anti-NXP2 antibody, anti-SAE antibody, anti-SRP antibody, anti-HMGCR antibody, anti-cN1A
Antibody；MAAs have anti-PM-Scl antibody, anti-Ku antibody, anti-SSA antibody, anti-Ro50 antibody, anti-U1-RNP antibody, anti-SSB antibody,
AECA.MSAs can be related to specific clinical subtype, helps to predict complication, assists the diagnosis of myositis, Index for diagnosis and mentions
Show the correct therapeutic strategy of selection of clinical.
A kind of chronic auto-immune inflammatory myopathy that childhood that JDM is occurs, with striated muscle and skin purulent inflammation
For main feature, clinical manifestation is muscle weakness proximal and various fash, and the internal organs such as alimentary canal and lung can also be involved.Foreign countries' report
JDM disease incidence is 2-4 people/million children, and the onset age is mostly at 5-14 years old.This disease etiology unknown, it is now recognized that by inheritance susceptible
Property and the morbidity of environmental factor (as infect) collective effect.The appearance sequence and disease incidence of the clinical manifestation of JDM and ADM have certain
Difference.Children are similar to adult symptom, but often onset is anxious by children, and adult is mostly concealment morbidity.Infant usually first has skin disease
Become, muscle weakness proximal then occurs, constitutional symptom then occur, be frequently accompanied by fever.Atopy calcium deposition is in children
Disease incidence is common in end-stage disease compared with adult's height.JDM it may also occur that internal organ similar with ADM involvement performance, but more tend to have
On the one hand gastrointestinal disease, the on the one hand gastrointestinal ulceration due to caused by vasculitis, bleeding or perforation weaken with smooth muscle function
It is related.Heart involves rare compared with adult, and part infant can also have hydropericardium and pleural effusion, and electrocardiogram may occur in which conduction resistance
Stagnant equal change.In the past, the prognosis of JDM is excessively poor, and case fatality rate accounts for the 1/3 of all infants, separately has 1/3 infant to leave residual throughout one's life
Disease.In recent years, as the understanding to JDM is more and more deep, treatment means are increasing, and the case fatality rate of JDM declines to a great extent to 2%
~3%, prognosis is also significantly improved.More and more clinicians tend to detect using atraumatic, as muscle nuclear-magnetism is swept
It retouches and replaces electromyogram and Muscle biospy.It is noted that in MSAs research, the new development of acquirement provides for the diagnosis of JDM
Great help.However, autoantibody diagnostic value relevant to JDM reported at present is relatively low, new marker is found
It is still the emphasis and clinic urgent problem to be solved of research.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of LIM Zinc finger domain albumen 1, i.e. LIMS1 or its segment exist
Prepare the purposes in the reagent for diagnosing juvenile dermatomyositis (JDM).
In one embodiment of the invention, described diagnose includes：Measurement is obtained from the patient's that juvenile dermatomyositis is presented
To the level of the reactive antibody of LIMS1 or its segment in biological sample；Optionally,
Number is compareed with adult polymyositis/dermatomyositis contrasting data, other autoimmunity disease contrasting datas and Healthy People
According to the level of antibody in the biological sample, wherein be anti-to LIMS1 in the sample relative to the contrasting data
The detectably raising of the antibody of answering property shows a possibility that suffering from juvenile dermatomyositis.
Wherein, the biological sample is blood serum sample.
Wherein, the level of anti-LIMS1 antibody is measured by following steps, including：
Contact the biological sample from patient with LIMS1 or its segment；
B. antibody-protein complex is formed between existing antibody and LIMS1 or its segment in the biological sample；
C. washing is to remove any unbonded antibody；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. washing is to remove any unbonded labeled detection antibody；With
F. detectable signal is converted by the marker of the detection antibody；Wherein the presence of detectable signal shows described
There are anti-LIMS1 antibody in patient.
Wherein, the LIMS1 or its segment are deposited or are fixed on solid support.
Wherein, the solid 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 for detecting and/or quantifying the diagnostic kit of anti-LIMS1 antibody in biological sample, packet
It includes：A kind of solid support, wherein the LIMS1 or its segment deposition are fixed on solid support, and described is anti-
Biomarker of the LIMS1 autoantibody as diagnosis juvenile dermatomyositis.
In one embodiment of the invention, the diagnostic kit further includes labeled and to from biology
The antibody of sample is reactive detection antibody.
Wherein, the solid support is the form of latex pearl, porous flat plate or film item.
The present invention using high-density protein chip technology to 40 PM/DM patients, 30 autoimmunity diseases control patients and
20 normal healthy controls carry out the screening of PM/DM related auto-antibodies target antigen, for statistical analysis to screening results, determine 91
PM/DM related auto-antibodies.Then target antigen building preparation PM/DM correlation is corresponded to certainly using 91 PM/DM related auto-antibodies
Body antibody target antigen protein chip, to 100 APM, 262 ADM, 35 JDM, 200 disease control patients (including 40
RA, 40 SLE, 40 SS, 40 SSc, 40 chronic diseases) and the serum of 100 normal healthy controls carry out clinical enlarged sample
Verifying.Result of study shows that anti-LIMS1 antibody in the positive rate of JDM group is 31.4%, and it is positive to be higher than disease and healthy control group
9.0% (P=2.4 × 10 of rate-4), meanwhile, higher than 8.6% (P=3.3 × 10 of positive rate of APM/ADM group-4), result difference is equal
With conspicuousness.Show that anti-LIMS1 antibody can be used as diagnosis and the detection marker of juvenile dermatomyositis.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
The doctor that 437 PM/DM patients that this research is included in support from BJ Union Hospital and sector fund project
Institute.Wherein male patient 131, female patient 306, the range of age 2-79 years old, average age (44.07 ± 16.57) year.Institute
There is PM/DM patient to meet the diagnostic criteria of Bohan/Peter in 1975, and except other autoimmunity diseases, as rheumatoid is closed
Save scorching (rheumatoid arthritis, RA), systemic loupus erythematosus (systemic lupus erythematosus,
SLE), Sjogren syndrome (Syndrome, SS), systemic sclerosis (systemic sclerosis, SSc)
Deng.Disease control group 220, including 46 RA, inclusion criteria：ACR classification diagnosis standard in 1987；47 SLE enter a group mark
It is quasi-：ACR classification diagnosis standard in 1997；47 SS, inclusion criteria：AECG classification diagnosis standard；50 SSc, inclusion criteria：
SSc classification diagnosis standard in 2013；40 other chronic diseases controls.Healthy control group 120, derive from BJ Union Hospital
Medical center.Sample strictly is left and taken by proteomics research requirement, acquires peripheric venous blood, is divided after serum is separated in 2 hours
Dress, -80 DEG C freeze it is spare.
1 high-density protein cDNA microarray PM/DM related auto-antibodies of embodiment
All proteantigen N-terminals have the GST label for protein purification on high-density protein chip.Make first
With rabbit-anti GST monoclonal antibody and chip protein hybridization proofing chip quality.Then 40 PM/DM patients serums, 30 are chosen
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 data analyze and identify candidate PM/DM correlation autoantigen.
The extraction of 1.1 high-density protein chip scanning data
High-density protein chip scanning is carried out using GenePix 4000B chip scanner, obtains tiff format grayscale image
As after, according to the operation manual of 6.0 software 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 and extracts, while the artificial letter for checking all protein sites on chip one by one
The offset of the point position of protein signal caused by the human factors such as impurity, scratch and size is avoided in number intensity, manual setting, last complete
At the extraction of proteantigen point signal strengths all on every high-density protein chip, and data file is generated, it is subsequent further
Analysis uses.
1.2 high-density protein chips detect quality evaluation
Gray level image after hybridizing 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.
1.3 high-density protein chips detect autoantibodies
Serum specimen for the detection of high-density protein chip includes PM/DM patient 40, and autoimmunity disease compares patient
30, normal control 20.Concrete operation step is as follows：
(1) after the high-density protein chip for saving -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 specimen according to 1：In lml 3%BSA PBST, concussion mixes 1000 dilution proportion,
10000rpm*lOmin centrifugation, supernatant are the serum diluted.By protein chip after being taken out in confining liquid, if albumen core
There is bubble on piece surface, then is drawn with lml sample loading gun and be incubated for sealing liquid in box and rinse from top, with blotting paper by extra closing
Liquid blots as far 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, slowly covers slide closing, is avoided generating bubble, is incubated at room temperature 1h；
(3) protein chip being incubated for is placed in 10ml PBST washing lotion, carefully removes coverslip, chip, which is put into, washes box
The middle PBST 40rpm with room temperature is rinsed 3 times, 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, concussion mixes, and 10000rpm*10min centrifugation, supernatant is the secondary antibody diluted.Protein chip is taken out in box from washing, if
Chip surface has bubble, then is drawn with 1ml sample loading gun and be incubated for liquid in box and rinse from top, it is extra to be blotted with blotting paper from side
Washing lotion, be placed in wet box.The 150 μ l fluorescence secondary antibody diluted is added on chip, slide closing is slowly covered, avoids generating
Bubble, room temperature, which is protected from light, is incubated for secondary antibody 1h；
(5) protein chip being incubated for is placed in PBST washing lotion, carefully removes coverslip, chip is put into wash in box and use
PBST 40rpm is protected from light rinsing 3 times, each 10min；
(6) 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 tube, have antigen protein one is put on chip and is faced outwardly, 1000rpm
It is centrifuged 2min, dries ddH remaining on chip20；
(8) GenePix 4000B chip scanner scans protein chip, and identical scanning is all arranged after the completion of hybridization every time
Parameter scanning chip.
The determination of 1.4PM/DM related auto-antibodies target antigen
By 6.0 software collection of GenePix Pro to every high-density protein chip on all protein sites signal strength
Information imports in Excel table.With the foreground signal intensity (F635median) of each protein site divided by being carried on the back around the protein site
Signal value of the scape signal strength (B635median) as the protein site.That is Iij=F635median/B635median (Iij generation
The signal value of protein i point 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
The signal strength of protein site on the chip.When the average value >=2, it is believed that the protein site is the positive.Then every part of blood is counted
The information of positive rate is immunoreacted with proteantigen each on high-density protein chip clearly.Using high-throughput chip biological information point
Common genetic chip significance analysis (SAM) method is to PM/DM patient group, autoimmunity disease control group and normal in analysis technology
Control group carries out data analysis.
1.5 result
91 total through the determining PM/DM associated target antigens of analysis, these target antigens will bring PM/DM correlation into and itself resist
The small chip preparation of body target antigen, see Table 1 for details.
Table 1 prepares the albumen of PM/DM related auto-antibodies target antigen protein chip
The building of 2 PM/DM related auto-antibodies target antigen protein-chip of embodiment and serum screening are verified
Serum of Patients With Autoimmune Diseases is due to the difference of genetic background and the difference of environmental impact factor, autoimmunity disease of the same race
Generated autoantibody Species differences are larger between different individual patients.Use the progress of high-density protein chip small to confirm
The value that diagnoses to all ages and classes layer PM/DM patient of PM/DM related auto-antibodies that screening sample goes out, this research is by high density egg
The PM/DM related auto-antibodies target antigen that white cDNA microarray goes out is prepared into PM/DM related auto-antibodies target antigen protein-chip
Carry out clinical enlarged sample verifying.Big Clinical Samples verifying inspection is carried out for PM/DM related auto-antibodies target antigen protein chip
The serum specimen of survey includes APM/ADM 362, wherein APM 100, and ADM 262, JDM 35, disease control patient
200, normal healthy controls 100.Specific step is as follows：
(1) standing 30min (avoids condensing after taking out the PM/DM related auto-antibodies target antigen protein chip 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 in each fence containing 3%BSA is added
In battle array, closed 1 hour at 37 DEG C；
(3) protein chip is placed in 50ml centrifuge tube, there is the one of proteantigen to face outwardly on chip, 1000rpm
2min is centrifuged to remove the liquid in each small square matrix；
(4) take 1 μ l serum according to 1：In lml 3%BSA PBST, concussion mixes 1000 dilution proportion,
10000rpm*10min centrifugation, supernatant are the serum diluted；
(5) it is separately added into what 50 μ l had diluted in each small square matrix of PM/DM related auto-antibodies target antigen protein chip
Serum 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 placed in 50ml centrifuge tube, there is proteantigen on chip
One faces outwardly, and 1000rpm is centrifuged 2min, and centrifugation removes the liquid in each small square matrix；
(8) goat anti-human IgG antibodies for taking Alexa 647 to mark, according to 1：1000 dilution proportion is in 3%BSA PBST
In, concussion mixes, and 10000rpm*10min centrifugation, supernatant is the secondary antibody diluted；
(9) 50 microlitre 1 is added in each small square matrix of protein chip:1000 diluted A1exa647 mark Goat anti-Human
IgG antibody, 37 DEG C are protected from light incubation 1 hour；
(10) after carefully 14 hole fences being pasted 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) 10ml ddH is then used20 is protected from light rinsing protein chip 3 times, each 10min.Protein chip is placed in
There is the one of protein probe to face outwardly in 50ml centrifuge tube, on chip, 1000rpm is centrifuged 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.
By 6.0 software collection of GenePix Pro to every protein chip on the signal strength informations of all protein sites lead
Enter in Excel table.With the foreground signal intensity (F635median) of each protein site divided by the protein site ambient background signal
Signal value of the intensity (B635median) as the protein site.That is (Iij is represented Iij=F635median-B635median
The signal value of protein i point in block j).The signal value Iij of proteantigen protein site more levels off to 0, illustrates in serum
Corresponding autoantibody content is lower, which is less susceptible to be detected by chip.Iij signal value is higher to be illustrated in serum specimen
The ability of the combination target antigen protein of the autoantibody is stronger.The average value of different two signal strengths of the same albumen is made
For the signal strength of the protein site on the chip.Calculate separately 100 each albumen of healthy control group being included in this research
The average signal strength Iij (Averagenormal) and Std of point target antigen, Iij (Averagenormal)+3Std is used as should
The positive reaction Cutoff value of protein site.Then each study group's serum and PM/DM related auto-antibodies target antigen albumen core are counted
The positive rate information of each proteantigen immune response of on piece.Autoantibody markers sun between carrying out different groups using Chi-square Test
The comparison of property rate, works as P<When 0.01, it is believed that difference has conspicuousness.
91 kinds of target antigens are detailed in table to the testing result of APM/ADM and JDM on PM/DM related auto-antibodies target antigen chip
2。
Testing result of the 2 PM/DM related auto-antibodies target antigen of table in all ages and classes layer PM/DM
Result of study shows that anti-LIMS1 (LIM Zinc finger domain albumen 1) antibody in the positive rate of JDM group is 31.4%, high
In 9.0% (P=2.4 × 10 of disease and healthy control group positive rate-4), meanwhile, higher than the 8.6% (P of positive rate of APM/ADM group
=3.3 × 10-4), result difference all has conspicuousness.Show that anti-LIMS1 antibody can be used as the diagnosis and detection of juvenile dermatomyositis
Marker.
Claims (10)
1.LIM Zinc finger domain albumen 1, i.e. LIMS1 or its segment are preparing the use in the reagent for diagnosing juvenile dermatomyositis
On the way.
2. purposes as described in claim 1, which is characterized in that the diagnosis includes：Measurement, which is obtained from, is presented juvenile dermatomyositis
To the level of the reactive antibody of LIMS1 or its segment in the biological sample of patient；Optionally,
With adult polymyositis/dermatomyositis contrasting data, other autoimmunity disease contrasting datas and Healthy People contrasting data ratio
The level of antibody in the biological sample, wherein be reactivity to LIMS1 in the sample relative to the contrasting data
The detectably raising of antibody show a possibility that suffering from juvenile dermatomyositis.
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 anti-LIMS1 antibody is measured by following steps, including：
Contact the biological sample from patient with LIMS1 or its segment；
B. antibody-protein complex is formed between existing antibody and LIMS1 or its segment in the biological sample；
C. washing is to remove any unbonded antibody；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. washing is to remove any unbonded labeled detection antibody；With
F. detectable signal is converted by the marker of the detection antibody；Wherein the presence of detectable signal shows the patient
In there are anti-LIMS1 antibody.
5. purposes as claimed in claim 4, wherein the LIMS1 or its segment deposition are fixed on solid support.
6. purposes as claimed in claim 5, wherein the solid support is latex pearl, porous flat plate or film item
Form.
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. it is a kind of for detect and/or quantitative biological sample in anti-LIMS1 antibody diagnostic kit, including：A kind of solid phase branch
Hold object, wherein the LIMS1 or its segment deposition are fixed on solid support, and the anti-LIMS1 autoantibody is made
For the biomarker for diagnosing juvenile dermatomyositis.
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 solid support is latex pearl, porous flat
The form of plate or film item.
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