CN104777300A - Biomarker for Behcet disease (BD) detection and use thereof - Google Patents

Abstract

The invention provides a use of RNA polymerase II subunit A C-terminal domain phosphatase (CTDP1) or a fragment thereof in preparation of a reagent used for diagnosing Behcet disease (BD). The positive rate of the CTDP1 or the fragment thereof in a BD patient is remarkably higher than that of normal control, a Western blot verification result displays that an anti-CTDP1 antibody can be detected in the serum of the BD patient, which proves that the CTDP1 or the fragment thereof can be used for a detection marker of BD.

Description

Biomarker that a kind of Behcet's disease detects and uses thereof

Technical field

The invention belongs to field of biological detection, be specifically related to biomarker of a kind of Behcet's disease detection and uses thereof.

Background technology

Behcet's disease (Behcet's disease, BD) also known as Behcet syndrome (Behcetsyndrome, BS), be a kind of etiology unknown, the scorching and ubiquitous system autoimmune disease of depositing of high incidence, recurrent, big and small vessel.Nineteen thirty-seven Hulusi Behcet describes three patients of oral cavity and the complicated triad shape of genital ulcer, hypopyon and uveitis 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.Usually there is oral cavity repeatedly and genital ulcer, arthritis, uveitis, cutaneous lesions, thrombophlebitis and intestines and stomach and central nervous system and get involved in patient.BD is found in the whole world, and the Middle East be mainly apt to occur between north latitude 30 degree to 45 degree and mediterranean country, and rare and American continent, Oceania and areas to the south of African the Sahara, 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 disease is relevant to specific tumor susceptibility gene.

It is 16-50 year that Behcet's disease sends out well the age, with canker sore, genital ulcer, ophthalmia, and then causes the serious consequence principal characters 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, in recent years along with autoimmune disease popularization of knowledge, the raising of clinician's cognitive ability and the deterioration of environmental factor, the patient being diagnosed as BD constantly increases.The morbidity of Behcet's disease is the coefficient result of inherent cause and environmental factor.He result of investigation display BD is at the Hesperian incidence of disease far below Asian countries, and the incidence of disease as U.S. BD is 8.6/ hundred ten thousand, Britain is 0.64/ hundred ten thousand, Spain is 6.4/ hundred ten thousand, Germany is 4.2/ hundred ten thousand, France is 7.2/ hundred ten thousand.And the incidence of disease of Turkey BD be 20-421/ hundred ten thousand, Iran is 80,/00 ten thousand, Israel is 120/ hundred ten thousand.In inherent cause, HLA B51 is considered to the closest with BD relation, and MHC I chain related gene A (MIC-A) exists significant linkage disequilibrium with HLA-B51.Up-to-date research data show BD patient HLA-B51/B5 and clinical manifestation closely related.Research shows, China Behcet's disease patient has self genetic characteristics, and BD apparently higher than other areas, is 14 times of area, the America incidence of disease at the incidence of disease of China.The pathogenesis of BD is not still illustrated so far completely.Although this disease has genetic predisposition, environmental factor and dysimmunity also play a key effect in morbidity.In environmental factor, various pathogenic infections etc. make immunity of organism tolerance disintegrate by molecular simulation mechanism, cause T cells with antigenic specificity to activate the reaction of generation abnormal immune and cause body injury, the Th1 relevant cell factor raised is found in Behcet's disease patient body, as IFN-γ, IL-12 and TNF (TNF)-α.

The modal clinical manifestation of Behcet's disease is that canker sore repeatedly accounts for, and this clinical symptoms can appear at the BD patient of 98%, and lists in the diagnosis guide of Behcet's disease.Canker sore often appearance more Zao than other clinical manifestations repeatedly, but this symptom also can appear at other diseases patient, and be not the specific clinical symptoms of Behcet's disease.Genital ulcer is also the common sympton of BD, usually occurs in males scrotum, and rare on glans penis.And Behcet's disease patient is different with std patient from Reiter syndrome, BD patient is not with urethritis or dysuria.For female patient, ulcer often occurs in large nymphae simultaneously.It is also the common clinical manifestation of Behcet's disease that eyes are got involved, and incidence up to 30%-70%, and often affects patient's vision, and the patient of 25% even causes blind serious consequence.Skin damages and reaches 38-99% in the incidence of BD patient, and neurological involvement is 5-10%, and affected persons often mortality ratio is higher.The vascular injury of Behcet's disease patient often adds up arteriovenous simultaneously, and vein is impaired more common.Behcet's disease patient's gastrointestinal tract incidence of getting involved is 3-26%, comprises apocleisis, vomiting, indigestion, diarrhoea, stomachache.Acupuncture reaction is clinical in diagnosing a common test of BD patient, and its essence is the reaction of skin unspecific allergic, and namely after acupuncture, 12-48h starts the red grouper papule occurring grain of rice size, then develops into blister, warts and incrustation, and about 1-2 week disappears.The positive rate 57.9%-70% of acupuncture reaction in Behcet's disease, higher than normal population, the male sex apparently higher than women.Have certain correlativity with state of an illness activity, when the state of an illness is heavy, positive rate is high, degree weight.

Because Behcet's disease lacks specific clinical manifestation, scholars deepens continuously to the research of Behcet's disease blood serum designated object in recent years.AECA is the common autoantibody of Behcet's disease patient, and it is at the positive rate 13% of BD patient, but the autoimmune disease patient of other multiple vascular injury also can appear in this antibody, also can reach 5% at its positive rate of normal person.The positive pulmonary hypertension that is normal and BD patient of anti-endothelial cell antigen exists and necessarily contacts.Along with going deep into of research, researcher constantly identifies the related auto-antibodies for target antigen multiple on endothelial cell in BD patient.Lee etc. adopt two dimensional gel electrophore-sis and mass spectrometric hyphenated technique to study BD patients serum, and in the positive BD patient of AECA, identify target antigen is α enolase.Anti alpha enolase antibody is 45% at the positive rate of BD patient, at wegener granulomatosis (Wegener's granulomatosis, etc. WG) also there is more positive rate, WG (100%), RA (16.7%) in other immunological diseases patient.Cho etc. study BD patient, 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, be 13.3% at the positive rate of SLE patient, be 26.7% at the positive rate of RA patient, the positive rate of TA patient is 30%, and the positive rate in normal person is 20%.Therefore, the diagnostic value of anti-core heterogeneity nucleoprotein A2/B1 antibody to BD is limited, 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%, IgM type be 41%, IgA type is 17%.This antibody is rare in SLE, SSc, IBD, Uveitis Patients, but at primary angiitis patient 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 endothelial cell 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 two dimensional gel electrophore-sis and mass spectrometric hyphenated technique finds the target antigen that inhibin (prohibitin) is BD in BD patient.It is 28% that ELISA result shows anti-inhibin antibody 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 identifies BD target antigen Heat shock protein 27 (HSP27) in BD patient, antigen Heat shock protein 27 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 lower.This research team anti-ANX2L4 antibody that adopted similar investigative technique also to find in BD patient is 34% at its positive rate of BD patient, be 7%, SS patient is 8% SLE patient.As can be seen here, endothelial cell 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 injury of blood vessel of patient, can come across the Serum of Patients With Autoimmune Diseases of various vascular injury, as RA, SLE, SS, SSc etc.

Except endothelial cell related auto-antibodies, various countries researcher many autoantibodies for other target antigens that adopted various different investigative technique also to find in BD patient.The scholars such as Okunuki adopt classical two-way cohesion electrophoretic techniques to carry out blood serum designated object research to the concurrent Uveitis Patients serum of BD, after Mass Spectrometric Identification, at BD Finding case 3 kinds of BD related auto-antibodies: anti alpha-enolase S-antigen-antibody, anti-selenium associated protein (SBP) antibody, wherein anti-SBP antibody is newfound blood serum designated object, be a retina autoantigen, its positive rate BD patient is 16%.The scholars such as Yu Lu utilize cDNA library to screen BD related auto-antibodies, and research finds that the positive rate of anti-kinectin antibody BD patient is up to 23%.But research subsequently finds that the positive rate of this antibody in other autoimmunity disease pSS, SLE, MCTD and RA patient reaches 12.5% to 25%, and in addition, this antibody exists higher positive rate in tumor patient is as patients with hepatocellular carcinoma.2014, Morishima adopts the protein chip technology comprising 9000 human gene encoding proteins to carry out examination research to BD patients serum, white-1 antibody of anti-claudin-3 is there is in result of study display BD patient, 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.

Injury of blood vessel is the important clinical manifestation of BD patient, and research finds to there is anti-oxidant modified low density lipoprotein antibody BD patient.Therefore this antibody appearance and the impaired and dysfunction of vascular endothelial cell closely related, take part in, BD patient, atherosclerotic pathologic process occurs, play a role in BD patient vessel damaged process.Although the vascular injury clinical manifestation that to be BD patient common, but not its specific clinical performance, therefore, anti-oxidant modified low density lipoprotein antibody also extensively comes across SLE, SV patient of other vascular injury, the appearance of this antibody is only show relevant to specific clinical, 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 injury of blood vessel up to 42%, far above the BD patient without injury of blood vessel.Thrombosis is the common clinical symptoms of BD patient, but the result of study of Aslan shows that anti-AnnexinⅤ antibody is between the concurrent thrombosis group of BD and simple BD patient's group and there was no significant difference, shows that anti-AnnexinⅤ antibody meaning in BD patient's thrombosis is less thus.

Except research is used for the relevant autoantibody of BD clinical diagnosis, the autoantibody also having scholar relevant to BD treatment is studied.IFN-α 2a is the common drug for the treatment of BD patient, but the patient of about 26.6% can produce anti-IFN-α 2a antibody, the incidence of this antibody and the using dosage non-correlation of IFN-α 2a in the process using this drug therapy.Meanwhile, the possibility that the BD patient producing anti-IFN-α 2a antibody produces other autoantibodies is also higher.

Because BD patient lacks specific clinical manifestation and laboratoary markers, the diagnosis of current BD is still based on the diagnosis of clinical experience, and the diagnosis and differential diagnosis therefore clinically for BD is usually very difficult.BD patient's such as not positive treatment effectively can cause considerable distress, have a strong impact on patients ' life quality, even cause the serious consequences such as infection, blind, aneurysm, eye, central nervous system and trunk affected individual prognosis are not good, have caused showing great attention to of clinical workers.BD brings huge misery to patient, deepen continuously along with to the research of BD, the active drug of various treatment BD completely newly constantly occurs, the optimum choice that the Therapeutic mode of BD changes multiple therapeutic scheme into gradually by pasting medical help, changed into the early treatment preventing serious consequence, improve patients ' life quality gradually by the symptomatic treatment in late period, therefore clinical early diagnosis active treatment can allow BD patient reap no little benefit, and the quality of life of BD patient is 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 pathogenesis is failed to understand, although have genetic predisposition, environmental factor and dysimmunity also play a key effect in morbidity.Although BD patient HLA-B*51 and serum anti-endothelial cell antibody positive, but it is not special, therefore the clinical serological index that there is no reliably diagnosis BD at present, the diagnosis of BD, mainly by there is no specific clinical manifestation, needs to differentiate with various autoimmune disease.Many BD patients can not get correct Diagnosis and Treat in time.And BD lacks the index of condition assessment.These clinical Problems existing and facing challenges are all need at present to deepen continuously to study to BD progressively to solve.

Behcet's 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, alimentary canal, joint, lung, kidney, epididymis can be involved, major part patient prognosis bona, eye, central nervous system and trunk affected individual prognosis are not good.At present because the diagnosis of Behcet's disease lacks specificity experiments room Index for examination, the clinical criteria that the diagnosis of BD patient mainly relies on formulate for 1989.Although BD patient HLA-B*51 and serum anti-endothelial cell antibody positive, not special, these indexs also have similar positive rate other autoimmune disease patient.Therefore, the diagnosis of Behcet's disease mainly relies on clinical manifestation, as canker sore, genital ulcer, ophthalmia and skin lesion, but these Clinical symptoms are not special, and sometimes patient need experience the several years even longer time just in succession there are these Clinical symptoms, therefore, owing to lacking specific clinical manifestation and specific laboratoary markers, the clinical diagnosis of Behcet's disease is very difficult, many Behcet's 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 produce low titre) antibody for autoantigen.When body is affected by infection, damage or other chemical factors, autoantigen exposure, sex change, damage, displacement is made to cause Immune discrimination disorderly, immune dysfunction, when immune tolerance is disintegrated, body can produce the antibody for autoantigen composition, if cause autoimmune pathology to damage or dysfunction, be then called autoimmune disease.The specific autoantibody of high titre is usually one of Serological Characterization of autoimmune disease, so autoantibody detection has become autoimmune disease and has diagnosed one of indispensable effective means, and listed the diagnosis guide of various autoimmune disease in, become one of diagnostic criteria of various autoimmune disease.Autoantibody testing result is also the efficiency index that autoimmune disease condition assessment and disease prognosis judge.

Serum antibody mark produces in the pathogenic process of autoimmune disease, and often in disease, there is typical clinical manifestations and any laboratory or iconography and detect the abnormal front several years and even occurred before more than 10 years, autoantibody appearance is in vivo 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 the AMA-M2 in PBC patient body 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 appears in SLE patient, and RA antiCCP antibody of being correlated with can detect for average 4.5 years before clinical symptoms appears in RA patient.Because the morbidity of autoimmune disease and disease progression are processes relatively slowly, all there is longer Patients with Subclinical in many autoimmune diseases.Be in the autoimmune disease patient of Patients with Subclinical, without any typical clinical symptom and other Testing index exceptions of any laboratory except the autoantibody of disease association being detected.Therefore, clinically usually there will be the positive and patient of some autoantibody testing result of a certain proportion of Care cause and there is no clinical symptoms or abnormal without any other testing results.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 research to autoimmune disease of autoantibody is emphasis and the focus of the clinical and fundamental research of autoimmunity disease in recent years.

Therefore autoantibodies detects the early stage correct diagnosis contributing to autoimmune disease.Previously due to the restriction of investigative technique, think that Behcet's disease is seronegative autoimmunity disease, but along with deepening continuously of research, scholar is constantly had to find autoantibody in Behcet's disease patients serum, as AECA, Anti-C1q antibodies, anti-Heteronuclear ribonucleoprotein A2/B1 antibody, anti-carboxyl terminal subunit SIP1 antibody and anti-kinectin antibody etc.Behcet's disease patient can be seen thus and the seronegative autoimmune disease of really, but due to the restriction of the past investigative technique, not yet find valuable Behcet's disease serodiagnosis mark.In these autoantibodies, clinical practice is in the blood serum designated object only AECA of BD auxiliary diagnosis, but that studies along with various clinical disease gos deep into, find that AECA is not the specific antibody of BD gradually, extensively be present in the various autoimmune diseases such as ANCA relevant blood vessel inflammation, systemic loupus erythematosus, systemic sclerosis, especially in the patient of vasculitic damage, the positive rate of AECA is up to 50%.Even if therefore the BD patient of a lot of AECA positive still can not get early diagnosis, the BD patient diagnosis said nothing of up to the AECA feminine gender of more than 50% is more difficult.

The early lesion of Behcet's disease generally belongs to clinical treatment reversible refunding or part reversible refunding, and result for the treatment of is better; And later stage or whole latter stage belong to the clinical treatment irreversible refunding, disable lethal, not good more, consequence is serious.That studies BD along with scholars deepens continuously, many new medicines constantly for Behcet's disease treatment and achieve good effect, cause BD treatment new round revolution.Only have the early stage understanding early diagnosis and therapy constantly paying attention to BD, the disability rate of Behcet's disease and mortality ratio just can be made to improve gradually.But research confirms that current various means only can make the BD patient of part obtain Diagnosis and Treat timely, only have early stage treatment in time that BD conditions of patients just can be made to be eased, effectively stop disease progression, prevent from disabling, improve the quality of living.The early stage correct diagnosis in time of BD seems most important in the diagnosis and treatment process of BD.But BD incidence of occult, current detection means is difficult to early diagnosis BD correct in time, and the BD patient of current clinical diagnosis often blood vessel and eyesight has started 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.Because the treatment of BD patient is different from other diseases, and the incidence of the scorching disease of ANCA relevant blood vessel in crowd is higher, also indefinite to the pathogenesis of BD at present, the clinical manifestation of BD patient is very complicated and not special, therefore, BD patient is distinguished from this kind of crowd in clinic diagnosis, with wanting timely entirely accurate and seem very difficult but extremely important.Along with the present understanding to this disease improves, find that existing means have a lot of limitation (as acupuncture reaction test and AECA) for the diagnosis and treatment of BD.Current research shows that the diagnosis of BD seems still very imperfect if rely on clinical symptoms, image check and the inspection of patients serum's autoantibody only can find and timely correct diagnosis of partial BD patient.Serum antibody mark produces in the pathogenic process of autoimmune disease, extremely important to the early stage correct diagnosis of autoimmunity disease, therefore thoroughly to accomplish practical significance carries out early diagnosis and effective early treatment to BD, with serum antibody mark on the front burner in pathogenesis as a whole, the antibody marlcers overall picture of further comprehensive system research BD, finds the BD mark of being correlated with for Clinics and Practices to be the research emphasis of current BD and clinical problem anxious to be resolved.

Summary of the invention

In order to solve the problem, the invention provides a kind of BD biomarker, for detecting BD.

First, the invention provides phosphatase (CTDP1) or the purposes of its fragment in the reagent for the preparation of diagnosis Behcet's disease (BD) of rna plymerase ii subunit AC-terminal domains.

In one embodiment of the invention, described diagnosis comprises: 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 Behcet's disease symptom; Optionally,

Level with antibody in the more described biological sample of contrasting data, wherein shows the possibility of Behcet's disease relative to the detectable raising being reactive antibody to CTDP1 in sample described in described contrasting data.

Wherein, described biological sample is blood serum sample.

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

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 to which and CTDP1 or its fragment;

C. washing removes any unconjugated antibody;

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

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

F. described label is converted into detectable signal; Wherein the existence of detectable signal shows the possibility 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, the label that has the label of fluorescent chemicals or metal or have a chemiluminescence compound marks.

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

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, comprise detection antibody further, it is reactive antibody that wherein said detection antibody to be specific in the sample of described patient 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 routine BD patients, 15 routine autoimmunity disease contrast patients and 20 routine normal controls carry out the examination of BD related auto-antibodies target antigen, statistical study is carried out to screening results, determines 36 BD related auto-antibodies.In order to confirm Sensitivity and Specificity and the clinical value of these BD related auto-antibodies, the corresponding target antigen of 36 BD related auto-antibodies is adopted to build preparation BD related auto-antibodies target antigen protein chip to 130 routine BD patients subsequently, from exempt from disease contrast patient 103 example (comprise aorto-arteritis patient 40 example, ANCA relevant blood vessel scorching patient 40 example and SS patient 23 routine) and the routine serum of normal control 110 carry out clinical enlarged sample checking.It is 23.85% (31/130) be significantly higher than autoimmunity disease control group 4.85% (5/103) (X that result of study shows anti-CTDP1 antibody [phosphatase (RNA polymerase II subunit A C-terminal domain phosphatase) of the rna plymerase ii subunit AC-terminal domains] positive rate in BD patient ²=15.87, P=6.79x10 ^-5) and normal control 6.4% (7/110) (X ²=13.67, P=0.000218) in positive rate, result difference has conspicuousness.Anti-CTDP1 antibody can be detected in Western blot the result display BD patients serum, the protein chip testing result of this mark is reliable, has reliable methodology repeatability.

Accompanying drawing explanation

Figure 1 shows that BD group and oneself exempt from disease control group and Normal group average positive albumen is counted.

Figure 2 shows that the typical consequence that BD patient, autoimmunity disease contrast 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 research object serum and CTDP1.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.

Research object used in following embodiment and material.

Research object derive from February, 2012 to 2014 year BJ Union Hospital's outpatient service in January and be in hospital BD patient 170 example, wherein the male sex 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 the international Behcet's disease council (ISG) in 1989 is determined, other autoimmune diseases (as systemic loupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, diabetes, ankylosing spondylitis etc.) except all BD patients.Disease control group comprises: aorto-arteritis patient 40 example, wherein the male sex 6 example, women 34 example, the range of age 14 ~ 52 years old, the mean age (29.4 ± 8.8) year; Enter group standard: the aorto-arteritis criteria for classification adopting nineteen ninety U.S.'s rheumatology association.Scorching patient 40 example of ANCA relevant blood vessel, the wherein male sex 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 scorching Chapel Hill diagnosis common recognition in 1993; SS patient 28 example, wherein the male sex 0 example, women 28 example, the range of age 14 ~ 76 years old, the mean age (50.8 ± 15.2) year; Normal group 130 example, derive from BJ Union Hospital's MEC, wherein the male sex 89 example, women 41 example, the range of age 21 ~ 70 years old, the mean age (37.6 ± 9.0) year;

Strictly leave and take sample by proteomics research requirement, gather peripheric venous blood, packing after separation of serum in 2 hours ,-80 DEG C frozen for subsequent use.

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

Proteantigen N all on high-density protein chip holds all with the GST label for protein purification.First the anti-GST monoclonal antibody of rabbit and chip protein hybridization proofing chip quality is used.Then choose 40 parts of BD patients serums, 15 routine Serum of Patients With Autoimmune Diseases control serums and 20 routine normal healthy controls serum and 75 high-density protein microarray biochips are hybridized, to be correlated with autoantigen by signals collecting and data analysis qualification candidate BD.

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

Adopt GenePix 4000 B chip scanner scanning high flux protein chip, obtain tiff format gray level image, according to the operation manual of GenePix Pro 6.0 software, according to high flux chip loading dot matrix parameter, automatically complete each protein site signal pixels by software to split, the artificial letter intensity checking all protein sites on chip one by one simultaneously, impurity is avoided in manual setting, the protein signal point position that the human factors such as cut cause and the skew of size, finally complete the extraction of often opening each antigen protein particle signal intensity on high flux protein chip and generate data file and use for subsequent analysis.

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, just think that when the signal to noise ratio (S/N ratio) (Signal to Noise Ratio, SNR) of 2 parallel points of probe on chip is greater than 2 simultaneously this protein site can be detected on chip.The correlativity of signal intensity between the ratio of the protein spots that then computing chip can be detected and the parallel point of two, each albumen.

3, high-density protein chip detection autoantibodies

Serum specimen for high-density protein chip detection comprises BD patient 40 example, certainly exempts from disease contrast 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

Grouping	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 control 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 GenePix Pro 6.0 software collection to the signal strength information often opening all protein sites on high-density protein chip import in Excel form.By the foreground signal intensity (F635median) of each protein site divided by the signal value of this protein site ambient background signal intensity (B635median) as this protein site.I.e. Iij=F635median/B635median (Iij represents the signal value of the protein i point in block j).The signal value of proteantigen protein site more levels off to 1, and illustrate that in serum, corresponding autoantibody concentrations is lower, this antibody is more not easy to be detected.In signal value higher explanation serum specimen, the ability in conjunction with target antigen protein of this autoantibody is stronger.Using the mean value of different for same albumen two some signal intensities as the signal intensity of this protein site on this chip.When these mean value >=2, think that this protein site is for positive.Then the information of each proteantigen immune response positive rate on every part of serum and high-density protein chip is added up.Adopt genetic chip significance analysis (SAM) method conventional in high flux chip biological information analysis techniques to BD patient's group, certainly exempt from disease control group and Normal group carries out data analysis, enter to elect as BD candidate with Q value <35% and to be correlated with autoantigen.BD patient's erythrocyte sedimentation rate increases group and directly compares with erythrocyte sedimentation rate normal group, enters to elect as BD candidate to be correlated with autoantigen with Q value <5%.BD candidate autoantigens in order to ensure screening has clinical value, the positive rate positive rate >25% of all selected preparation BD related auto-antibodies target antigens.

Result:

The high-density protein chip comprising 47616 protein sites is adopted to carry out screening BD related auto-antibodies to 40 parts of BD patients serums, 15 routine Serum of Patients With Autoimmune Diseases control serums and 20 routine normal healthy controls serum.Result of study shows, all serum specimens can only react with the raw antigen and antibody specific of a few eggs white hair 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 of exempting from disease control 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 sample positive protein point confirms, adopt genetic chip significance analysis (SAM) method conventional in high flux chip biological information analysis techniques to BD patient's group, certainly exempt from disease control group and Normal group carries out data analysis, enter to elect as BD candidate with Q value <35% and to be correlated with autoantigen.BD patient's erythrocyte sedimentation rate increases comparing between group with erythrocyte sedimentation rate normal group, enters to elect as BD candidate to be correlated with autoantigen with Q value <5%.BD candidate autoantigens in order to ensure screening has clinical value, the positive rate >25% of all selected preparation BD related auto-antibodies target antigens.The BD associated target antigens confirmed by analysis amounts to 36, and these target antigens will bring the little chip preparation of BD associated antibodies target antigen into, refer to table 2.The BD associated antibodies typical figure of high-density protein cDNA microarray is as Fig. 2:

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

Structure and the serum screening of embodiment 2 BD related auto-antibodies target antigen protein-chip are verified

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

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

The serum specimen that the checking of table 3 Big Clinical Samples detects

To the 130 routine BD patients that BD related auto-antibodies target antigen protein chip carries out, 103 examples exempt from disease contrast patient certainly and 110 routine 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 autoimmunity disease control group 4.85% (X ²=15.87, P=6.79x10 ^-5) and normal healthy controls group 6.36% (X ²=13.67, P=0.000218), result difference has conspicuousness.BD related auto-antibodies target antigen chip Shang Ge seminar's serum specimen and CTDP1 hybridize typical figure as Fig. 3.The BD associated target antigens that all the other 35 kinds of high-density protein chips sift out passes through on little chip after enlarged sample detection validation, and result is presented at BD group, positive rate there was no significant difference between autoimmunity disease control group and normal healthy controls group.

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

Testing result on table 4 BD 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, and we adopt western blotting method the most frequently used in proteomics research method to detect CTDP1 antibody anti-in BD patients serum.The anti-CTDP1 antibody capable that the display of immune-blotting method result adopts the screening of BD autoantibody target antigen chip large sample to confirm well is repeated in WB detects, there is the autoantibody with CTDP1 specific reaction in BD patients serum, its reaction signal intensity is higher than autoimmunity disease control group and normal controls group.The result of immune-blotting method is 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 prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. The phosphatase (CTDP1) of 1.RNA polymerase II subunit AC-terminal domains or the purposes of its fragment in the reagent for the preparation of diagnosis Behcet's disease.
2. 2. purposes as claimed in claim 1, it is characterized in that, described diagnosis comprises: 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 Behcet's disease symptom; Optionally,

   Level with antibody in the more described biological sample of contrasting data, wherein shows the possibility of Behcet's disease relative to the detectable raising being reactive antibody to CTDP1 in sample described in described contrasting data.
3. 3. purposes as claimed in claim 1 or 2, wherein, described biological sample is blood serum sample.
4. 4. purposes as claimed in claim 1 or 2, wherein, measuring horizontally through following steps of CTDP1 antibody, comprising:

   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 to which and CTDP1 or its fragment;

   C. washing removes any unconjugated antibody;

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

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

   F. described label is converted into detectable signal; Wherein the existence of detectable signal shows the possibility of CTDP1 in described patient.
5. 5. as purposes as claimed in claim 4, wherein, described CTDP1 or its fragment deposit or are fixed on solid support.
6. 6. the purposes of claim 5, wherein, described holder is the form of latex pearl or porous flat plate.
7. 7. purposes as claimed in claim 4, wherein, described detection antibody by being covalently attached to enzyme, the label that has the label of fluorescent chemicals or metal or have a chemiluminescence compound marks.
8. 8., for the identification of from being the existence of reactive antibody or an equipment for level to CTDP1 in the sample of patient, comprising:

   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.
9. 9. equipment as claimed in claim 8, comprises detection antibody further, and it is reactive antibody that wherein said detection antibody to be specific in the sample of described patient CTDP1, and described detection antibody produces detectable signal.
10. 10. equipment as claimed in claim 9, wherein, the sample of described patient is blood serum sample.