CN104777300B - A kind of biomarker of behcets disease detection and application thereof - Google Patents

Abstract

The present invention provides phosphatase (CTDP1) or its fragment purposes in preparing the reagent for diagnosing behcets disease of rna plymerase ii subunit A C-terminal domains。The CTDP1 of the present invention or its fragment positive rate in BD patient are significantly higher than normal control, can detect that anti-CTDP1 antibody, it was demonstrated that CTDP1 or its fragment can be used as the detection mark of BD in Western blot the result display BD patients serum。

Description

A kind of biomarker of behcets disease detection and application thereof

Technical field

The invention belongs to field of biological detection, be specifically related to biomarker of a kind of behcets disease detection and application thereof。

Background technology

Behcets disease (Behcet'sdisease, BD), also known as behcet syndrome (Behcetsyndrome, BS), is a kind of etiology unknown, high incidence, recurrent, big and small vessel inflammation the ubiquitous system autoimmune disease deposited。Nineteen thirty-seven HulusiBehcet describes oral cavity and three patients of genital ulcer, hypopyon and uveitis complexity triad shape first。Research subsequently shows that this disease is that a kind of multisystem is got involved, the Autoimmune diseases that complicated clinical manifestation is changeable。Patient is usually present oral cavity repeatedly and genital ulcer, arthritis, uveitis, dermatosis, thrombophlebitis and gastrointestinal tract and central nervous system gets involved。BD is found in the whole world, and mainly it is apt to occur in the Middle East between north latitude 30 degree to 45 degree and mediterranean country, and rare and American continent, Oceania and areas to the south of the Sahara, Africa, because its distribution is consistent with ancient " Silk Road ", therefore be also called " Silk Road disease ", the obvious region of this Disease Distribution shows that this sick and specific tumor susceptibility gene is relevant。

It is 16-50 year that Behcet disease sends out well the age, with oral ulcer, genital ulcer, ophthalmia, and then causes the serious consequence principal character such as infection, blind, aneurysm。Scholars is through further investigation for many years, the research in BD field achieves many important achievements, previously think that BD is a kind of rare autoimmune disease, recently as the deterioration of autoimmune disease popularization of knowledge, the raising of clinician's cognitive ability and environmental factors, diagnose the patient for BD and be continuously increased。The morbidity of Behcet disease is inherited genetic factors and the coefficient result of environmental factors。He result of investigation display BD is at Hesperian sickness rate far below Asian countries, and the sickness rate such as U.S. BD is 8.6/ million, Britain is 0.64/ million, Spain is 6.4/ million, Germany is 4.2/ million, France is 7.2/ million。And the sickness rate of Turkey BD be 20-421/ million, Iran be 80/,000,000, Israel be 1,20/,000,000。In inherited genetic factors, HLAB51 is considered as the closest with BD relation, and MHC I chain related gene A (MIC-A) and HLA-B51 exists significant linkage disequilibrium。Up-to-date research data shows that BD patient HLA-B51/B5 and clinical manifestation are closely related。Research shows, China Behcet disease patient has the genetic characteristics of self, and BD is regional apparently higher than other at the sickness rate of China, for 14 times of area, America sickness rate。The pathogenesis of BD is so far still without illustrating completely。Although this disease has genetic predisposition, but environmental factors and dysimmunity also function to pivotal role in morbidity。In environmental factors, various pathogenic infections etc. make immunity of organism tolerance disintegrate by molecular simulation mechanism, cause that T cells with antigenic specificity activation produces abnormal immune reaction and causes body injury, Behcet disease finds the Th1 relevant cell factor raised in the patient, such as IFN-γ, IL-12 and tumor necrosis factor (TNF)-α。

The modal clinical manifestation of Behcet disease is that oral ulcer repeatedly accounts for, and this clinical symptoms may alternatively appear in the BD patient of 98%, and lists in the diagnosis guide of Behcet disease。Oral ulcer is often Zao than other clinical manifestations repeatedly occurs, but this symptom may also appear in other diseases patient, and is not the specific clinical symptoms of Behcet disease。Genital ulcer is also the common sympton of BD, usually occurs in males scrotum, and rare on glans penis。And Behcet disease patient is different from Reiter syndrome and std patient, BD patient is also not accompanied by urethritis or dysuria。For female patient, ulcer often simultaneously occurs at big nympha。It is also the common clinical manifestation of Behcet disease that eyes are got involved, and incidence rate is up to 30%-70%, and often affects patient's vision, and the patient of 25% even results in blind serious consequence。Skin lesion reaches 38-99% in the incidence rate of BD patient, and neurological involvement is 5-10%, and affected persons's often mortality rate is higher。The vascular injury of Behcet disease patient often adds up arteriovenous simultaneously, and vein is impaired more common。Behcet disease patient's gastrointestinal tract incidence rate of getting involved is 3-26%, including anorexia, vomiting, dyspepsia, diarrhoea, stomachache。Acupuncture reaction is the common test being clinically used for diagnosis BD patient, and it is the reaction of skin unspecific allergic substantially, and namely after acupuncture, 12-48h starts red grouper pimple grain of rice size occur, then develops into vesicle, pustule and incrustation, and about 1-2 week disappears。Acupuncture reaction positive rate 57.9%-70% in Behcet disease, higher than normal population, male apparently higher than women。Having certain dependency with state of an illness activity, during state of an illness weight, positive rate is high, degree weight。

Owing to Behcet disease lacks specific clinical manifestation, the research of Behcet disease blood serum designated object is deepened continuously by scholars in recent years。AECA is the autoantibody that Behcet disease patient is common, and it is at the positive rate 13% of BD patient, but this antibody also may occur in which the autoimmune disease patient of other multiple vascular injury, at its positive rate of normal person also up to 5%。Anti-endothelial cell antigen is positive often necessarily to be contacted with the pulmonary hypertension existence of BD patient。Along with going deep into of research, researcher constantly identifies the related auto-antibodies for target antigen multiple on endotheliocyte in BD patient。Lee etc. adopt two dimensional gel electrophore-sis and mass spectrometric hyphenated technique that BD patients serum is studied, and identifying target antigen in AECA positive BD patient is α Enolase。Anti alpha Enolase antibody is 45% at the positive rate of BD patient, there is also more positive rate, WG (100%), RA (16.7%) other immunological diseases patients such as wegener granulomatosises (Wegener'sgranulomatosis, WG)。BD patient is studied by Cho etc., find anti-core heterogeneity nucleoprotein A2/B1 antibody (hnRNP-A2/B1), IgA type anti-core heterogeneity nucleoprotein A2/B1 antibody is 83.3% at the positive rate of BD patient, positive rate SLE patient is 13.3%, positive rate RA patient is 26.7%, the positive rate of TA patient is 30%, and the positive rate in normal person is 20%。Therefore, anti-core heterogeneity nucleoprotein A2/B1 antibody is limited to the diagnostic value of BD, and clinical meaning is little。2007, Federica etc. found anti-Sip-1 antibody in BD patient, its positive rate in BD patient, and IgG type is 20%, and IgM type is 41%, and IgA type is 17%。This antibody is rare in SLE, SSc, IBD, Uveitis Patients, but at primary angiitis patient's IgM type positive rate up to 45%。Anti-Sip-1 antibody is the mark of activated endothelial cell and inflammatory reaction, and its diagnostic value in BD patient is limited。It is BD patient's target antigen that Paola adopts Human umbilical vein endothelial cells cDNA library technology to identify RLIP-76 in endotheliocyte antibody positive BD patients serum, the positive rate of its IgG type autoantibody is 30%, this antibody is 17% at the positive rate in SS, positive rate in SLE is 25%, and the appearance of this antibody is relevant with endothelial cell damage。Yiping etc. adopt Human umbilical vein endothelial cells lysate to carry out the target antigen that two dimensional gel electrophore-sis and mass spectrometric hyphenated technique find that in BD patient inhibin (prohibitin) is BD。ELISA result shows that anti-inhibin antibody is 28% at the positive rate of BD patient, at the positive rate 6% of SLE patient。Peng adopts Human umbilical vein endothelial cells lysate to carry out two dimensional gel electrophore-sis and mass spectrometric hyphenated technique and identifies BD target antigen Heat shock protein 27 HSP27 (HSP27) in BD patient, antigen Heat shock protein 27 HSP27 antibody is at BD patient's positive rate 57%, SLE patient's positive rate 24%, RA patient's positive rate 72%, SS patient's positive rate 36%, its diagnostic value is relatively low。This research team adopts similar investigative technique to be also found that anti-ANX2L4 antibody in BD patient, is 34% at its positive rate of BD patient, is 7% SLE patient, and SS patient is 8%。As can be seen here, endotheliocyte antibody and autoantibody corresponding to associated target antigens thereof are not BD patient-specific autoantibody, the appearance of this antibody-like is relevant to the blood vessel injury of patient, may occur in which in the Serum of Patients With Autoimmune Diseases of various vascular injury, such as RA, SLE, SS, SSc etc.。

Except endotheliocyte related auto-antibodies, various countries' researcher adopts various different investigative techniques to be also found that many autoantibodys for other target antigens in BD patient。The scholars such as Okunuki adopt the two-way cohesion electrophoretic techniques of classics that the concurrent Uveitis Patients serum of BD is carried out blood serum designated object research, after Mass Spectrometric Identification, at 3 kinds of BD related auto-antibodies of BD Finding case: anti alpha-enolase S-antigen-antibody, anti-selenium associated protein (SBP) antibody, wherein anti-SBP antibody is newfound blood serum designated object, being a retina autoantigen, it is 16% at the positive rate of BD patient。The scholars such as YuLu utilize cDNA library screening BD related auto-antibodies, and research finds that the anti-kinectin antibody positive rate BD patient is up to 23%。But research subsequently finds that this antibody positive rate in other autoimmune disease pSS, SLE, MCTD and RA patient reaches 12.5% to 25%, and in addition, this antibody exists higher positive rate in tumor patient such as patients with hepatocellular carcinoma。2014, Morishima adopts the protein chip technology comprising 9000 human gene's encoding proteins that BD patients serum is carried out examination research, result of study display BD patient exists white-1 antibody of anti-claudin-3, the research specimen amount related to due to this research is not enough, still can not specify this antibody at the positive rate of BD patient and clinical value。

Blood vessel injury is the clinical manifestation that BD patient is important, and research finds there is antioxidation modified low density lipoprotein antibody BD patient。Therefore this antibody appearance and vascular endothelial cell impaired and dysfunction is closely related, take part in, BD patient, atherosclerotic pathological process occurs, play a role in BD patient vessel's damaged process。Although the vascular injury clinical manifestation that to be BD patient common, but not its specific clinical performance, therefore, antioxidation modified low density lipoprotein antibody also extensively comes across SLE, SV patient of other vascular injury, the appearance of this antibody is only relevant to specific clinical performance, and is not the specific autoantibody of BD。Bassyouni adopts ELISA method to detect Anti-C1q antibodies in BD patient, and its positive rate is 18%, and at its positive rate of BD patient of blood vessel injury up to 42%, far above the BD patient without blood vessel injury。Thrombosis is the clinical symptoms that BD patient is common, but the result of study of Aslan shows that anti-AnnexinⅤ antibody and there was no significant difference between the concurrent thrombosis group of BD and simple BD patient's group, is indicated above anti-AnnexinⅤ antibody meaning in BD patient's thrombosis less。

Except the autoantibody that research is correlated with for BD clinical diagnosis, also have scholar that the autoantibody that BD treatment is relevant is studied。IFN-α 2a is the common drug for the treatment of BD patient, but the patient of about 26.6% can produce the using dosage non-correlation of anti-IFN-α 2a antibody, the incidence rate of this antibody and IFN-α 2a in the process using this Drug therapy。Meanwhile, producing the BD patient of anti-IFN-α 2a antibody, to produce the probability of other autoantibodys also higher。

Lack specific clinical manifestation and laboratoary markers due to BD patient, the diagnosis of current BD still is based on the diagnosis of clinical experience, therefore usually extremely difficult for the diagnosis and differential diagnosis of BD clinically。BD patient can cause considerable distress if actively do not effectively not treated, have a strong impact on patients ' life quality, even causing the serious consequence such as infection, blind, aneurysm, eye, central nervous system and big vascular involvement person's prognosis are not good, have caused showing great attention to of clinical workers。BD brings huge misery to patient, along with the research of BD is deepened continuously, the active drug of various brand-new treatment BD constantly occurs, the Therapeutic mode of BD is gradually converted into the optimized choice of multiple therapeutic scheme, is gradually converted into the early treatment preventing serious consequence, improving patients ' life quality by the symptomatic treatment in late period by pasting medical help, therefore clinical early diagnosis active treatment can allow BD patient reap no little benefit, and makes the quality of life of BD patient be greatly improved in recent years。

BD pathogenesis is failed to understand, clinical early diagnosis active treatment can allow BD patient reap no little benefit。BD pathogeny is failed to understand, although have genetic predisposition, but environmental factors and dysimmunity also function to pivotal role in morbidity。Though BD patient HLA-B*51 and serum anti-endothelial cell antibody positive, not special, and therefore clinic there is no the serological index diagnosing BD reliably at present, and the diagnosis of BD is mainly by there is no specific clinical manifestation, it is necessary to differentiates with various autoimmune disease。Many BD patients can not get correctly diagnosing and treating in time。And BD lacks the index of condition assessment。These clinical Problems existing and facing challenges are all be presently required research that BD is deepened continuously progressively to solve。

Behcet disease is a kind of chronic generalized vascular inflammatory pathologies, main manifestations is recurrent oral ulceration, genital ulcer, ophthalmia and skin lesion, also the organs such as blood vessel, nervous system, digestive tract, joint, lung, kidney, epididymis can be involved, major part patient prognosis bona, eye, central nervous system and big vascular involvement person's prognosis are not good。At present owing to the diagnosis of Behcet disease lacks specificity experiments room Index for examination, the diagnosis of BD patient relies primarily on the clinical criteria formulated in 1989。Though BD patient HLA-B*51 and serum anti-endothelial cell antibody positive, not special, and these indexs also have similar positive rate other autoimmune disease patient。Therefore, the diagnosis of Behcet disease relies primarily on clinical manifestation, such as oral ulcer, genital ulcer, ophthalmia and skin lesion, but these Clinical symptoms are not special, and sometimes patient need to experience the several years even longer time these Clinical symptoms just occur in succession, therefore, owing to lacking specific clinical manifestation and specific laboratoary markers, the clinical diagnosis of Behcet disease is extremely difficult, many Behcet disease patients can not get diagnosis correct in time, and delay treatment causes irreversible damage and misery to patient。

Autoantibody refers to the general name of the antibody for individual autologous tissue, organ, cell and cell component produced by immune system。Generally, body self does not produce (or only producing low titre) antibody for autoantigen。When body is affected by infection, damage or other chemical factors, autoantigen exposure, degeneration, damage, displacement is made to cause that Immune discrimination is disorderly, immune dysfunction, when immunologic tolerance is disintegrated, body can produce the antibody for autoantigen composition, if causing autoimmune pathology damage or dysfunction, then it is called autoimmune disease。One of specific autoantibody Serological Characterization that may often be such that autoimmune disease of high titre, then autoantibody detection has become one of indispensable effective means of autoimmune disease diagnosis, and listed the diagnosis guide of various autoimmune disease in, become one of diagnostic criteria of various autoimmune disease。Autoantibody testing result is also autoimmune disease condition assessment and the efficiency index of disease prognosis judgement。

Serum antibody mark produces in the pathogenic process of autoimmune disease, and often in disease, there is typical clinical manifestations and any laboratory or occurred before iconography detection abnormal even more than 10 years front several years, autoantibody appearance in vivo is early than the appearance of autoimmune disease clinical symptoms。Existing result of study shows that autoantibody is the early sign thing of autoimmune disease。In various autoimmune Disease body, the appearance of autoantibody is often early than the appearance of patient clinical symptom, as PBC AMA-M2 in the patient can occur in any clinical symptoms of patient or other laboratory parameters for abnormal first 10 years, SLE related auto-antibodies can detect for average 9.4 years before clinical symptoms occurs in SLE patient, and the relevant antiCCP antibody of RA can detect for average 4.5 years before clinical symptoms occurs in RA patient。Owing to morbidity and the disease progression of autoimmune disease are relatively slow processes, all there is longer Patients with Subclinical in many autoimmune diseasees。It is in the autoimmune disease patient of Patients with Subclinical, abnormal without any typical clinical symptom and other Testing index of any laboratory except the autoantibody of disease association being detected。Therefore, clinic usually there will be that some autoantibody testing result of a certain proportion of Care cause is positive and patient does not have clinical symptoms or do not have any other testing result abnormal。Along with developing rapidly for the treatment of of autoimmune diseases, the early diagnosis of autoimmune disease and early treatment drastically increase quality of life and the disease prognosis of patient。Due to autoantibody can autoimmune disease patient's typical clinical symptom occur before the several years detect, therefore, autoantibody examination seems most important in the early diagnosis process of autoimmune disease, and the early diagnosis of autoimmune disease is emphasis and the focus of autoimmune disease clinic and basic research in recent years by the research of autoantibody。

Therefore autoantibodies detection contributes to early stage of autoimmune disease and correctly diagnoses。Previously due to the restriction of investigative technique, think that Behcet disease is seronegative autoimmune disease, but it is as deepening continuously of research, scholar is constantly had to find autoantibody in Behcet disease patients serum, such as AECA, Anti-C1q antibodies, anti-Heteronuclear ribonucleoprotein A2/B1 antibody, anti-carboxyl terminal subunit SIP1 antibody and anti-kinectin antibody etc.。Thus can see Behcet disease patient non-real seronegative autoimmune disease, but due to the restriction of the past investigative technique, not yet find valuable Behcet disease serum diagnosis mark。In these autoantibodys, clinical practice is in the blood serum designated object only AECA of BD auxiliary diagnosis, but it is as going deep into of various clinical disease research, find that AECA is not the specific antibody of BD gradually, it is widely present in the various autoimmune diseases such as ANCA relevant blood vessel inflammation, systemic lupus erythematosus (sle), systemic sclerosis, especially in the patient of vasculitic damage, the positive rate of AECA is up to 50%。Even if the BD patient that therefore a lot of AECAs are positive still can not get early diagnosis, the BD patient diagnosis saying nothing of the AECA up to more than 50% negative is more difficult。

The early lesion of Behcet disease typically belongs to clinical treatment reversible refunding or part reversible refunding, and therapeutic effect is better；And later stage or whole latter stage belong to the clinical treatment irreversible refunding, disabling lethal, not good after more, consequence is serious。Along with scholars is to deepening continuously that BD studies, many new medicines are constantly used for the treatment of Behcet disease and achieve good effect, cause the new round revolution of BD treatment。Only constantly pay attention to the early stage understanding early diagnosis and therapy of BD, the disability rate of behcets disease and mortality rate just can be made to improve gradually。But, research confirms that current various means are only capable of making the BD patient of part be diagnosed and treated timely, and only early stage treatment in time just can make BD conditions of patients be eased, and effectively stops disease progression, it is prevented that disable, improves the quality of living。The early stage of BD correctly diagnoses in time and seems most important in the diagnosis and treatment process of BD。But BD incidence of occult, current detection means is difficult to correct in time early diagnosis BD, and the BD patient of current clinical diagnosis often blood vessel and vision have begun to impaired, misses optimal treatment period。Therefore, the early diagnosis of BD has become the important bottleneck of BD central aspects, seriously governs the raising of BD treatment level。Owing to the treatment of BD patient is different from other diseases, and incidence rate that ANCA relevant blood vessel inflammation disease is in crowd is higher, at present that the pathogenesis of BD is also indefinite, the clinical manifestation of BD patient is extremely complex and not special, therefore, clinic diagnosis is wanted timely entirely accurate BD patient is distinguished from this kind of crowd and seem very difficult but extremely important。Along with now the understanding of primary disease being improved, it has been found that existing means have a lot of limitation (such as acupuncture reaction test and AECA) for the diagnosis and treatment of BD。Current study show that the diagnosis of BD seems still very imperfect if relying on clinical symptoms, image check and patients serum's autoantibody inspection to be only capable of finding and timely correct diagnosis of partial BD patient。Serum antibody mark produces in the pathogenic process of autoimmune disease, the early stage of autoimmune disease is correctly diagnosed extremely important, therefore thoroughly to accomplish BD is carried out early diagnosis and effective early treatment by practical significance, in pathogenesis, serum antibody mark on the front burner is as entirety, the antibody marlcers overall picture of further comprehensive system research BD, finding for the BD mark that Clinics and Practices is relevant is the research emphasis of current BD and problem clinical anxious to be resolved。

Summary of the invention

In order to solve the problems referred to above, the present invention provides a kind of BD biomarker, is used for detecting BD。

First, the present invention provides the phosphatase (CTDP1) of rna plymerase ii subunit AC-terminal domains or its fragment to be used for the purposes diagnosing in the reagent of behcets disease (BD) in preparation。

In one embodiment of the invention, described diagnosis includes: measure the level to the phosphatase (CTDP1) of rna plymerase ii subunit AC-terminal domains or reactive antibody of its fragment in the biological sample available from the patient presenting behcets disease symptom；Optionally,

Compare the level of antibody in described biological sample with contrasting data, wherein show the probability of behcets disease relative to the detectable raising in sample described in described contrasting data to CTDP1 being reactive antibody。

Wherein, described biological sample is blood serum sample。

In one embodiment of the invention, measuring horizontally through following steps of CTDP1 antibody, including:

A. the sample from patient is made to contact with CTDP1 or its fragment；

B. antibody-protein complex is formed between the antibody existed in the sample and CTDP1 or its fragment；

C. washing removes any unconjugated antibody；

D. add labeled and be reactive detection antibody to the antibody from sample；

E. washing removes the detection antibody of any unconjugated labelling；With

F. described label is converted into detectable signal；Wherein the existence of detectable signal shows the probability of CTDP1 in described patient。

Wherein, described CTDP1 or its fragment deposit or are fixed on solid support。

Wherein, described holder is preferably the form of latex pearl or porous flat plate。

In one embodiment of the invention, described detection antibody by being covalently attached to enzyme, there is fluorescent chemicals or the label of metal or there is the label of chemiluminescence compound carry out labelling。

Further, the present invention also provides for a kind of in identifying in the sample of patient the equipment to the existence that CTDP1 is reactive antibody or level, including:

A. at least one CTDP1 protein or its fragment；With

B. at least one solid support, wherein said CTDP1 protein or its fragment are deposited on described holder。

In an embodiment of present device, farther including detection antibody, it is reactive antibody that wherein said detection antibody is specific in the sample of described patient to CTDP1, and described detection antibody produces detectable signal。

Wherein, the sample of described patient is blood serum sample。

The present invention adopts the high flux protein chip technology comprising 47616 protein sites to 40 example BD patients, 15 example autoimmune disease comparison patients and 20 example normal controls carry out the examination of BD related auto-antibodies target antigen, screening results is carried out statistical analysis, it is determined that 36 BD related auto-antibodies。In order to confirm Sensitivity and Specificity and the clinical value of these BD related auto-antibodies, 36 BD related auto-antibodies correspondence target antigens are adopted to build preparation BD related auto-antibodies target antigen protein chip to 130 example BD patients subsequently, from exempting from disease comparison patient 103 example (including Takayasu arteritis patient 40 example, ANCA relevant blood vessel inflammation patient 40 example and SS patient 23 example) and normal control 110 example serum carries out clinical enlarged sample and verifies。Result of study shows that the anti-CTDP1 antibody [phosphatase (RNApolymeraseIIsubunitAC-terminaldomainphosphatase) of rna plymerase ii subunit AC-terminal domains] positive rate in BD patient is 23.85% (31/130) be significantly higher than autoimmune disease matched group 4.85% (5/103) (X²=15.87, P=6.79x10^-5) and normal control 6.4% (7/110) (X²=13.67, P=0.000218) in positive rate, result difference has significance。Can detect that anti-CTDP1 antibody in Westernblot the result display BD patients serum, the protein chip testing result of this mark is reliable, has reliable methodology repeatability。

Accompanying drawing explanation

Fig. 1 show BD group and oneself exempts from disease control group and Normal group average positive albumen is counted。

Fig. 2 show the typical consequence that BD patient, autoimmune disease comparison and normal control serum react on high-density protein chip。

Fig. 3 BD related auto-antibodies target antigen chip Shang Ge seminar's serum specimen and CTDP1 hybridize typical figure。

The immune-blotting method result of Fig. 4 part object of study serum and CTDP1。

Detailed description of the invention

Following example are used for illustrating the present invention, but are not limited to the scope of the present invention。

Object of study used in following embodiment and material。

Object of study derive from February, 2012 in January, 2014 BJ Union Hospital's outpatient service and be in hospital BD patient 170 example, wherein male 91 example, women 79 example, the range of age 14～66 years old, the mean age (37.1 ± 11.6) year。Enter group standard: all patients all meet the BD classification diagnosis standard that international Behcet disease committee (ISG) in 1989 is determined, other autoimmune diseasees (such as systemic lupus erythematosus (sle), inflammatory bowel, rheumatoid arthritis, diabetes, ankylosing spondylitis etc.) except all BD patients。Disease control group includes: Takayasu arteritis patient 40 example, wherein male 6 example, women 34 example, the range of age 14～52 years old, year mean age (29.4 ± 8.8)；Enter group standard: adopt the Takayasu arteritis criteria for classification of nineteen ninety U.S.'s rheumatology association。ANCA relevant blood vessel inflammation patient 40 example, wherein male 21 example, women 29 example, the range of age 17～85 years old, the mean age (54.9 ± 16.0) year, the diagnosis of all patients meets ANCA relevant blood vessel inflammation ChapelHill in 1993 diagnosis common recognition；SS patient 28 example, wherein male 0 example, women 28 example, the range of age 14～76 years old, year mean age (50.8 ± 15.2)；Normal group 130 example, derives from BJ Union Hospital's MEC, wherein male 89 example, women 41 example, the range of age 21～70 years old, year mean age (37.6 ± 9.0)；

Strictly leaving and taking sample by proteomics research requirement, gather peripheric venous blood, subpackage after separation serum in 2 hours ,-80 DEG C frozen standby。

Embodiment 1 high-density protein cDNA microarray BD related auto-antibodies

On high-density protein chip, all of proteantigen N end is all with the GST label for protein purification。First by the anti-GST monoclonal antibody of rabbit and chip protein hybridization proofing chip quality。Then choose 40 parts of BD patients serums, 15 example Serum of Patients With Autoimmune Diseases control serums and 20 example normal healthy controls serum and 75 high-density protein microarray biochip hybridization, identify the relevant autoantigen of candidate BD by signals collecting and data analysis。

1, the extraction of high-density protein chip scanning data

Adopt GenePix4000B chip scanner scanning high flux protein chip, obtain tiff format gray level image, workbook according to GenePixPro6.0 software, according to high flux chip loading dot matrix parameter, it is automatically performed the segmentation of each protein site signal pixels by software, the letter intensity of all protein sites on the artificial chip of inspection one by one simultaneously, impurity is avoided in manual setting, protein signal point position that the anthropic factors such as cut cause and the skew of size, finally complete the extraction of each antigen protein particle signal intensity on every high flux protein chip and generate data file for subsequent analysis use。

2, high-density protein chip detection quality evaluation

Use the gray level image after anti-GST antibody hybridization high-density protein chip, after completing each protein site signal intensity collection of chip, when on chip, the signal to noise ratio (SignaltoNoiseRatio, SNR) of the two of probe parallel points just thinks when being simultaneously greater than 2 that this protein site can be detected on chip。The dependency of signal intensity between ratio and the parallel point of two, each albumen of the protein spots then can being detected on computing chip。

3, high-density protein chip detection autoantibodies

Serum specimen for high-density protein chip detection includes BD patient 40 example, certainly exempts from disease comparison patient 15 example, normal control 20 example。Related data is in Table 1, and concrete operation step is as follows:

Table 1 is for the serum specimen of high-density protein chip detection

Packet	Number of cases	M-F	The range of age	Mean age
					BD	40	26/14	15-66	40.0±12.2
From exempting from disease matched group	15	1/14	32-65	45.0±9.7
					Normal group	20	12/8	23-67	41.5±11.9

4, the determination of BD related auto-antibodies target antigen

By GenePixPro6.0 software collection to every high-density protein chip on the signal strength information of all protein sites import in Excel form。By the foreground signal intensity (F635median) of each protein site divided by this protein site ambient background signal intensity (B635median) signal value as this protein site。The i.e. Iij=F635median/B635median signal value of the protein i point in blockj (Iij represent)。The signal value of proteantigen protein site more levels off to 1, illustrates that in serum, corresponding autoantibody concentrations is more low, and this antibody is less susceptible to be detected。In the more high explanation serum specimen of signal value, the ability in conjunction with target antigen protein of this autoantibody is more strong。Using the meansigma methods of different for same albumen two some signal intensitys as this protein site signal intensity on this chip。When these meansigma methods >=2, it is believed that this protein site is positive。Then every part of serum and the information of each proteantigen immunoreation positive rate on high-density protein chip are added up。Adopting gene chip significance analysis (SAM) method conventional in high flux chip biological information analysis techniques to BD patient's group, certainly exempt from disease matched group and Normal group carries out data analysis, with Q-value, < 35% enters to elect as BD candidate is correlated with autoantigen。BD patient's erythrocyte sedimentation rate increases group and directly compares with erythrocyte sedimentation rate normal group, and with Q-value, < 5% enters to elect as the relevant autoantigen of BD candidate。BD candidate autoantigens in order to ensure screening has clinical value, all selected positive rate positive rates preparing BD related auto-antibodies target antigen > 25%。

Result:

Adopt the high-density protein chip comprising 47616 protein sites that 40 parts of BD patients serums, 15 example Serum of Patients With Autoimmune Diseases control serums and 20 example normal healthy controls serum carry out screening BD related auto-antibodies。Result of study shows, all serum specimens can only react with the raw antigen and antibody specific of a few eggs poliosis on high-density protein chip, it is 244.05 ± 206.05 that the average positive albumen of BD group is counted, counting from the average positive albumen exempting from disease matched group is 261.45 ± 249.2, and it is 220.6 ± 265.4 (Fig. 1) that the average positive albumen of Normal group is counted。Protein positive reflecting point sum there was no significant difference (F=0.139, P=0.87 > 0.05) between three groups。

On the basis that each seminar specimen positive protein point confirms, adopting gene chip significance analysis (SAM) method conventional in high flux chip biological information analysis techniques to BD patient's group, certainly exempt from disease matched group and Normal group carries out data analysis, with Q-value, < 35% enters to elect as BD candidate is correlated with autoantigen。BD patient's erythrocyte sedimentation rate increases the comparison between group and erythrocyte sedimentation rate normal group, and with Q-value, < 5% enters to elect as the relevant autoantigen of BD candidate。BD candidate autoantigens in order to ensure screening has clinical value, all selected positive rates preparing BD related auto-antibodies target antigen > 25%。The BD associated target antigens confirmed by analysis amounts to 36, and these target antigens will be brought the BD little chip of associated antibodies target antigen into and prepare, and refer to table 2。BD associated antibodies typical figure such as Fig. 2 of high-density protein cDNA microarray:

The albumen of BD related auto-antibodies target antigen protein chip prepared by table 2

The structure of embodiment 2BD related auto-antibodies target antigen protein chip and serum screening checking

The difference of the Serum of Patients With Autoimmune Diseases difference due to genetic background and environmental impact factor, the produced autoantibodies rate between different individual patients differs greatly。In order to confirm to adopt high-density protein chip to carry out whether the BD related auto-antibodies that small sample filters out has clinical meaning and using value, and the Sensitivity and Specificity that these antibody are to BD patient diagnosis, the BD related auto-antibodies target antigen that high-density protein cDNA microarray is gone out by this research is prepared into BD related auto-antibodies target antigen protein chip and is carried out clinical enlarged sample checking。

The serum specimen carrying out Big Clinical Samples checking detection for BD related auto-antibodies target antigen protein chip includes BD patient 130 example, certainly exempts from disease comparison patient 103 example, normal control 110 example。Related data is in Table 3。

The serum specimen of table 3 Big Clinical Samples checking detection

The 130 example BD patients that BD related auto-antibodies target antigen protein chip is carried out, 103 examples exempt from disease comparison patient certainly and 110 example normal control testing results are analyzed。On BD related auto-antibodies target antigen chip, the testing result of 36 kinds of target antigens refers to table 4。The testing result display CTDP1 of 36 kinds of target antigens is 23.85% at the positive rate of BD group, higher than autoimmune disease matched group 4.85% (X²=15.87, P=6.79x10^-5) and normal healthy controls group 6.36% (X²=13.67, P=0.000218), result difference has significance。BD related auto-antibodies target antigen chip Shang Ge seminar's serum specimen and CTDP1 hybridize typical figure such as Fig. 3。The BD associated target antigens that all the other 35 kinds of high-density protein chips sift out is by after the detection checking of enlarged sample on little chip, and result is shown between BD group, autoimmune disease matched group and normal healthy controls group positive rate and there was no significant difference。

Analyze CTDP1 positive events on high-density protein chip further, the positive rate of CTDP1 is for being 22.5% (9/40) in BD group, positive rate at autoimmune disease matched group is 6.67% (1/15), and the positive rate in normal healthy controls group is 5.0% (1/20)。This result is closely similar with its result on little chip。

Testing result on table 4BD related auto-antibodies target antigen chip

The newfound BD autoantibody immunoblotting checking of embodiment 3

In order to verify whether the testing result that the screening of BD autoantibody target antigen chip Big Clinical Samples confirms can obtain reproducible results in other detection methods, we adopt western blotting method the most frequently used in proteomics research method that CTDP1 antibody anti-in BD patients serum is detected。The display of immune-blotting method result adopts the anti-CTDP1 antibody that the screening of BD autoantibody target antigen chip large sample confirms well can be repeated in WB detects, there is the autoantibody with CTDP1 specific reaction in BD patients serum, its reaction signal intensity is higher than autoimmune disease matched group and normal controls group。The result of immune-blotting method such as Fig. 4。

The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the technology of the present invention principle; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention。

Claims (7)

1.RNA polymerase II subunit AC-terminal domains phosphatase, i.e. CTDP1, or the purposes that its fragment is in preparing the reagent for diagnosing behcets disease, described diagnosis includes: measure in the biological sample available from the patient presenting behcets disease symptom the level that rna plymerase ii subunit AC-terminal domains phosphatase is reactive antibody。

2. purposes as claimed in claim 1, it is characterised in that also include:

The level of antibody in described biological sample is compared with contrasting data, relative to described contrasting data, CTDP1 is by described biological sample the detectable raising of the reactive antibody probability that shows behcets disease。

3. purposes as claimed in claim 1 or 2, wherein, described biological sample is blood serum sample。

4. purposes as claimed in claim 1 or 2, wherein, measuring horizontally through following steps of CTDP1 antibody, including:

A. the biological sample from patient is made to contact with CTDP1 or its fragment；

B. antibody-protein complex is formed between the antibody and CTDP1 or its fragment that exist in biological sample；

C. washing removes any unconjugated antibody；

D. add labeled and be reactive detection antibody to the antibody carrying out biological sample；

E. washing removes any unconjugated labeled described detection antibody；With

F. the label of described detection antibody is converted into detectable signal；Wherein the existence of detectable signal shows there is anti-CTDP1 antibody in described patient。

5. purposes as claimed in claim 4, wherein, described CTDP1 or its fragment are fixed on solid support。

6. purposes as claimed in claim 5, wherein, described holder is the form of latex pearl or porous flat plate。

7. purposes as claimed in claim 4, wherein, described detection antibody by being covalently attached to enzyme, there is fluorescent chemicals or the label of metal or there is the label of chemiluminescence compound carry out labelling。