CN104292323B - Primary biliary cirrhosiss specificity autoantigen and its application - Google Patents

Abstract

The invention discloses primary biliary cirrhosiss（PBC）Specificity autoantigen, its KLHL7.The present invention screens 6 new PBC autoantigens.In detection PBC patients serums, the positive rate of these autoantigens has all reached more than 15%, therefore, these newfound autoantigens are the biomarkers for potentially distinguishing other autoimmune diseasees and Accurate Diagnosis PBC.

Description

Primary biliary cirrhosiss specificity autoantigen and its application

The application is the applying date for 26 days 03 year 2012, and Chinese Application No. 201210080385.8 is entitled The divisional application of the patent application of " primary biliary cirrhosiss specificity autoantigen and its application ".

Technical field

The invention belongs to biomarker field, and in particular to primary biliary cirrhosiss antigen markers and its should With.

Background technology

Primary biliary cirrhosiss（PBC）It is a kind of chronic cholestatic hepatic disease, with medium and small bile duct in liver Progressive destruction for principal character, ultimately result in liver cirrhosis and liver failure a kind of disease (Poupon, R 2010, Selmi, C. 2011).Its Pathological is portal area inflammation, lymphocyte around impaired bile duct, and infiltrate to In bile duct basement membrane and bile duct epithelial cell, bile duct epithelial cell is in empty balloon-shaped degeneration necrosis, and then causes Epithelial tissue thin Born of the same parents' hypertrophy, forms granuloma, and this Morphological Characteristics prompting epithelial duct is the target of PBC immune attacks.PBC is modal to be faced Bed symptom is weak and skin pruritus.The special complication of the PBC relevant with cholestasis mainly has osteoporosises, fat-soluble fiber Element shortage, hyperlipemia and steatorrhea etc..In end-stage disease, a series of and other reasonses of liver cirrhosis and portal hypertension can occur The substantially similar complication of person caused by caused liver cirrhosis, such as ascites, treatment for esophageal varices bleeding and hepatic encephalopathy Deng.

The topmost Biochemical Indices In Serums of PBC are exactly serum alkaline phosphatase（ALP）And gamma glutamyl transpeptidase （Gamma-glutamyltransferase, γ-GT）Raise, it is general to raise 3-4 times, and it is found in early stage and the nothing of disease The symptom phase.But glutamate pyruvate transaminase（ALT）And glutamic oxaloacetic transaminase, GOT（AST）It is generally normal or slight to moderate rising.Early stage patient Without jaundice, but with the progress of the state of an illness, in relatively late patient, serum bilirubin is significantly raised, and abnormal level of serum total bilirubin has Help judge the prognosis of PBC patient and determine the opportunity of liver transplantation.During the thrombin of high bilirubin, hypoalbuminemia and prolongation Between be all prognosis malas index.Additionally, the serum Ievel of total bile acids of PBC patient would generally be raised.

There is anti-mitochondrial antibody in serum（AMA）It is the important symbol of PBC.AMA not unique specificitys of PBC itself Antibody, also can detect that antinuclear antibody in PBC serum（ANA）, have presentation nuclear model（M-ANA）, have presentation multinuclear point-type （Multiple nuclear dots, MND）.Additionally, part PBC patient is presented with the smooth muscle antibody positive, antithyroid resisting The autoantibodys such as body, anti-DNA antibody and rheumatoid factor.

After medical worker has found that the serum of some Diseases is presented special immunofluorescence dyeing pattern, explore The targeted autoantigen of these autoantibodys and the work of correlation detection technology is set up with regard to carrying out always.Identification resists itself Former aspect, is originally gained knowledge by immunofluorescence technique and cell biological and primarily determines that the cellular localization of target antigen, then by dividing From different organelles are extracted, the finer positioning in cell of the protein of autoantibody identification is confirmed.With protein electricity The development of swimming skills art, immunoblot assay and tree species for bio-energy source, increasing autoantigen are identified.In recent years, with The utilization of cDNA expression library technology and high flux expression of recombinant proteins purification technique, particularly high throughput protein chip skill The development of art, the appraisal of autoantigen become increasingly simpler, convenient and quick.

On the other hand, clinical laboratory's detection technique of autoantibody is also achieved and is developed rapidly, from originally based on group Knit and/or cell immunohistochemistry technology and immunofluorescence technique, to traditional precipitation, immuno-electrophoresis, solidifying Collection test, complement fixation test technology etc..With the development of molecular biology, the particularly development of expression of recombinant proteins technology, Various label immunoassay technologies (include enzyme-linked immunoassay technology, radioimmunoassay technique, fluorescence immunoassay, chemiluminescence Immunoassay and golden immunological technique etc.) become main immunoassay.Additionally, immunoblotting has also been played significantly Effect.In recent years, with the development of genomics and proteomic techniques, protein biochip technology is with its higher detection spirit Sensitivity has played important function in terms of autoantigen identification, and being no less than 21 kinds of protein chips at present is used for clinic （Hartmann, M., J 2009）, wherein at least have 5 kinds of Jing FDA authentication approvals for autoimmune disease diagnosis.

At present, the method for autoantigen identification mainly includes following five kinds：1）SEREX technologies；2）Display technique of bacteriophage； 3）SERPA technologies；4）Protein biochip technology；5）MAPPing technologies.Wherein SEREX technologies, display technique of bacteriophage need structure Expression library is built, through the Biopanning of number wheel, the final identity for determining candidate autoantigens, program are loaded down with trivial details, take time and effort.This Also suffer from the drawback that outward, 1）The epi-position for screening is mainly based on the escherichia coli easily linear epitope of expression；2）Due to big Partial library is to extract mRNA to prepare cDNA from tumor tissues or cell, and expression library tends to be exactly compared with Gao Feng originally The intracellular protein of degree, and the autoantigen major part of autoantibody identification is all the relatively low protein of expression.

SERPA technologies be with middle proteomic techniques development and produce, after Identification protein translation There is the advantage of uniqueness in terms of modification autoantibody, and also broken away from the work that construction expression library is wasted time and energy, but due to Bidirectional electrophoresis technique separate cell whole protein ability limited, particularly extremist amount and isoelectric point, IP proteantigen divide From, and native protein conformation is destroyed in electrophoresis process, the autoantibody for screening is still based on identification linear epitope.

MAPPing technologies are in advance to combine non-diseases specific recognition first by control serum using the method for affine in immunity Native protein autoantigen, then reuses the lower native protein special with reference to disease of disease blood serum affine in immunity effect certainly Body antigen.The method is easier to identify the autoantibody for being capable of identify that native protein.Its techniqueflow closer to and SEREX Technology and display technique of bacteriophage, are all, through the multiple process eluriated, simply to need exist for identifying itself through biological mass spectrometry The identity of antigen.Its shortcoming is exactly to identify that the identity of autoantigen is cumbersome, especially because existing in n cell a large amount of The complex of protein protein interaction, is possible to be combined with non-specific autoantigen during preadsorption The special autoantigen of disease remove together, cause subsequently find the special autoantigen of disease.

Protein biochip technology be a kind of high flux, high sensitivity, miniaturization analytical technology, be detection serum and other The effective tool of various autoantibodys in clinical sample.It is used not only for quickly finding that new diagnosis and prognosis to disease has weight Want the disease related auto-antibodies label of using value, and for high-volume, disease known to low cost detection it is related from Body antibody.As on protein chip, target antigen is all known, therefore it is easy to judge the new autoantigen of identification Identity.But prepare on protein chip thousands of proteantigen probe but not a duck soup.Current chip antigen can be with Using the detached cell whole protein component of Jing proteomic techniques, the native protein of various known antibodies affinity captures, with And the recombiant protein of foreign host expression.The recombiant protein of foreign host expression may face the conformation of Non natural proteins, The shortcomings of clonal expression recombiant protein task is heavy.Detached cell whole protein component and with the natural egg of known antibodies affinity capture The method of white matter essentially eliminates the misgivings of Non natural proteins, but also faces various conditions and limit, such as proteomics skill The separating effect of the detached whole protein component of art has much room for improvement；Identification purpose target antigen is cumbersome；The preparation of known antibodies Also it is comparatively laborious and be difficult obtain etc. significant deficiency.In addition also have using construction cDNA gene chip, using external in situ without thin Cellular expression system prepares the report of corresponding protein chip, and this method eliminates the trouble of a large amount of recombiant proteins of purification, but The effective expression of recombinant protein on chip cannot be guaranteed.

As it was previously stated, PBC is a kind of autoimmunity hepatic disease, the diagnosis of the detection of autoantibody marker in disease In have important function.AMA is one of major criterion of PBC clinical diagnosises, and can be before PBC asymptomatic stages or clinical symptoms Phase is occurred in patients serum.AMA（Mainly II types anti-mitochondrial antibody, AMA-M2）Sensitivity in PBC diagnosis is 90% Left and right.With going deep into that various clinical diseases are studied, it is found that AMA can be widely present in other various autoimmune diseases, Such as primary Sjogren's syndrome, scleroderma, autoimmune hepatitiss and viral hepatitis etc..Discovery 40.9% is had been reported that even （28/69）Acute hepatic failure patient present AMA-M2 it is positive（leung 2007）.Additionally, still having 10% or so to be that AMA is negative PBC patient, the diagnosis of this some patients can only be confirmed with reference to liver biopsy by other various clinical symptoms.

In addition to AMA, anti-gp210 antibody, anti-p62 antibody, anti-sp100 antibody, anti-sp140 antibody and anti-LBR antibody etc. are certainly Body antibody also has important clinical value in the diagnosis of PBC, but universal positive rate is low（1%-30%）, and most of and AMA- M2 occurs simultaneously.

PBC is the autoimmune disease under a kind of inherited genetic factorss and environmental factorss interaction, and its pathogenesis is not Clearly.Effect of the autoantibody in PBC pathogenesis understands not enough and there is dispute.Although AMA possesses higher in PBC Sensitivity and specificity, have experiment to show, the titre of AMA may be related to Disease Activity, point out AMA take part in liver The damage of tissue.But also there is contrary viewpoint, dispute on a lot of.Therefore identify that more autoantibodys potentially contribute to disclose PBC Pathogenesis, particularly autoantibody effect wherein.

As the treatment of PBC is more special, other diseases, particularly autoimmune hepatitiss and viral are totally different from Other hepatic disease such as hepatitis.Therefore in the larger China of hepatitis radix, the Differential Diagnosiss of PBC are extremely important.It is therefore desirable to The related autoantibody markers of more PBC are identified, to serve the clinical diagnosises of PBC.

The content of the invention

In order to solve the above problems, the present invention provides primary biliary cirrhosiss（PBC）Specificity autoantigen and its Using.

The present invention is by high-density protein matter chip and small sample serum screening by hybridization candidate PBC autoantigens and follow-up The scheme for preparing PBC chips and combining with large sample serum screening by hybridization filters out 6 and primary biliary cirrhosiss altogether (PBC) height correlation and sensitivity are all higher than 15% specificity autoantigen, respectively HK1 hypotypes 1, HK1 hypotypes 2, KLHL12, KLHL7, ZBTB2 and EIF2C1.

Further, the present invention also provides described autoantigen and is preparing primary biliary liver as biomarker Application in hardening detectable.

Wherein, the reagent is the reagent based on radioimmunity, fluorescence immunoassay, chemiluminescence immunoassay or golden immunological technique, Immunofluorescence test paper strip, the immune radiating test strips of the antigen, immune gold test paper strip etc., but not limited to this are coated with such as.

Wherein, described autoantigen can be the antigen of two or more autoantigen fusions.

In a currently preferred embodiment, described detectable be coated with described in one or more from The primary biliary cirrhosiss detection chip of body antigen.

Wherein, described detection chip can also be coated with BCOADC-E2, PDC-E2, OGDC-E2, E3BP, PDC-E1 β, PDC- One or more primary biliary cirrhosiss specificity in E1 α, Gp210, p62, LBR, CENP-B, Sp100 and Sp140 is certainly Body antigen.

In another embodiment of the invention, the detection chip is coated with KLHL12, ZBTB2 and gp210.

Wherein, described detection chip, can also be coated with Quality Control antigen.Wherein, described Quality Control antigen can be in this area Suitable any Quality Control antigen, one or more in such as Mus IgG, human IgG and bird flu viruss nucleoprotein.

Further, the present invention also provides a kind of primary biliary cirrhosiss ELISA detection kit, and which contains enzyme mark Plate, described ELISA Plate are coated with the autoantigen described in one or more.

Preferably, described ELISA Plate can also be coated with BCOADC-E2, PDC-E2, OGDC-E2, E3BP, PDC-E1 β, PDC- One or more primary biliary cirrhosiss specificity in E1 α, Gp210, p62, LBR, CENP-B, Sp100 and Sp140 is certainly Body antigen, to improve the sensitivity of detection PBC.

The present invention by high-density protein matter chip and PBC patients serums screening by hybridization to 23 candidate PBC it is related itself Antigen.In order to further verify the specificity of these autoantigens, construct and itself resist comprising 21 candidate PBC therein correlations Former and 9 PBC specificitys autoantigens for clinically having used or having reported in interior PBC chips, then by large sample Serum（Including 191 parts of PBC serum, and 321 parts of control serums：43 AIH, 55 HBV, 31HCV, 48 RA, 45 SLE, 49 SSc and, 50 Healthy Peoples）With PBC chip hybridizations, analyzed by data result, 13 autoantigens are identified altogether in PBC There is in serum higher sensitivity and specificity, data analysiss show sensitivity of 13 kinds of protein in PBC reach 15% with On, wherein 6 is newfound, respectively HK1（Isoforms I and isoforms II）, KLHL7, KLHL12, ZBTB2, and EIF2C1.Wherein, HK1 hypotypes I, HK1 hypotype II, KLHL12, the positive rate of KLHL7 have reached more than 35%. As a result also find, combine KLHL12, ZBTB2 and gp210 detection and the AMA-M2 negative important auxiliary of PBC patient diagnosis is made With detection sensitivity is 47.8%, and specificity is 89.4%, basically identical with AMA-M2 detection PBC（89.7%）.For convenience in Clinical detection, 297 parts of PBC serum and 637 parts of control serums resist for the anti-KLHL12 and ZBTB2 itself based on elisa technique Health check-up is surveyed, and as a result finds that its positive rate is respectively 29.6% and 11.2%, and specificity is respectively 98% and 99.7%.Other Western Blot results confirm that the autoantibody of anti-HK1 and KLHL7 in PBC serum not only can be can detect by chip, Western blot It is detectable.

Description of the drawings

Fig. 1 show PBC serum and control serum hybridization high-density protein matter chip partial result figure.A, B and C figure shows Be three parts of PBC serum and chip hybridization design sketch；D, E and F figure is it is shown that three parts of matched group serum and chip hybridization Design sketch.In green box, two parallel point protein probes are HK1 hypotypes I（isoform I）；Yellow box middle probe is KLHL12；It is ZBTB2 in blue square frame.

The number of the positive control probe that Fig. 2 is recognized by every part of serum after high-density protein matter chip and the hybridization of 46 parts of serum samples Amount compares.A is the distribution that 26 parts of PBC serum recognize positive control probe quantity；B is 20 parts of control serum identification positive control probe quantity Distribution.

The layout of Fig. 3 PBC autoantigen protein matter chips and anti-GST antibody detection.30 of A expression and purifications again PBC autoantigens and positive control（Red filling）And negative control（Lycoperdon polymorphum Vitt is filled）Distribution in the chips.B anti-GST antibodies Testing result, all 30 recombinant antigens carry GST labels, and in control, AIV-NP is escherichia coli expression, is marked with 6*His Sign.

Fig. 4 large samples serum and PBC autoantigen chip hybridization partial result figures.A-D is shown four parts of PBC serum With the design sketch of chip hybridization；The control serum that E-I shows and chip hybridization design sketch.

Fig. 5 PBC autoantigens and 191 parts of PBC serum and 321 parts known to 7 kinds more than 15% of sensitivity in PBC serum Signal distributions after the hybridization of matched group serum.

Fig. 6 in PBC serum sensitivity more than 6 kinds of 15% new identifications PBC autoantigens and 191 parts of PBC serum and Signal distributions after 321 parts of matched group serum hybridization.

Fig. 7 ELISA method detections KLHL12 and ZBTB2 signal distributions respectively with 934 parts of various serum hybridizations.

Fig. 8 parts serum and HK1（isoform I）With the immune-blotting method of KLHL7.

Specific embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.

The diagnosis of PBC is that (Heathcote, E. J. is 2000) for the standard diagnostics established according to AASLD in 2000.It is all Serum is collected from 2006 to 2010 by rheumatism immunity section of BJ Union Hospital, and all disease blood serums are all from clinical definite trouble Person.All PBC serum have carried out the detection of AMA, ANEA, ACA and epipole type antibody and AMA-M2.AMA, ANEA, ACA and The detection of epipole antibody is based on Hep2 cells（German Euroimmune companies）Immunofluorescence technique.And anti-AMA-M2 antibody Detection be that the ELISA carried out using anti-MIT3E kit (German Euroimmune companies) test kit is detected.All AMA Or/and the serum of anti-M2 negative antibodies is satisfied by PBC and punctures liver biopsy and the diagnostic criteria of biochemistry detection.Relate in following embodiments And each test obtain Ethics Committee of BJ Union Hospital examination ＆ verification approval.

Embodiment 1 is using high-density protein matter cDNA microarray candidate PBC related autoantigen

High-density protein matter chip is provided by Johns Hopkins University of U.S. teacher Zhu Heng laboratory.Every high density Contain 48 matrixes in protein chip, each matrix includes 800 probes, the arrangement of the array in 32X25.It is every kind of on chip Protein probe has a parallel point, and the protein chip includes 16,368 kinds of recombinant proteins.Most of recombiant protein from Corresponding gene total length ORF of Saccharomyces cerevisiae host expression, and there is GST labels in N-terminal（Jeong, 2012）.The high density egg White matter chip has been disclosed in Jeong et al., Rapid Identification of Monospecific Monoclonal Antibodies Using a Human Proteome Microarray, Molecular ＆ Cellular Proteomics (2012.2.3 is disclosed online), those skilled in the art can be prepared accordingly.

As the protein probe N-terminal on all chips carries GST labels, therefore first by the anti-GST monoclonals of Mus Antibody and chip hybridization proofing chip quality.Then 26 parts of PBC patients diagnosed serum of picking and 20 parts of control serums（5 parts certainly Body autoallergic (AIH) serum, 5 parts of hepatitis B (HBV) serum and 10 parts of Healthy Human Serums）It is miscellaneous with 46 chips Hand over, the PBC correlation autoantigens of candidate are identified by signals collecting and data analysiss.

1）Chip quality is verified

Using anti-GST antibody hybrid proteins chip, the quantity of effective protein probe and parallel spy on proofing chip Dependency between pin, concrete operation step are as follows：

It is directly immersed in the confining liquid of 3% BSA after the high-density protein matter chip for taking out -80 DEG C of preservations, 37 DEG C of closing 1h；

The anti-GST monoclonal antibodies of 0.5 μ l mices are taken according to 1:In 3% BSA, concussion is mixed 1000 dilution proportion, 10000rpm*10min is centrifuged, and supernatant is for having diluted and resists.After chip is taken out from confining liquid, will be unnecessary with absorbent paper Confining liquid as far as possible blot from side, be placed in wet box.Then draw that 180 μ l have diluted one it is anti-be added on chip, it is slow plus Coverslip, it is to avoid bubble is produced, 37 DEG C of incubation 1h；

The chip being incubated is placed in PBST, coverslip is carefully removed, chip is put into be washed in box with 37 DEG C of preheatings PBST 40rpm are rinsed 3 times, every time 10 minutes；

The goat anti-mouse IgG antibody of 0.5 μ l cy5 labellings is taken, according to 1:1000 dilution proportion is shaken in 3% BSA Mixing, 10000rpm*10min centrifugations are swung, supernatant is two for having diluted and resists.Chip take out in box from being washed, with absorbent paper from one Unnecessary washing liquid is blotted in side, is placed in wet box.Draw that 180 μ l have diluted two it is anti-be added on chip, slowly add a cover slide, keep away Exempt from bubble generation, 37 DEG C of lucifuges are incubated two anti-1h；

The chip being incubated is placed in PBST, coverslip is carefully removed, chip is put into be washed in box with 37 DEG C of preheatings PBST 40rpm lucifuges are rinsed 3 times, every time 10 minutes.Then rinsed 3 times with the pure water 40rpm lucifuges of 37 DEG C of preheatings again, 10 minutes every time；

Chip is taken out, being placed in has the side of probe outwardly on chip in 50ml centrifuge tubes, 2000rpm*3min centrifugations, Pure water remaining on chip is dried, LuxScan-10K/A chip scanners scanning chip all arranges identical after the completion of hybridizing every time Sweep parameter scanning chip.The parameter setting of scanner is as follows：Photomultiplier tube（PMT）850 are set to, laser energy （Power）95 are set to, the pixel of scanning is 5um,

When the signal to noise ratio of probe on chip（SNR）During more than 3, it is believed that the probe can be detected on chip, core is then calculated Dependency between two parallel points of the ratio and each probe of the protein probe that can be detected on piece.

Amount to 38400 probe points on every high-density protein matter chip（Including positive control point and negative control point；Often Plant probe and there are two parallel points）, including 16368 kinds of nonredundancy recombination human source protein.

On every chip, all probes constitute 48 matrixes altogether, and microarray of each matrix in 32X25 is arranged.Due to all The N-terminal of recombinant protein probe carries GST labels, therefore carries out institute in detection chip using the anti-GST monoclonal antibodies of mice There is probe, it is ensured that can be detected for most recombiant proteins on the chip of serum screening.With the 2 of each protein probe The signal intensity of individual parallel point does scatterplot, and the correlation coefficient between parallel point is 97.8%, shows There is good repeatability.

2）Screening candidate PBC related autoantigen

The composition of screening serum：26 parts of PBC serum and 20 parts of control serums（5AIH serum, 5HBV serum and 10 are good for Health human serum）.Per portion serum with the hybridization of protein chip, resist for PBC patients serums or compare disease blood serum except one And two resist outside the goat anti-human igg antibody for cy5 labellings, hybridizing method such as step 1）Middle 1-6.

According to the workbook of 6.0 softwares of GenePix Pro, will be the brightness and contrast of all chip scanning pictures equal 99 are set to, are then analyzed using chip image collecting software GenePix Pro 6.0 and is obtained each target egg on every chip The signal intensity of white matter dot blot.

By 6.0 software collections of GenePix Pro to every chip on all probes signal message import Excel tables In lattice.The foreground signal intensity of each probe points（F635median）It is respectively divided by its ambient background signal intensity （B635median）As the signal value of the point.

Iij= F635median/ B635median

Iij represents the signal value of the protein i points in block j.

The signal value of proteantigen probe more levels off to 1, illustrate that autoantibody cannot more be detected accordingly in serum. The ability of the combination target proteins matter antigen probe of the higher explanation autoantibody of signal value is stronger.

In order to eliminate the difference that different spaces are caused to hybridization on different batches chip and same batch chip, chip data Process using normalization in chip（within-chip normalization）Method the signal on every chip is carried out Normalization.Assume in chip all target proteinses be random point system on substrate, and only little part（Less than 5%）'s Target proteins matter is detected by corresponding target autoantibody identification in serum as autoantigen, therefore signal on chip Distribution be random, different matrixes（block）Between be consistent.This research sets all probe points in each block The median of signal value is 1, carrys out the signal value of difference block internal probe points on normalization chip with this.

That is ij=Iij-median (Ij)+1.

Median (Ij) represents the median of had signal value in block j, and ij is represented in the block j after normalization Protein i points signal value.

On this basis, the article delivered according to teacher's Zhu Heng laboratory（Hu, 2009）Described in method setting Cutoff values judge whether all probe points are positive on chip.The average of had signal value on whole chip is calculated I_average, and all signal values are less than standard deviation SD of 1 signal value, with I_average+ 6SD is cutoff values, judges chip On probe points be whether positive.Then count the positive reaction of chip of every part of serum hybridization proteantigen probe Number, using non parametric testss X 2 test（Chi-square test, X²）Or Fisher is accurately checked（Fisher exact test）Determine the PBC correlation autoantigens of candidate.Meanwhile, specificity is reached into 100%, antigen of the sensitivity not less than 15% As the PBC autoantigens of candidate.

High-density protein matter chip is as shown in Figure 1 with the representative topography after seroreaction（It is difference in square frame Proteantigen probe）.On the whole, either PBC patients serums, or hepatopathy matched group（AIH serum and HBV serum）With it is strong Health human serum, albumen that all can only be little on identification chip.

The signal on every chip is normalized using normalized method in chip, then statistical analysiss determine every Open the positive control probe on chip.Can be not quite similar by the number of the protein of every part of serum identification on chip, as shown in Fig. 2 The identified number of proteantigen PBC with compare in be not different substantially.The proteantigen of 26 parts of PBC serum identifications Number is 70-709（Average ± standard deviation：347±191）, the proteantigen data of 20 parts of control serum identifications are 74- 1440（429±306）.

For on chip, whether each protein probe is the related autoantigens of PBC, using X²Inspection or Fisher are smart Really inspection determines target proteins matter antigen of 7 protein for PBC specific reactioies.Meanwhile, specificity is also reached by this research 100%, sensitivity is standard not less than 15%, has the special autoantigens of PBC that 16 antigens also serve as candidate.Details are such as Table 1.

1 small sample serum of table and high-density protein matter chip hybridization screen the special autoantigen of 23 candidate PBC.

Protein title is write	GenBank accession number	Antigen	26 parts of PBC serum recognize positive control probe number	20 parts of control serums recognize positive control probe number
					BCOADC-E2	NM_001918	M2 antigens	17	2
PDC-E1α	NM_000284	M2 antigens	16	0
					DR1	NM_001938	It is new to identify	6	0
TRA16	NM_176880	It is new to identify	6	0
					ANXA10	NM_007193	It is new to identify	6	0
KLHL12	NM_021633	It is new to identify	6	0
					ZBTB2	NM_020861	It is new to identify	6	0
EIF4H	NM_031992	It is new to identify	5	0
					IL1A	NM_000575	It is new to identify	5	0
KLHL7	NM_018846	It is new to identify	5	0
					SPATA5	NM_145207	It is new to identify	5	0
EIF2C1	NM_012199	It is new to identify	5	0
					ATCAY	NM_033064	It is new to identify	5	0
NXN	NM_022463	It is new to identify	4	0
					KEAP1	NM_012289	It is new to identify	4	0
DDX55	NM_020936	It is new to identify	4	0
					SNX9	NM_016224	It is new to identify	4	0
PDC-E1β	NM_000925	M2 antigens	4	0
					RPS19	NM_001022	It is new to identify	4	0
HK1- hypotypes I	NM_000188	It is new to identify	4	0
					HK1- hypotypes II	NM_033496	It is new to identify	4	0
TPM1	NM_000366	It is new to identify	4	0
					CHRNA3	NM_000743	It is new to identify	4	0

The structure of 2 PBC autoantigen protein matter chips of embodiment is verified with serum screening

23 candidate PBC autoantigens and 9 clinically known or document reports recent years are obtained using the screening of embodiment 1 When the PBC specific autoantibodies label in road prepares PBC chips, the recombinant protein antigen for planning to express in chip is 32 It is individual.But due to some uncontrollable factors, the present embodiment carries on research with 30 PBC therein correlation autoantigens （Table 2）.

Above-mentioned 30 proteantigens again expression and purification are used to prepare PBC associated antigen protein matter chips, Fig. 3-A are 30 PBC correlation autoantigens and dot matrix layout of the control probe on little chip.Except 30 PBC correlation autoantigen probes Outward, also including positive control probe 1）AIV-NP（Avian influenza virus- Nucleoprotein, bird flu viruss Nucleoprotein）；2）Mus IgG（Mus anti-human IgG antibodies）；3）Human IgG and negative control probe 1）Buffer（Recombiant protein eluting Liquid）；2）GST label proteins.Fig. 3-B are the knot of all PBC correlations autoantigens in a dot matrix in anti-GST antibody detection chip Really.

On PBC autoantigen protein matter chips, all 35 probes all have the two point for repeating.Concurrent system on every substrate Each dot matrix, before hybridization of the serum with chip, is kept apart, so each dot matrix by 12 dot matrixs with fence An independent space is formed, therefore every chip can detect 12 parts of serum simultaneously.

Include 43 parts of AIH serum with the large sample serum of PBC autoantigen protein matter chip hybridizations, 55 parts of HBV serum, 31 Part HCV serum, 50 parts of Healthy Human Serums, 191 parts of PBC serum, 48 parts of rheumatoid arthritiss（rheumatoid Arthritis, RA）Serum, 45 parts of systemic lupus erythematosus (sle) (systemic lupus erythematosus, SLE) serum, 49 Part systemic sclerosiss（Systemic Sclerosis, SSc）Serum.Fig. 4 is PBC autoantigen protein matter chips and PBC blood The presentation graphics of clear and control serum reaction.In therefrom can significantly seeing the result of PBC serum hybridization, signal intensity is big （It is red）Number of probes substantially than AIH, SSc, SLE etc., other autoimmune diseasees are more.

The meansigma methodss and its standard deviation of 50 parts of Healthy Human Serum hybridization signals for each probe, are calculated first, are adopted It is cutoff values judging whether all serum are positive with the probe higher than the numerical value of five times of standard deviations of meansigma methodss.Then Count positive rate of each probe in 191 parts of PBC serum and individual matched group serum.

Concrete each serum group is shown in Table 2 with the number of cases and its positive rate of all 30 kinds of protein antigen positives reactions on chip（1 Proteantigen known to kind）With table 3（18 kinds of candidate's PBC proteantigens）.

2 each serum group of table is presented positive case load and its sun with PBC autoantigens hybridization known to 12 kinds on chip Property rate

* non-PBC includes health, AIH, HBV, HCV, RA, SLE and SSc

3 each serum group of table is presented positive case load and its sun with 18 kinds of candidate's PBC autoantigens hybridizations on chip Property rate

* non-PBC includes health, AIH, HBV, HCV, RA, SLE and SSc

30 autoantigen probes and healthy human blood from the point of view of the data results of table 2 and table 3, on all little chips The positive rate of clearance response is respectively less than 2%., there are 13 kinds of protein amounting to more than 15% with the positive rate of PBC seroreactiones, is respectively： PDC-E2、BCOADC-E2、gp210、E3BP、CENPB、LBR、sp140、HK1（isoform I）、HK1（isoform II）、 KLHL7, KLHL12, ZBTB2 and EIF2C1.The positive ratio of all this 13 kinds of antigens has statistics between PBC and Healthy People Significant difference on（p<0.05）.

In autoantigen of this 13 kinds of positive rates more than 15%, first 7 is clinically to have used or document report PBC correlation autoantigens, in each serum group with chip, the signal distributions of correspondent probe hybridization are shown in Fig. 5；Send out for new for 6 afterwards The PBC correlation autoantigens now and with large sample verified, the signal point of correspondent probe hybridization in each serum group with chip Cloth is shown in Fig. 6.

Can see from Fig. 5 and Fig. 6, in the result of little chip detection, PDC-E2 and BCOADC-E2 in M2 antigens Signal in PBC serum signal apparently higher than control serum and Healthy Human Serum, another gp210 performances are also fine.In new identification In antigen, HK1（isoform I）、HK1（isoform II）, the hybridization of the protein in PBC serum such as KLHL7 and KLHL12 Signal is also significantly better than control serum and Healthy Human Serum.

12 kinds of known antigens, including 6 hatching egg recognized by AMA-M2 type autoantibodys are added up on PBC autoantigen chips White matter；The protein on nuclear membrane such as gp210, p62 and LBR that ANEA is recognized；Multinuclear point-type fluorescence mode antibody is known Two kinds of protein of other Sp100 and Sp140；And the major antigen protein C ENP-B of ACA.

For the special autoantigens of 6 known PBC of M2 complexs, wherein anti-BCOADC-E2, PDC-E2 and The positive rate of the autoantibody of E3BP is higher, respectively 62.3%, 51.83% and 33.51%.And for other 3 members, resist The positive rate of OGDC-E2, PDC-E1 α and PDC-E1 β autoantibodys is than relatively low, respectively 8.4%, 9.4% and 12.6%.

In this experiment, all PBC serum in addition to using immunofluorescence technique detection AMA, also using originating from Ou Meng（North Capital）The commercial ELISA kit detection AMA-M2 type autoantibodys of medical diagnostic techniqu company limited.Commercial kit It is entitled：Anti- M2-3E IgG antibody detection kit（Anti-M2-3E ELISA）.As a result show that AMA-M2 type autoantibodys exist Positive rate in PBC serum is 84.8%（162/191）.The coated antigen of commercial ELISA kit microwell plate is from Cor Sus domestica In isolated ketoacid dehydrogenase complex and recombination fusion protein mixture.Recombinant fusion protein is escherichia coli table Reach, contain tri- kinds of epitopes of BCOADC-E2, PDC-E2 and OGDC-E2.Therefore, in serum is for M2 complexs When one autoantigen composition reacting positive of any of which, this research is considered as the anti-M2 complexs positive of the serum, little core Piece detects 191 parts of PBC serum, it is found that 157 parts is that anti-M2 complexs are positive, and positive rate is 82.2%.Homemade chip in this research The matching rate detected with the test kit of Ou Meng companies（Detect that the serum of positive or negative accounts for overall ratio simultaneously）For 91.6%. As a result show that PBC associated antigen protein matter chips prepared by this laboratory are reliable.

It is worth joyful, in 13 protein with PBC seropositivities rate more than 15%, 6 is newfound PBC autoantigens, they are HK1 respectively（Hexokinase isoform I, positive rate is 46.6%）, HK1（Hexokinase Isoform II, positive rate are 44.0%）, KLHL12（Kelch-like protein 12, positive rate are 40.3%）, KLHL7 （Kelch-like protein 7, positive rate are 35.1%) ZBTB2（Zinc finger and BTB domain- Containing protein 2, positive rate are 16.8%）And EIF2C1（Eukaryotic translation initiation Factor 2C, subunit 1, positive rate are 15.2%）.

As PBC is a kind of special autoimmune disease of liver, the clinical symptoms and biochemical indicator of its early stage and its His hepatic disease is than relatively similar, therefore this research is conceived to newfound autoantigen first in PBC and hepatic disease matched group Between difference.Hepatic disease matched group includes 43 AIH serum, 55 HBV serum and 31 HCV serum.With another Autoimmune liver disease AIH is compared, and in 6 newfound autoantigens in addition to EIF2C1, significantly can be suffered from PBC Person's seroreaction is related（p<0.01）.The viral hepatitis common with China（86）If comparing, all 6 it is newfound from Body antigen can be significantly related to PBC patients serums reaction（p<0.01）.Accordingly, with respect to AIH and viral hepatitis etc. For other common hepatic disease, these newfound autoantigens are PBC patients serums institute specific recognition.

, used as a kind of autoimmune disease, the autoantigen for newly identifying is excellent between difference PBC and AIH for PBC Performance promotes the difference that this research is absorbed between PBC and other autoimmune diseasees.In this research it is actually used from The control of body immune disease includes 48 RA, 45 SLE and 49 SSc serum cases.Compare with RA with SSc serum samples, All 5 autoantigens for newly identifying（HK1 isoform I, HK1 isoform II, KLHL12, KLHL7 and ZBTB2） The positives significant reaction of PBC serum increases.Compared with SLE serum samples, HK1 isoform I, KLHL12, KLHL7 etc. The ratio that autoantigen is recognized by PBC is apparently higher than SLE serum（p<0.05）.Therefore, these newfound autoantigens are latent Difference other autoimmune diseasees Accurate Diagnosis PBC biomarker.

Although AMA-M2 is extremely important in the diagnosis of PBC, but still there is about 10% PBC patient as AMA-M2 is cloudy Property must particularly have traumatic biological biopsy to detect ability by means of more biochemistry detection and the investigation of clinical symptoms Make a definite diagnosis.Due in known PBC autoantibody markers in addition to AMA-M2 the sensitivity of remaining autoantibody all than relatively low（1- 30%）.Therefore find the difficult point that the PBC special label of M2 feminine genders is also always PBC researchs.Sieved based on PBC autoantigens chip Select 191 parts of PBC serum and 321 parts of each matched group serum cross experiment research show joint-detection KLHL12, ZBTB2 and Gp210 is for the diagnosis of M2 feminine gender PBC patients is with important using value.The sensitivity of joint-detection is 47.8%, and special Property is 89.4%（The positives rate of 321 parts of control serums is 10.3%）, it is similar to the specificity that PBC is detected to M2（89.7%）.Therefore The autoantigen for newly screening will be helpful to the diagnosis to AMA-M2 feminine gender PBC patients with gp210 joint-detection.

3 ELISA of embodiment verifies newfound autoantigen

In view of HK1 isoform I in little chip results, HK1 isoform II, KLHL12, KLHL7 and ZBTB2 for The sensitivity and specificity of PBC diagnosis is higher（EIF2C1 is similar to detection sensitivity in SLE in PBC, 15.2%vs15.6%）, certainly The fixed autoantigen using clinically conventional these new identifications of ELISA method detection.Additionally, due to HK1 isoform I and HK1 Isoform II only have 21 aminoacid of N-terminal in sequence and have differences, and both performances in little chip are basically identical, therefore The antigen of elisa plate prepared by here includes HK1 isoform I, KLHL12, KLHL7 and ZBTB2, and coating concentration is respectively 200ng/ holes, 75ng/ holes, 200ng/ holes and 50ng/ holes.

For KLHL12 and the detection of two autoantigens of ZBTB2（Fig. 7）.The sample of ELISA detections includes 934 parts of blood Clearly：53 parts of AIH patients serums, 112HBV patients serums, 54 parts of HCV patients serums, 87 parts of Healthy Human Serums, 297 parts of PBC patients Serum, 122 parts of RA patients serums, 86 parts of SLE patients serums, 123 parts of SSc patients serums.

In the testing result of ELISA, every part of serum carries out Parallel testing, using the meansigma methodss of the OD values of parallel holes as This part of serum and the signal value of envelope antigen immunoreation.Setting OD450>0.4 is cutoff values, when OD values are more than 0.4, should Serum and the autoantigen reacting positive for detecting.The number of anti-KLHL12 and ZBTB2 Positive Seras is as shown in table 4.

Positive number and positive rate of the 4 anti-KLHL12 and ZBTB2 autoantibodys of table in each group serum.

Disease	Sample number	KLHL12	ZBTB2
				PBC	297	88(29.6%)	35(11.2%)
Non-PBC	637	13(2.0%)	2(0.3%)
				Healthy	87	1(1.15%)	0(0.0%)
AIH	53	7(13.2%)	0(0.0%)
				HBV	112	0(0.0%)	1(0.9%)
HCV	54	0(0.0%)	0(0.0%)
				RA	122	1(0.8%)	0(0.0%)
SLE	86	0(0.0%)	1(1.2%)
				SSc	123	4(3.3%)	0(0.0%)

Learn that the positive rate in PBC serum of anti-KLHL12 and ZBTB2 autoantibodys is respectively 29.6% He from table 4 11.5%, specificity is 98% and 99.7%.Compared with the result of PBC autoantigen chip detection, sensitivity is low（29.6% Vs40.3%, 11.5%vs16.8%）Specificity is higher（98%vs95%, 99.7%vs98.4%）.

Embodiment 5 detects anti-HK1 and KLHL7 autoantibodys using Western blot methods

Itself the resisting of anti-HK1 and KLHL7 is shown with the result of PBC autoantigen protein matter chip hybridizations in large sample serum The signal of body is significantly greater than a negative control group, and the signal value of chip hybridization is very high（At least 25% serum hybridization signal value More than 5000）, but have been found that positive serum compares similar with the OD values of negative control sera in ELISA detections.In order to detect Anti- HK1 and KLHL7 autoantibodys necessary being in serum in serum, this research have used Western blot methods to be examined Survey.

As found from the results, when being detected with anti-label protein antibody, HK1 and KLHL7 antigens and label are found There is band in corresponding molecular weight, and the results of hybridization of the positive of 4 selected respectively anti-HK1 antibody and anti-KLHL7 antibody Respectively there is the autoantibody of anti-HK1 and anti-KLHL7 in showing these PBC serum（Fig. 8）.

The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, on the premise of without departing from the technology of the present invention principle, some improvements and modifications can also be made, these improvements and modifications Also should be regarded as protection scope of the present invention.

Claims (7)

1. primary biliary cirrhosiss specificity autoantigen KLHL7 is preparing primary biliary liver as biomarker Application in hardening detectable.

2. application according to claim 1, it is characterised in that described detectable is to be coated with autoantigen KLHL7 Primary biliary cirrhosiss detection chip.

3. application according to claim 2, it is characterised in that described detection chip is also coated with BCOADC-E2, PDC- One kind or many in E2, OGDC-E2, E3BP, PDC-E1 β, PDC-E1 α, Gp210, p62, LBR, CENP-B, Sp100 and Sp140 Plant primary biliary cirrhosiss specificity autoantigen.

4. the application according to Claims 2 or 3, it is characterised in that described detection chip is also coated with Quality Control antigen.

5. application according to claim 4, it is characterised in that described Quality Control antigen is Mus IgG, human IgG and bird flu One or more in virus nucleoprotein.

6. application according to claim 1, it is characterised in that described detectable is ELISA detection kit, described ELISA detection kit contain ELISA Plate, the ELISA Plate is coated with autoantigen KLHL7.

7. application according to claim 6, it is characterised in that described ELISA Plate be also coated with BCOADC-E2, PDC-E2, One or more in OGDC-E2, E3BP, PDC-E1 β, PDC-E1 α, Gp210, p62, LBR, CENP-B, Sp100 and Sp140 Primary biliary cirrhosiss specificity autoantigen.