CN105527435B - Protein chip, protein-chip diagnostic kit method of preparation and use - Google Patents

Abstract

The present invention relates to protein chips, protein-chip diagnostic kit method of preparation and use, and the protein marker comprising following autoantigen protein: P53 antigen fragment, characteristic amino acid sequence is as shown in ID.1sequence；SOX2 antigen fragment, characteristic amino acid sequence is as shown in ID.2sequence；COPB1 antigen fragment, characteristic amino acid sequence is as shown in ID.3sequence；EFHD2 antigen fragment, characteristic amino acid sequence is as shown in ID.4sequence；EIF4G3 antigen fragment, characteristic amino acid sequence is as shown in ID.5sequence；PCNA antigen fragment, characteristic amino acid sequence is as shown in ID.6sequence.The present invention very convenient, quick, noninvasive, efficiently can early diagnose or generally investigate screening lung cancer suspected patient or High Risk of Lung Cancer crowd.

Description

Protein chip, protein-chip diagnostic kit method of preparation and use

Technical field

The present invention relates to a kind of for early diagnosing the autoantibody detection based on 6 kinds of lung cancer correlation autoantigens of lung cancer Protein-chip, and thus diagnostic kit derived from protein-chip.Invention further provides the protein cores Morning of the diagnostic kit derived from piece and thus protein-chip in lung cancer early diagnosis, healthy population or High Risk of Lung Cancer crowd Application in phase screening lung cancer.

Specifically, the present invention relates to a kind of protein-chip that can be used for Serologic detection early diagnosis human lung cancer, the egg White matter chip can pass through respective corresponding IgG type of the relevant autoantigen of 6 kinds of lung cancer of detection in tested person's blood sample With the relative amount of IgM type autoantibody, and Conjoint Analysis this 6 kinds of autoantigen corresponding antibodies detection as a result, to determine quilt Whether inspection people suffers from lung cancer.Further, the invention further relates to diagnostic kit derived from thus protein-chip, these diagnosis examinations Agent box is all made of or contains this protein-chip.In addition, the invention further relates to the protein-chip and derivative diagnostic reagents Application of the box in the early stage of lung cancer screening of early stage of lung cancer diagnosis, healthy population or High Risk of Lung Cancer crowd.

The present invention relates to a kind of protein chips, protein-chip diagnostic kit method of preparation and use.

Background technique

Lung cancer is one of the global death rate and the highest malignant tumour of disease incidence.According to the newest number at american cancer center According to, lung cancer mortality accounts for the 27% of cancer mortality total number of persons, than common colon cancer, breast cancer and cancer of pancreas death toll summation It is more.The World Health Organization in 2001 once publication warning: in past 10 years, the disease incidence of global lung cancer every year with 22% speed is rising very fast, and the annual whole world increases patients with lung cancer newly up to 1,200,000 people, dead 1,100,000 people.According to Beijing Municipal Health Bureau The statistical data of offer shows that lung cancer in 2010 occupies first of Beijing's household registration population's male malignancy morbidity, in female Second is occupied in property, is only second to breast cancer.2001 to 2010 years, Beijing's lung cancer morbidity rate increased 56%.The whole city newly diagnoses Having 1/5th in cancer out is lung cancer.It is predicted according to epidemiological specialist, if not controlling smoking and air pollution, to 2025 Year, China's patients with lung cancer will be more than 1,000,000, become the first in the world lung cancer big country.

Existing clinical related data shows that 5 annual survival rates of lung cancer are 16.6%, substantially less than other common cancers, such as Colon cancer (64.2%), breast cancer (89.2%) and prostate cancer (99.2%).The prognosis of lung cancer and the close phase of its developing stage It closes, limitation lung cancer five-year survival rate is 53%, and five-year survival rate is only 4% in the case where shifting.The morning of lung cancer It was found that the survival rate and quality of life of patient can be significantly improved with early prevention and treatment.

However, clinically only the lung cancer of 10%-15% can be in early stage due to lung cancer development early stage most non-evident sympton It is found.Due to early stage of lung cancer patient's Absence of physical signs, thus the patient overwhelming majority to go to see a doctor is middle and advanced stage patient.It improves early The diagnosis of phase lung cancer, lung cancer, by subclinical lung cancer or Occult Lung Cancer, screening is come out from crowd, for improving controlling for lung cancer More rate and the reduction death rate is of great significance.

The diagnosis of lung cancer at present is broadly divided into 2 classes: invasive diagnosis and non-invasive diagnostic.Invasive diagnosis includes puncturing The traumatic means such as biopsy, operations research；Non-invasive diagnostic includes various imaging diagnosis means (such as x-ray chest radiograph, CT, core Magnetic resonance, PET etc.), in cytolgical examination and various body fluid certain lung cancer or tumor markers detection.The intrusion of lung cancer Property diagnosis pain it is big, patient is unwilling to receive, and is normally only used for clinical antidiastole and makes a definite diagnosis.The most common diagnosis hand of lung cancer Section or non-invasive diagnostic are referring in particular to imaging diagnosis and cytodiagnosis at present.

40 years anterior part of chest X-rays are just as one of the diagnosis of the early stage of lung cancer and screening means, although this technology is time-consuming, Expense can find the negative early stage of lung cancer of some phlegm inspections also above cytolgical examination really, be better than sputum cytology.But X-ray It is typically only capable to the lung cancer that detection is greater than 1.5cm lesion, and it is equal to the lung cancer of more early stage, invisible, suspicious lung cancer and precancerous lesion It cannot detect.

From the British scientist Housfield seventies invention CT for lung cancer diagnosis since, advanced image documentation equipment and The application of technology is so that the recall rate to the early stage of lung cancer also increases.The U.S., some developed countries of Japan are by spiral in recent years CT technology is used for the screening of crowd's early stage of lung cancer.The early stage of lung cancer generaI investigation scheme in the U.S. in 1993 is to 1000 high-risk people of volunteer Group uses low-dose spiral CT screening, and discovery 233 has 1-6 non-calcified tubercles, wherein 27 are made a definite diagnosis lung cancer, the early stage of lung cancer 23 Example (85%), and same period X-ray rate of missed diagnosis is 83%.With the development of Medical Imaging, low-dose spiral CT is widely used in Detect lung's early stage tubercle.Faced according to the American National screening lung cancer that in June, 2011 is published on " New England Journal of Medicine " Bed test data shows that carrying out lung's screening using low-dose spiral CT makes the lung cancer lethality of people at highest risk reduce 20% (Aberle DR,et al.(2011)Reduced lung-cancer mortality with low-dose computed tomographic screening.N Engl J Med 365(5):395-409.).Therefore, National Cancer Institute pushes away Recommend the effective means that low-dose spiral CT is lung cancer early screening.Meanwhile National Cancer Institute also explicitly points out, low dose The problem of measuring spiral CT is the high false positive of its testing result, and repeatedly CT is detected it is also possible to accelerating the deterioration of lung cancer.

Another new technology PET to grow up in recent years has further the sensibility and accuracy of pulmonary cancer diagnosis again It improves.The various countries such as the U.S., Japan, Germany, Canada report the results show that PET differentiate it is pernicious with benign non-calcified tubercle Sensibility, specificity be above spiral CT, up to 85% and 90%.But PET cannot detect the pulmonary carcinosis less than 1.5cm Stove, without significant advantage in the diagnosis of the early stage of lung cancer, and equipment price is more expensive.

In conclusion existing iconography equipment and technology, can not be individually used for crowd under the current national conditions in China The screening and diagnosis of the early stage of lung cancer, it is high and sensitive to solve false positive rate to need to combine the early diagnosis of other lung cancer or screening technology The sometimes not high problem of property.But they have certain meaning in making a definite diagnosis the early stage of lung cancer.

Bronchofiberscope examines the application in pulmonary cancer diagnosis, is mainly used for phlegm inspection and x-ray chest radiograph notes abnormalities or CT It was found that the case of non-calcified tubercle, is examined by bronchial microscope and determines lesion, and take biopsy or washing lotion, be further carried out pathology Or cytology is made a definite diagnosis.Generally for experienced doctor, bronchial microscope examines the early carcinoma that can find about 30-40%, it is difficult to detect cancer Preceding lesion and carcinoma in situ.Occur fluorescence bronchoscope in recent years, aid in determining whether biopsy site, finds the early stage of lung cancer, and have Help judge range of operation, while precancerous lesion can also be detected.The U.S., Canada report the results show that its carcinoma in situ and cancer Preceding detection rate of pathological change is several times of common white light bronchial microscope.But this equipment price is expensive, to the requirement of operator's professional standards Very high, check fee is also expensive, is the invasive detection technique of pain, the central early stage of lung cancer and precancerous lesion, recall rate is higher, right Peripheral type carcinoma of lung recall rate is low.Therefore this technology and equipment is also impossible to early diagnose the technology with screening crowd as lung cancer Method.

Sputum cytology detection technique positive rate is low, and the recall rate of middle and advanced stage patient is 60-80%, and the early stage of lung cancer Recall rate only has 10-20%.In the lung cancer early detection experiment of U.S. NCI, the lung cancer less than 10% is individually sent out by cytology It is existing.Abroad in Recent Years develops a kind of new sputum cytology detection technique (ultra-thin cell cat scanner skill of ThinPrep i.e. liquid-based Art), third generation automation equipment AutocytePrep and AutoPap system has been developed at present, which can prepare pledge Very high ultra-thin cell sheet is measured, to improve the recall rate of phlegm inspection, 40- can be improved to the sensibility of early stage of lung cancer diagnosis 60%.But the technology needs expensive import equipment, and it is 100 yuans that the reagent of every part of sample disposal, which takes, operator Need higher professional standards.Foreign countries mainly apply in large hospital, and the report for crowd's early stage of lung cancer screening is very few.Domestic mesh There are this equipment in preceding only several metropolitan few hospitals, have carried out this technology.It therefore, can not under the existing national conditions in China For lung cancer early diagnosis and big screening.In addition, the fatal defects of the technology are for peripheral and peripheral type early stage lung Cancer recall rate is lower.

With the rapid development of molecular biology, study pulmonary carcinosis because while, also lung cancer is sent out on a molecular scale Hair tonic exhibition has more understandings.Existing research shows that during the multistage of lung cancer occurrence and development, the evil of cellular morphology Sexual biology, which changes, to be participated in and changes along with the several genes of its molecular level, they include the amplification of oncogene, excessive table It reaches, be mutated and the change of the missing of tumor suppressor gene, mutation, transposition, rearrangement etc. several genes.Up to the present, reported There are about more than ten for gene unconventionality change relevant to lung cancer generation.Most common oncogene change have ras family, myc family, Overexpression of HER2, Bcl-2 etc..The prominent of the tumor suppressor genes such as P53, Rb, CDKN2, DuTT1, FHIT, BAP-1 is found simultaneously Change, missing, transduction, transposition and methylation etc. change.The analysis of cytogenetics and Protocols in Molecular Biology, it was found that Duo Geran The change of certain genes and the occurrence and development of lung cancer are closely related on colour solid.In recent years it has also been found that Telomerase, hnRNPA2/B1 have Higher canceration prediction significance.Be also found in recent years some unconventionality expressions in lung cancer or existing miRNA (tiny RNA, MicroRNA, miRNA) and LncRNA (long-chain non-coding RNA, Long non-coding RNA, LncRNA)).To detect this The change of a little genes, is established and the different kinds of molecules Biological Detection technology that grows up in succession, such as various round pcr (RT- PCR.PCR-SSCP.PCR-RFLPs), various nucleic acid hybridization technique (Southern, Northern, Comparative genomic hybridization Deng) and direct Sequencing technology etc. and fluorescence in situ hybridization technique developed in recent years (FISH) and microsatellite polymorphism Analytical technology etc..But these technologies are both needed to the acquisition dependent on lesion tissue or sick cell, positive sample to the diagnosis of lung cancer Acquisition have biggish difficulty, and be difficult to collect required sample in the early stage of lung cancer, make these technologies in lung It is difficult to play corresponding effect in the early diagnosis of cancer.The diagnostic effect of the DNA or RNA marker of some blood types is not yet received The ability of the verifying of large-scale crowd detection, early diagnosis lung cancer remains very big query.In addition these technologies need special Reagent and instrument, complicated for operation, expensive, its sensibility, specificity and accuracy are still difficult to come to a conclusion, at present both at home and abroad not Using these technologies as the means and method of the mass survey screening early stage of lung cancer.

About have discovered that the blood type lung cancer biomarkers of more than 60 protides at present, they or it is straight with human lung cancer Correlation is connect, or related to different types of lung cancer or related to lung cancer for prognosis or related to lung cancer metastasis, or and lung cancer clinical It is by stages related or related to drug resistance of lung cancer etc..These markers include: ACE, ADH, AFP, AgNORs, ANP, AHH, blood group antigens、BN/GRP、CA125、CA19-9、CA242、CA50、Cadherins、Clcitonin、Cathepsin B、CC10、CEA、Cholecystokinin-B/gastrin receptor、CK-BB、c-N-L-myc、Cyclin D1、CYFRA 21-1、CYFRA21.1、CYP1A1/GSTM1、EGFR、Epithelial glycoprotein 1、Epithelial glycoprotein 2、Ferritin、Ganglioside fucosyl-GM1(FucGM1)、Glutathione-S- transferase、HER2、Heterogeneous nuclear ribonucleoprotein A2/B1、H-K-N-ras、IGF- I、IL-2R、Integrins、Ki-67、Lewis antigens、Le-y、MMP-9、MUC 1、NCAM、NeuAc、NSE、P53、 PCNA、Peptid hormones ACTH、Phosphopyruvate Hydratase、pro GRP、p21、SCC antigen、 sialyl SSEA-1、SLX、TA-4、Telomerase、TGF-a、Thymidine kinase、TPA、Transferrin、uPA Etc. (Kulpa, J., Wojcik, E., Radkowski, A., Kolodziejski, L., and Stasik, Z.CYFRA 21-1, TPA-M,TPS,SCC-Ag and CEA in patients with squamous cell lung cancer and in chemical industry workers as a reference group.Anticancer Res.,2000,20(6D): 5035-5040.Nguyen,V.N.,Mirejovsky,T.,Melinova,L.,and Mandys,V.CD44and its v6spliced variant in lung carcinomas:relation to NCAM,CEA,EMA and UP1and prognostic significance.Neoplasma,2000,47(6):400-408.Alatas,F.,Alatas,O., Metintas,M.,Colak,O.,Harmanci,E.,and Demir,S.Diagnostic value of CEA,CA 15-3, CA 19-9,CYFRA 21-1,NSE and TSA assay in pleural effusions.Lung Cancer,2001,31 (1):9-16.Foa,P.,Fornier,M.,Miceli,R.,Seregni,E.,Santambrogio,L.,Nosotti,M., Massaron,S.,Cataldo,I.,Oldani,S.,Iurlo,A.,Caldiera,S.,and Bombardieri, E.Preoperative CEA,NSE,SCC,TPA and CYFRA 21.1serum levels as prognostic indicators in resected non-small cell lung cancer.Int.J.Biol.Markers,1999,14 (2):92-98.Graziano,S.L.,Kern,J.A.,Herndon,J.E.,Tatum,A.,Brisson,M.L.,Memoli, V.,Sugarbaker,D.,Skarin,A.T.,Kreisman,H.,and Green,M.R.Analysis of neuroendocrine markers,HER2and CEA before and after chemotherapy in patients with stage IIIA non-small cell lung cancer:a Cancer and Leukemia Group B study.Lung Cancer,1998,21(3):203-211.Abe,S.[Molecular biological prognostic markers in lung cancer].Nippon Geka Gakkai Zasshi,1997,98(1):2-7.Fukuda,M.and Oka,M.[Usefulness and limitation of serum tumor markers in diagnosis of lung cancer].Nippon Rinsho,1996,54(6):1610-1615.Moro,D.,Villemain,D.,Vuillez,J.P., Delord,C.A.,and Brambilla,C.CEA,CYFRA21-1and SCC in non-small cell lung cancer.Lung Cancer,1995,13(2):169-176.Giovanella,L.,Ceriani,L.,Bandera,M., Beghe,B.,and Roncari,G.Evaluation of the serum markers CEA,NSE,TPS and CYFRA 21.1in lung cancer.Int.J.Biol.Markers,1995,10(3):156-160.Niklinski,J.and Furman,M.Clinical tumour markers in lung cancer.Eur.J.Cancer Prev.,1995,4(2): 129-138. Chinese turns over pool: Kulpa, J., Wojcik, E., Radkowski, A., Kolodziejski, L., and Stasik, Z.CYFRA 21-1, TPA-M, TPS, SCC-Ag and CEA are in prognosis of squamous cell lung cancer patient and chemical industry worker control crowd The case where anticancer study, 2000,20 (6D): 5035-5040.Nguyen, V.N., Mirejovsky, T., Melinova, L., and Mandys, V.CD44 and its v6 splice variant are in lung cancer: with NCAM, CEA, EMA and UP1 and prognostic factor Related neoplasm, 2000,47 (6): 400-408.Alatas, F., Alatas, O., Metintas, M., Colak, O., Harmanci, E., and Demir, S.CEA, CA 15-3, CA 19-9, CYFRA 21-1, NSE and TSA are tested in hydrothorax Diagnostic value lung cancer, 2001,31 (1): 9-16.Foa, P., Fornier, M., Miceli, R., Seregni, E., Santambrogio,L.,Nosotti,M.,Massaron,S.,Cataldo,I.,Oldani,S.,Iurlo,A., Caldiera, S., and Bombardieri, the E. preoperative CEA in resectability non-small cell lung cancer, NSE, SCC, TPA and 21.1 serum levels of CYFRA can be used as prognostic indicator biomarker International Periodicals, 1999,14 (2): 92- 98.Graziano,S.L.,Kern,J.A.,Herndon,J.E.,Tatum,A.,Brisson,M.L.,Memoli,V., Before Sugarbaker, D., Skarin, A.T., Kreisman, H., and Green, M.R.IIIA phase Treatment of non-small-cell lung cancer with chemotherapy The analysis of neuroendocrine mark afterwards, HER2 and CEA: cancer and lymthoma group B study lung cancer, 1998,21 (3): 203- 211.Abe, S. [the molecular biology prognostic indicator of lung cancer] Japan Geka Gakkai magazine, 1997,98 (1): 2- 7.Fukuda, M.and Oka, M. [effect and deficiency of the blood serum tumor markers in pulmonary cancer diagnosis] Japan Rinsho, 1996,54(6):1610-1615.Moro,D.,Villemain,D.,Vuillez,J.P.,Delord,C.A.,and Brambilla, C.CEA, CYFRA21-1 and SCC the lung cancer in non-small cell lung cancer, 1995,13 (2): 169- 176.Giovanella, L., Ceriani, L., Bandera, M., Beghe, B., and Roncari, G. blood serum designated object CEA, The assessment biomarker International Periodicals that NSE, TPS and CYFRA 21.1 is applied in lung cancer, 1995,10 (3): 156- The Europe the clinical tumor marker cancer periodical Prev. of 160.Niklinski, J.and Furman, M. lung cancer, 1995,4 (2):129-138.)。

In these lung cancer associated biomarkers, majority belongs to the type positioned at endochylema, AFP, CA125, CA19- in addition 9、CA242、CA50、Cadherins、Cholecystokinin-B/gastrin receptor、Epithelial glycoprotein 1、Epithelial glycoprotein 2、HER2、CEA、Integrins、Lewis antigens、 The markers such as MUC 1, NCAM are located at lung carcinoma cell surface, are roughly divided into three categories, receptor class, cell surface adhesion molecule and thin Cellular surface glycoprotein.Generally speaking, the meaning of the independent diagnosing of these markers is not too high, but joint-detection still has The meaning of certain diagnosis, judging prognosis.For example, positive rate of the NSE in Small Cell Lung Cancer is 60-85%, CEA reaches 50- 60%, and HER2 only up to 30-50%.

Above method is the antigen, albumen or gene to change in direct test sample.It is bright with the above detection method It is aobvious different, the antibody for tumour antigen or autoantigen, the phase of certain antigens are contained in discovered in recent years tumour patient blood It answers distribution of the antibody in normal person and tumour patient that certain specificity is presented, may have certain diagnostic value.Early in The sixties in last century, Robert W.Baldwin just have confirmed that early stage cancer development, human immune system can produce Autoantibody, but due to the limitation of technology, autoantibody is difficult to be detected early stage cancer development.It has been found that by In the sensibility and stability of immune system, lung cancer autoantibody can before imaging diagnosis goes out lung cancer 5 years in blood by Detect that (autoantibody molecule applies Jia Jing, Ren Juan, Huang Xiaoming, Ma Shenglin in lung cancer early diagnosis, and it is practical that is lifted in king's filial piety Medical journal the 2nd phase of volume 29 in 2013,319-321.).Chip is made with the protein isolate of lung cancer cell line A549 in Qiu etc., Serum sample in first 1 year is made a definite diagnosis to lung cancer and normal control serum sample detects, finds annexin I, 14-3- The autoantibody of 3theta and LAMR1 just has existed in asymptomatic stage, and autoantibody detection is prompted to can be used as High Risk of Lung Cancer people A kind of method (Qiu J, Choi G, Li L, et al.Wang H, Occurrence of of group's screening early diagnosis Autoantibodies to Annexin I,14-3-3Theta and LAMR1in Prediagnostic Lung Cancer Sera.J Clin Oncol.Nov 1,2008；26(31):5060–5066.).Chapman etc. is compared using enzyme-linked immunization Using 6 (p53, NY-ESO-1, CAGE, GBU4-5, Annexin I and SOX2) and 7 (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, HuD and MAGE A4) autoantibody detects corresponding autoantibody in patients with lung cancer and healthy control group serum Content.Monospecific antibody detects positive rate between 5~36% as the result is shown, and specificity is 96~100% (Autoantibodies in lungcancer:possibilities for early detection and subsequent cure.Thorax.2008；63:228–233.).Although the antigen of these presently found types is in neoplastic disease Positive rate is low in people, and the sensitivity and accuracy diagnosed when being used alone is all inadequate, cannot function as a kind of new diagnostic method, Meaning without individual diagnosing.But while joint-detection lung cancer related auto-antibodies marker spectrum in human body Expression, but can achieve improve pulmonary cancer diagnosis sensibility and accuracy purpose.The research and development of Univ Nottingham UK OutAnd the diagnostic products for becoming registration are released by Oncimmune company.It is one group of 7 correlation The product of autoantibody tumor markers (p53, NY-ESO-1, CAGE, GBU4-5, SOX2, HuD and MAGE A4) joint-detection, From 2009 since U.S.'s listing, used in more than 100,000 High Risk of Lung Cancer patients via 2000 doctors, the product 2010 U.S. clinical tumour association (ASCO) with 2010 American Thoracic doctor annual meeting (ACCP) on be used for by keypoint recommendation Lung cancer early stage auxiliary diagnosis.Sensibility be reported in early stage small-scale clinical examination according to 2008 Reach 76% in testing, specificity is 92% (Chapman CJ, et al. (2008) Autoantibodiesin lung cancer:possibilities for early detection and subsequentcure.Thorax 63(3):228- 233.).In the detection of 1600 people at highest risk delivered in 2013, sensibility 46%, and it is quick in I phase and II phase patient Perception reaches 57%, specificity for 91% (Jett JR, et al. (2014) Audit of the autoantibody test, EarlyCDT(R)-lung,in 1600 patients:an evaluation of itsperformance in routine clinical practice.Lung cancer 83(1):51-55.).Therefore,Sensibility have Wait further increase.Other one product for carrying out detection of early lung cancer using autoantibody is GENESYS BIOLABS Paula ' the s test of HAWAII company, withIt is significantly different, contained by detecting 3 kinds of tumor antigen proteins Amount and a kind of lung cancer autoantibody content carry out diagnosing, and the sensibility of Preliminary report reaches 74%, and specificity is 80%, should It still needs further improvement for the specificity of kit.

In conclusion common lung cancer screening diagnostic techniques external at present is phlegm in above-mentioned numerous pulmonary cancer diagnosis technology The technologies such as cytolgical examination, X-ray, recall rate be not still high；And low-dose spiral CT false positive rate is excessively high, needs other joints The diagnostic method or means of application.The technical method of various molecular markers is detected from blood, have it is easy, economical, quickly, It is noninvasive it is painless be easy to the characteristics of receiving, but still lack the higher marker of sensibility and specificity or marker combination at present Spectrum.The change of several genes and nucleic acid molecules that lung cancer is related to is difficult to detect in serum.Have at present some about use Multiple proteins antigen detects the content of corresponding antibodies in serum, and judges whether tested person suffers from the report of lung cancer with this, but There is not yet the protein-chip of polyprotein sample is used to detect the report of corresponding antibodies content in human serum, it is most important that Autoantibody markers spectrum is needed to be optimized to improve the sensibility or specificity of the checkout and diagnosis early stage of lung cancer, is allowed to Conscientiously for clinical early diagnosis lung cancer or the screening lung cancer in High Risk of Lung Cancer crowd.

Summary of the invention

In view of the deficiency of the prior art, the present invention one kind is provided can be very convenient, quick, noninvasive, efficiently Protein chip, the protein-chip diagnostic kit system of early diagnosis or generaI investigation screening lung cancer suspected patient or High Risk of Lung Cancer crowd Standby and application method.

The technical solution used in the present invention is:

A kind of protein-chip includes but not only comprising the protein marker of following 6 kinds of lung cancer autoantigen proteins: (1) P53 antigen fragment, characteristic amino acid sequence is as shown in ID.1sequence；(2) SOX2 antigen fragment, feature amino Acid sequence is as shown in ID.2sequence；(3) COPB1 antigen fragment, characteristic amino acid sequence is as shown in ID.3sequence； (4) EFHD2 antigen fragment, characteristic amino acid sequence is as shown in ID.4sequence；(5) EIF4G3 antigen fragment, feature Amino acid sequence is as shown in ID.5sequence；(6) PCNA antigen fragment, characteristic amino acid sequence such as ID.6sequence institute Show.

The protein marker of the autoantigen protein matter is the protein for recombinating expression and purification.

A kind of protein-chip diagnostic kit, comprising: detection slide, detection correction slide, sample diluting liquid, washing Liquid, fluorescence secondary antibody；

In the recombinant protein or again that expression in escherichia coli includes 6 kinds of antigen property amino acid sequences of the present invention Group fusion protein, these target proteins of purifies and separates make its purity reach 80% or more, in each antigen and human IgG, people IgM Middle that suitable biotinylated BSA is added, with suitable concentration point sample in the glass substrate handled through aldehyde radical, antigen will be total to Valence is crosslinking on substrate, closes remaining crosslink sites using cow's serum, and low temperature drying saves after drying, obtains detection glass Piece；

Human IgG and human IgM antibody standard items are subjected to doubling dilution, select suitable concentration gradient point sample in through aldehyde radical On the glass chip of processing, covalent cross-linking on substrate, is closed remaining crosslink sites using cow's serum by antibody standard substance, Low temperature drying saves after drying, obtains detection correction slide；

PBS (0.01mol/Lm, pH7.4) containing 10% cow's serum is sterile filtered and is bottled, sample diluting liquid is obtained；

PBS (0.01mol/Lm, pH7.4) containing 0.02%Tween20 is sterile filtered and is bottled, cleaning solution is obtained；

By Streptavidin-Cy5 fluorescent marker secondary antibody, Streptavidin-Cy3 fluorescent marker two containing acceptable diluent degree Anti-, goat anti-human igg's-Cy5 fluorescent marker secondary antibody and the bottling of goat-anti people's IgM-Cy3 fluorescent marker secondary antibody, obtain fluorescence secondary antibody.

A kind of preparation method of protein-chip diagnostic kit, comprising:

Detect slide preparation step；Detection correction slide preparation step；Sample diluting liquid preparation step；Cleaning solution preparation step Suddenly；Fluorescence secondary antibody preparation step；

The detection slide preparation step is as follows；

In the recombinant protein or again that expression in escherichia coli includes 6 kinds of antigen property amino acid sequences of the present invention Group fusion protein, these target proteins of purifies and separates make its purity reach 80% or more, in each antigen and human IgG, people IgM Middle that suitable biotinylated BSA is added, with suitable concentration point sample in the glass substrate handled through aldehyde radical, antigen will be total to Valence is crosslinking on substrate, closes remaining crosslink sites using cow's serum, low temperature drying saves after drying；

Sample diluting liquid preparation step；

Human IgG and human IgM antibody standard items are subjected to doubling dilution, select suitable concentration gradient point sample in through aldehyde radical On the glass chip of processing, covalent cross-linking on substrate, is closed remaining crosslink sites using cow's serum by antibody standard substance, Low temperature drying saves after drying；

Cleaning solution preparation step；

PBS (0.01mol/Lm, pH7.4) containing 0.02%Tween20 is sterile filtered and is bottled；

Sample diluting liquid preparation step；

PBS (0.01mol/Lm, pH7.4) containing 10% cow's serum is sterile filtered and is bottled；

Fluorescence secondary antibody preparation step；

By Streptavidin-Cy5 fluorescent marker secondary antibody, Streptavidin-Cy3 fluorescent marker two containing acceptable diluent degree Anti-, goat anti-human igg's-Cy5 fluorescent marker secondary antibody and the bottling of goat-anti people's IgM-Cy3 fluorescent marker secondary antibody.

The chain of fluorescent marker is added in the point sample area of detection slide in a kind of application method of protein-chip diagnostic kit Moisturizing is incubated for 1 hour at 37 DEG C of mould Avidin, is washed 3 times using cleaning solution；

Tested human serum is taken into 2 μ l, 20 μ l are diluted to sample diluting liquid, takes 10 μ l to be added on detection slide and covers point sample Area, moisturizing is incubated for 1 hour at 37 DEG C；

It takes the dilution of 10ul to be added on detection correction slide and covers point sample area；

It is washed 3 times using cleaning solution, the anti-human igg or people IgM fluorescence two different from marked by streptavidin fluorescence is added Anti-, moisturizing is incubated for 1 hour at 37 DEG C, is washed 5 times using cleaning solution, is read on laser co-focusing chip projection reading set after dry each The color and intensity of point of sample fluorescence.

Carcinomebryonic antigen (CEA) is also put on detection slide.

The present invention compared with the prior art the utility model has the advantages that

Protein chip of the present invention, protein-chip diagnostic kit method of preparation and use, for being clinically suspect to be The examinee of lung cancer can extract its blood sample, using the protein-chip according to the present invention for diagnosing human lung cancer and its spread out Raw diagnostic kit, detects the content of 6 kinds of antigen corresponding antibodies described in tested human blood, and then whether judges examinee For patients with lung cancer.Due to the difference of the various controls of detection method or kit, judge examinee whether be the standard of lung cancer simultaneously It is not unique, once examinee is used the diagnostic method that present invention judgement is related to or the judgement of the kit as derived from this method It is practical patients with lung cancer with lung cancer, then to answer strong suspicion examinee, and carries out various clinics really using various detection means It examines, if clinic cannot find lesion, then the case where answering close follow-up visit monitoring examinee lung cancer occurrence and development.

The High Risk of Lung Cancer crowd for common healthy population or medically defined, then can use according to the present invention The protein-chip and its derivative diagnostic kit for diagnosing human lung cancer carry out generaI investigation screening to them, extract the blood of tested crowd Liquid sample, determines whether it suffers from lung cancer according to the above method, once examinee is used the diagnostic method that present invention judgement is related to Or the kit as derived from this method is judged as with lung cancer, then should carry out various clinical definites using various detection means, such as The case where clinic cannot find lesion, then answer close follow-up visit monitoring examinee lung cancer occurrence and development.

The protein-chip and its derivative diagnostic kit of new diagnosis human lung cancer provided by the invention, then can be very Convenient, fast, noninvasive, efficiently early diagnosis or generaI investigation screening lung cancer suspected patient or High Risk of Lung Cancer crowd, and participation inspection The normal health crowd of survey.

Specific embodiment

The present invention will be described in detail for following embodiment:

The first purpose of this invention is to provide a kind of new protein-chip that can be used for early diagnosing human lung cancer, be somebody's turn to do Protein-chip includes but is not that only can detecte by the protein-chip tested comprising 6 kinds of specific proteantigens In human blood (serum, blood plasma or blood components) the corresponding IgG type of this 6 kinds specific lung cancer correlation autoantigens and IgM type from The content of body antibody is that this 6 kinds of antigen corresponding antibodies of Conjoint Analysis detect as a result, can be used to determine whether tested person suffers from lung Cancer.

Second object of the present invention is to provide a kind of examination for early diagnosing lung cancer as derived from above-mentioned protein-chip Agent box.

Third object of the present invention be the early diagnosis human lung cancer protein-chip and its derivative diagnosis Application of the kit in lung cancer early diagnosis, healthy population or High Risk of Lung Cancer crowd's screening lung cancer.

According to the first aspect of the invention, the present invention provides it is a kind of early diagnose human lung cancer protein-chip,

1) include but not only include following 6 kinds of lung cancer autoantigen proteins protein marker: (1) P53 antigen fragment, Its characteristic amino acid sequence is as shown in ID.1sequence；(2) SOX2 antigen fragment, characteristic amino acid sequence is such as Shown in ID.2sequence；(3) COPB1 antigen fragment, characteristic amino acid sequence is as shown in ID.3sequence；(4)EFHD2 Antigen fragment, characteristic amino acid sequence is as shown in ID.4sequence；(5) EIF4G3 antigen fragment, characteristic amino acid sequence Column are as shown in ID.5sequence；(6) PCNA antigen fragment, characteristic amino acid sequence is as shown in ID.6sequence；

2) test sample is the blood sample of tested person,

3) the corresponding IgG type and IgM type itself of 6 kinds of autoantigen proteins in sample of the detection as described in 2) as described in 1) The relative amount of antibody,

4) whether tested person may determine that by the testing result of the corresponding autoantibody of 6 kinds of autoantigen proteins of Conjoint Analysis For lung cancer.

In the present invention, the autoantigen protein refers to any polypeptide, egg comprising above-mentioned amino acid residue sequence White matter or fused protein.Because the present invention is antibody that can be in combination using the detection of these proteantigens, As long as being technically it will be apparent that using any polypeptide, protein or fusion protein comprising above-mentioned amino acid residue sequence Matter can have almost the same immunological characteristic, so that can be used for detection can be in conjunction with these proteantigens Antibody.

6 kinds of antigen of the present invention is through using mixing human lung cancer primary lesion tissue (including 2 adenocarcinomas of lung, 2 lungs Squamous carcinoma, 2 Small Cell Lung Cancer) expression cDNA library is established, and be immunized self with allosome lung cancer patient serum by 30 Trace screening, cut down inhibit screening by hybridization, go Ig background screen after obtain there are corresponding antibodies in lung cancer patient serum Antigen is screening 3 × 10⁶After the library of pfu, obtaining 102 altogether can be with the proteantigen of lung cancer patient seroreaction Clone.

The gene cloning of above-mentioned antigen is entered into the prokaryotic expression carrier pET30 (b) with correct reading frame, is lured by IPTG Expression is led, inclusion body and preliminary purification is extracted, obtains the recombinant protein of the LuCA.Using above-mentioned recombinant protein, with suitable Concentration point is measured on immunoblotting film, each 10 using lung cancer and normal human serum, and detected using Immunoblot, it chooses The positive rate of the positive rate and lung cancer patient that select normal person has the clone of larger difference.According to said method obtain 24 and normal person The positive rate and lung cancer patient of seroreaction have the antigen clone of larger difference.

By the recombinant protein of the antigen of aforementioned acquisition using biochip point sample machine point sample in the glass substrate of aldehyde radical, Every concentration about 1mg/ml, point sample amount about 8nl.It is detected in tested serum using the method for immunofluorescence for these antigens Human antibody.200 lung cancer patient serum and 200 normal human serums are had detected using the above method altogether, the results show that wherein 6 There are biggish difference, connection for corresponding IgG type and IgM type autoantibody of the kind antigen in lung cancer patient serum and normal human serum The sensitivity of analyzing and diagnosing lung cancer is closed up to 89.0%, and specificity is also up to 85.5%.This 6 kinds of antigens are respectively: (1) P53 Antigen fragment, (2) SOX2 antigen fragment, (3) COPB1 antigen fragment, (4) EFHD2 antigen fragment, (5) EIF4G3 antigen fragment, (6) PCNA antigen fragment.The process data for screening the marker spectrum of 6 kinds of lung cancer correlation autoantigens above shows in use It states 6 kinds of proteantigens and detects the content of corresponding IgG type and IgM type autoantibody in above-mentioned 6 kinds of antigen blood, and carry out Conjoint Analysis, judges whether tested person suffers from lung cancer sensitivity with higher for pulmonary cancer diagnosis and specificity, has very Important application value.Further, it is detected using serum of the same procedure to the patient of 200 lung's benign lesions, As a result autoantibody markers combination (spectrum) has preferable specificity, only 18% vacation to lung's benign lesion is distinguished Positive rate, antidiastole work well.

In conclusion the present invention provides a kind of new new protein-chip that can be used for diagnosing human lung cancer, the albumen Matter chip can be used for joint-detection and analyze containing for the corresponding IgG type of 6 kinds of specific antigens and IgM type autoantibody in tested human blood Amount, to judge whether tested person is patients with lung cancer.The protein-chip uses the blood sample of tested person for test sample, can Detect the corresponding IgG type and IgM type autoantibody in the sample opposite of 6 kinds of specific antigens respectively using various suitable methods Content, the relative amount of every kind of antigen corresponding antibodies have a corresponding threshold value, are detected blood sample and are higher than (or being lower than) this value It is judged as the positive, the sum of this corresponding autoantibodies of 6 kinds of antigens of Conjoint Analysis, when 12 kinds of autoantibodies of tested person When the value of (IgG type+IgM type) positive sum is greater than a corresponding threshold value, which is judged to the positive, that is, is diagnosed as lung Cancer patient.The protein-chip is verified in the patients with lung cancer of 200 people and the normal person of 200 people, sensitivity 89.0%, Accuracy is 85.5%.The innovation of the protein-chip is: 1) detect index be 6 kinds of antigens corresponding IgG type and IgM type autoantibody is for the content being detected in human serum, relatively direct detection antigen itself, and the corresponding antibodies of antigen are in lung The early stage of carcinogenesis can generate, and perhaps antigen cannot still be detected in blood at this time, thus than directly detecting antigen Say that diagnosable lung cancer may be smaller；The corresponding antibodies of antigen are easier to enter blood circulation and be detected, while human body can To generate considerable corresponding antibodies to trace antigen, there is certain amplification；In addition, it is very important that in addition to normal Except the IgG type autoantibody of rule, we have detected this 6 kinds of autoantigens generation corresponding IgM autoantibody the case where, this In the past without relevant report, our correlative study early period result is prompted, and detection IgM type autoantibody efficiently contributes to send out The now tumor patient of early stage, this makes this kit just have apparent novelty in principle.2) protein-chip is adopted There are 4 kinds (COPB1 antigen, EFHD2 antigen, EIF4G3 antigen, PCNA antigens) to have not yet to see pulmonary carcinosis in 6 kinds of antigens There are the reports of corresponding antibodies in human body, also there is not yet its report that is used to diagnose, detect lung cancer, so we itself is anti- Former marker marker combination (spectrum) has apparent innovative.3) result judgement is in 12 kinds of autoantibody (IgG of Conjoint Analysis Type+IgM type) relative amount testing result on the basis of make, there is better sensitivity and accuracy.The present invention is adopted 12 kinds of autoantibodies individually detect the sensitivity of lung cancer and accuracy is not very high, but this method is examined after Conjoint Analysis The sensitivity and accuracy of disconnected lung cancer all greatly improve, and have reached a clinically-acceptable level.

According to the second aspect of the invention, the reagent of a kind of diagnosing as derived from above-mentioned protein-chip is provided Box, the kit of this kind of diagnosing can be used for non-invasively diagnosing human lung cancer.This kind of diagnostic kit uses but is not Only with new protein-chip provided by the invention, in fact, these kits can in the known range in this field, In addition some other control judges to carry out the standardization of result, or included in protein-chip provided by the invention On the basis of 6 kinds of proteantigens, other Testing index are further added by combine judgement.In addition, lung cancer provided by the present invention Kit derived from diagnostic method can also detect each antigen phase using the multiple technologies means in the known range in this field The content for answering antibody, achieves the object of the present invention, such as immunofluorescence, immunoblotting, radio-immunity, Enzyme-linked Immunosorbent Assay etc., Detect antigen corresponding antibodies means difference will not influence to the tested person whether be patients with lung cancer judgement.

The protein-chip of the new diagnosis human lung cancer provided according to the present invention, it is easy to which acquisition is spread out by the protein-chip Raw various diagnostic kits.The core key of the protein-chip provided by the invention that can be used for pulmonary cancer diagnosis is the chip On included 6 kinds of proteantigens type (the specific amino acid sequence of protein), so as to pass through the protein core Piece detects the content of this corresponding IgG type of 6 kinds of specific antigens and IgM type autoantibody in tested human blood, from the art Common knowledge be easy to release, using the content of which kind of technological means detection antigen corresponding antibodies, and which kind of uses The existence form (native antigen, recombinant protein antigen, synthesis polypeptide) of antigen will not influence this method to tested person to detect Whether the judgement of lung cancer is suffered from；As long as also, containing the 6 of detection the method for the invention in any pulmonary cancer diagnosis kit The Testing index of the corresponding antibodies of a antigen, the sensitivity of kit detection lung cancer and specificity should just be at least up to this hair The sensitivity and accuracy of the bright method being related to, extraly increasing the relevant Testing index of other lung cancer generally can be in the present invention Diagnostic sensitivity and accuracy are further increased on the basis of the method being related to.Name it is some it is simple preparation the present invention relates to Method of lung cancer diagnosis derived from kit example, derivative reagent box can be prepared by the following, but be not limited solely to down Column method.

In the recombinant protein or again that expression in escherichia coli includes 6 kinds of antigen property amino acid sequences of the present invention Group fusion protein, these target proteins of purifies and separates make its purity reach 80% or more, in each antigen and human IgG, people IgM Suitable biotinylated BSA is added in (both the above be control, to carry out lateral reference), with suitable concentration point sample in In the glass substrate handled through aldehyde radical, covalent cross-linking on substrate, is closed remaining crosslinking position using cow's serum by antigen Point, low temperature drying saves after drying, and here it is the detection slides in kit；Human IgG and human IgM antibody standard items are carried out again Than dilution, select suitable concentration gradient point sample on the glass chip handled through aldehyde radical, antibody standard substance is by covalent cross-linking On substrate, remaining crosslink sites are closed using cow's serum, low temperature drying saves after drying, and here it is the inspections in kit Survey correction slide；PBS (0.01mol/Lm, pH7.4) containing 10% cow's serum is sterile filtered and is bottled, here it is sample dilutions Liquid；PBS (0.01mol/Lm, pH7.4) containing 0.02%Tween20 is sterile filtered and is bottled, here it is cleaning solutions；It will be containing suitable Streptavidin-Cy5 fluorescent marker secondary antibody, the Streptavidin-Cy3 fluorescent marker secondary antibody, goat anti-human igg-Cy5 of suitable dilution Fluorescent marker secondary antibody and the bottling of goat-anti people's IgM-Cy3 fluorescent marker secondary antibody, here it is fluorescence secondary antibodies.Above five kinds of ingredients can group A kind of pulmonary cancer diagnosis kit as derived from the method for the invention is dressed up,

In application, moisturizing is incubated for 1 hour at fluorescent marker is added in the point sample area of detection slide 37 DEG C of Streptavidin, It is washed 3 times using cleaning solution.Tested human serum is taken into 2 μ l, 20 μ l are diluted to sample diluting liquid, 10 μ l is taken to be added in detection slide Upper covering point sample area, moisturizing is incubated for 1 hour at 37 DEG C；And it takes the dilution of 10ul to be added on detection correction slide and covers point sample Area.It is washed 3 times using cleaning solution, the anti-human igg or people IgM fluorescence secondary antibody (example different from marked by streptavidin fluorescence is added Such as, Streptavidin is Cy-3 label, then goat anti-human igg's-Cy5 fluorescent marker secondary antibody is added), moisturizing is incubated for 1 hour at 37 DEG C, It is washed 5 times using cleaning solution, reads the color and intensity of each point of sample fluorescence after dry on laser co-focusing chip projection reading set. The fluorescence intensity of each antigen point of sample represents the relative amount of corresponding antibodies, and biotinylated BSA is reacted with Streptavidin Fluorescence intensity can be used as the internal reference of antigen-antibody reaction, human IgG, IgM fluorescence intensity can be used as each tested person and laterally join The control of ratio, and can be used as by the standard curve that the fluorescence intensity of the human IgG of different dilutions or IgM antibody standard items is drawn anti- The quantitative criteria of body content and batch between the control reacted.By each antigen fluorescence intensity of the Threshold Analysis of each antigen predefined Data, can determine whether the tested person antigen corresponding antibodies are positive, the sun for 12 kinds of autoantibodies (IgG type+IgM type) of adding up Property number, further according to predetermined number positive threshold value i.e. can determine whether the tested person is patients with lung cancer.In above-mentioned derivative reagent On the basis of box, it can be further added by Testing index, for example, carcinomebryonic antigen (CEA) is additionally also put as a kind of antigen on slide, Another kit can be derived, and the sensitivity and accuracy of the kit diagnosing can be can increase.

According to the third aspect of the invention we, the present invention also provides the protein-chip of the diagnosis human lung cancer and its spread out Application of the raw diagnostic kit in lung cancer early diagnosis, healthy population or High Risk of Lung Cancer crowd's screening lung cancer.

For the examinee for being clinically suspect to be lung cancer, its blood sample can be extracted, is examined using according to the present invention The protein-chip and its derivative diagnostic kit of disconnected human lung cancer detect 6 kinds of antigens described in tested human blood and accordingly resist The content of body, and then judge whether examinee is patients with lung cancer.Due to the difference of the various controls of detection method or kit, sentence It is not unique that whether disconnected examinee, which is the standard of lung cancer, once examinee is used the diagnostic method that present invention judgement is related to Or the kit as derived from this method is judged as with lung cancer, then answering strong suspicion examinee is practical patients with lung cancer, and is used Various detection means carry out various clinical definites, if clinic cannot find lesion, then close follow-up visit monitoring examinee lung cancer are answered to send out The case where hair tonic exhibition.

The High Risk of Lung Cancer crowd for common healthy population or medically defined, then can use according to the present invention The protein-chip and its derivative diagnostic kit for diagnosing human lung cancer carry out generaI investigation screening to them, extract the blood of tested crowd Liquid sample, determines whether it suffers from lung cancer according to the above method, once examinee is used the diagnostic method that present invention judgement is related to Or the kit as derived from this method is judged as with lung cancer, then should carry out various clinical definites using various detection means, such as The case where clinic cannot find lesion, then answer close follow-up visit monitoring examinee lung cancer occurrence and development.

Generally speaking, the protein-chip and its derivative diagnostic reagent of new diagnosis human lung cancer provided by the invention are utilized Box then very convenient, quick, noninvasive, efficiently can early diagnose or generally investigate screening lung cancer suspected patient or High Risk of Lung Cancer people Group, and participate in the normal health crowd of detection.

Illustrate how to implement the egg of new early diagnosis human lung cancer of the present invention below in conjunction with various embodiments How white matter chip by the protein-chip derives corresponding kit, and how with above-mentioned new diagnosis human lung cancer Protein-chip and its derivative diagnostic kit diagnosis generaI investigation screening lung cancer suspected patient, High Risk of Lung Cancer crowd and Healthy People Group.

Embodiment 1

The protein-chip of the new early diagnosis human lung cancer of preparation simultaneously detects tested serum sample.

The protein-chip of new diagnosis human lung cancer of the present invention can be implemented by a variety of specific technologies, below With regard to carrying out more detailed description:

The 6 kinds of Antigen adsorptions or covalent cross-linking that this method is related to are on suitable solid support.This 6 kinds of antigens can be with The native protein of purification, or the albumen of recombinant expression, or recombinant expression containing each antigen property amino acid sequence Recombination fusion protein, or the polypeptide of each antigen property amino acid sequence directly synthesized.Signified solid support can be Slide, immunoblotting film, various microwell plates used in immuno absorbence etc..Antigen can directly be inhaled by the way of electrostatic interaction It invests on solid support, can also be crosslinking on solid support by the way of covalent cross-linking.

Concrete example is that appropriate biotinylated BSA is taken to be added in this 6 kinds of antigen recombinant proteins after purification, then Final concentration of 0.05% Tween-20 is added, with the concentration point sample of 1mg/ml in the glass substrate table of aldehyde radical after mixing well Face, this 6 kinds of antigens are crosslinking in surface of glass slide in a manner of covalent cross-linking at this time, that is, have been successfully prepared and can be used for diagnosing people's lung The protein-chip of cancer.Then again using other uncrosslinked reaction sites of cow's serum closing surface of glass slide.

First by the solid phase of this 6 kinds of antigens of the Streptavidin Yu absorption or covalent cross-linking of Cy3 or Cy5 label after closing Support surface mixing is incubated for.After the various detergent washing used through usual immunological technique, tested blood sample and solid is added The mixing of phase support surface is incubated for, so that the corresponding antibodies of this 6 kinds of antigens in tested blood sample and solid support surface Antigen binding, to be adsorbed in solid support surface indirectly.It is washed again through the various detergent that usual immunological technique uses The foreign protein for removing non-specific adsorption leaves the Streptavidin of corresponding human antibody and fluorescent marker in solid support surface. Concrete example, by the Streptavidin of the Streptavidin of 1:100 diluted Cy5 label or the diluted Cy3 label of 1:600 with After 37 degree of moisturizings of closed slide are incubated for 1 hour, glass is rinsed using the phosphate buffer (PBS) of the pH7.4 of 0.01mol/L Piece 3 times.Then the diluted tested serum of 1:40 and the slide room temperature moisturizing after above-mentioned closed are incubated for 1 hour, abandon tested blood Clearly, it is rinsed slide 3 times using the phosphate buffer (PBS) of the pH7.4 of 0.01mol/L.

The above-mentioned corresponding human antibody for retaining in solid support surface, example can be detected using a variety of common immunological methods Such as immunofluorescence technique, immunoenzyme, chemoluminescence method, radioimmunology, enzyme linked immunosorbent assay, Western blot.Specifically Citing, can be used immunofluorescence technique.Goat anti-human igg-Cy5 secondary antibody is added in the slide after reacting with Cy3- Streptavidin, And goat-anti people IgM-Cy3 secondary antibody is added in the slide after reacting with Cy5- Streptavidin, moisturizing is incubated for 1 hour.Abandon fluorescence Secondary antibody liquid is rinsed slide 3 times using the PBS of the pH7.4 of 0.01mol/L, and deionized water is rinsed slide 1 time.It is air-dried, Yu Ji Light is copolymerized each point fluorescence intensity and area that 635nm and 530nm is read under burnt chip scanner.Wherein under 635nm the point it is total Fluorescence volume (or average fluorescent strength) represents the relative amount of the corresponding IgG antibody of the antigen in tested serum, should under 530nm Total fluorescence volume (or average fluorescent strength) of point represents the relative amount of the corresponding IgM antibody of the antigen in tested serum.So The processing of data is carried out afterwards: firstly, the fluorescence intensity that will test gained antigen point is reacted with biotin in point and Streptavidin Fluorescence intensity be divided by.Secondly, if the Antibody types of detection are IgG, this step will be by the first step treated data It is divided by with the numerical value of the IgG standard items through equally handling；If the Antibody types of detection are IgM, first will be passed through in this step Step treated data and the numerical value of IgM standard items in the hole through equally handling are divided by.Third, by IgG IgM standard film Data are handled also according to two above step.Then according to IgG or the dilution of IgM antibody, standard curve is drawn. By the difference of different batches standard curve, the experimental data of different batches is corrected.Then by the data generation of second step The formula for entering standard curve calculates corresponding antibodies content of the antigen in serum.It is marked referring to the control set up and with corresponding judgement Standard is compared, it can be determined that whether the corresponding antibodies of the antigen of the tested person are positive.

Determine whether single antigen is the knot of 2 kinds of the antigen 1 of Conjoint Analysis 6 autoantibody (IgG type+IgM type) after the positive Fruit calculates the number of total antigen positive, then is compared with corresponding judgment criteria, all to sentence greater than required standard value person Break as patients with lung cancer.

Embodiment 2

The preparation of 6 kinds of corresponding antigens.

(1) P53 antigen fragment of the present invention, (2) SOX2 antigen fragment, (3) COPB1 antigen fragment, (4) EFHD2 Antigen fragment, (5) EIF4G3 antigen fragment, the DNA fragmentation of (6) PCNA antigen fragment use EcoR I and Xho I enzyme in advance It is cloned in pBluescript SK (+/-) procaryotic clone carrier.EcoR I and Xho I double digestion recombinant expression carrier pET30b (+), the double digestion segment for recycling about 5.4kb are spare.EcoR I and Xho I double digestion is containing each antigen fragment gene PBluescript SK (+/-) carrier recycles about each corresponding double digestion segment of antigen, is cloned into the pET30b of recycling respectively In (+) expression vector.EcoR I and Xho I double digestion screening recombinant clone is respectively adopted again.It is largely extracted using alkaline lysis Plasmid converts BL21 Escherichia coli with the DNA of 0.1 μ g.Picking transformed clone is cultivated in LB culture medium to OD600 about 0.6, The IPTG inducing expression of final concentration of 2mM is added, thallus is harvested by centrifugation after 4 hours.Ultrasonic oscillation is crushed thallus, and centrifugation is received Collection precipitating, is precipitated as the inclusion body containing purpose antigen albumen.8M urea dissolution precipitating, 4 degree placements 12 hours or more, centrifugation is gone Except insoluble matter, combined 1 hour with the mixing of Ni affinity column.In conjunction with rear dress column, using the 8M urea washes chromatographic column 20 of pH6.5 Times column volume, then uses 8M urea elution chromatography 10 times of column volumes of column of pH5.9, and fraction collection finally uses the 8M of pH4.5 10 times of column volumes of urea elution chromatography column, fraction collection.It elutes each component difference loading and carries out SDS-PAGE analysis, select purpose Component where albumen carries out renaturation.Will component where destination protein merge after adjustment protein concentration to 0.25mg/ml, to containing The buffer of 2M urea is dialysed, and 4 degree are dialysed 12 hours, then is dialysed to the buffer of the urea containing 0.2M, and 4 degree are dialysed 12 hours, then Buffer dialysis to the urea containing 0.02M, 4 degree are dialysed 12 hours, then are dialysed to not urea-containing buffer, and 4 degree of dialysis 12 are small When.Each antigen is concentrated to ultimate density as 1mg/ml in the method for using PEG20000 water suction concentration.Centrifugation goes to precipitate, and measures albumen Content obtains the recombinant protein of each antigen.

Embodiment 3:

Early diagnose the preparation (protein chip diagnostic kit) of the derivative reagent box of the protein-chip of lung cancer

The composition of kit:

The protein chip of pre- point antigen；

Sample diluting liquid；

Wash concentrated liquor；

The concentrate of fluorescent marker secondary antibody；

The preparation of each composition of kit:

1, the preparation of the protein chip of point antigen in advance: the BSA coating of commercialization is bought and through aldehyde radicalization treated carrier Slide, such as the CSS-100 type slide of U.S. CE L Associates company.Using the purifying of 6 kinds of antigen prepared by embodiment 2 Suitable biotinylation is added as sample in recombinant protein and human IgG (0.2mg/ml), people IgM (0.2mg/ml) standard items BSA and tween-20, using biochip point sample machine by above-mentioned 14 kinds of sample spots in forming microarray, every glass on carrier slide 10 parallel microarrays of piece point, separated using the plastic foil for sticking suitable size between each microarray, each sample Every point sample about 8nl, 2 parallel points.Moisturizing is placed 4 hours promotion samples and is crosslinking on slide after point sample, then exists Surface of glass slide contains the PBS of 20% fetal calf serum plus one layer, stands overnight for 4 DEG C after room temperature moisturizing is placed 1 hour, with adequate closure Non-specific protein crosslinking or binding site on slide.Liquid is discarded, is air-dried, slide is placed in aluminium foil polybag and is filled The dry sealing of nitrogen, is placed at 4 DEG C and saves.Above procedure is successfully prepared the protein chip of pre- antigen.

2, the preparation of IgG/IgM antibody standard film: using 0.01mol/L pH7.4 PBS to the mark of IgG and IgM antibody Quasi- product are diluted, respectively prepare 6 dilutions antibody-solutions, including 0ug/ml, 10ug/ml, 20ug/ml, 40ug/ml, 80ug/ml and 160ug/ml.Suitable biotinylated BSA is added in every part of antibody samples, is then added final concentration of 0.05% Tween-20, mixes well.Using biochip point sample machine by above-mentioned antibody standard substance sample spot in carrier slide Upper formation microarray, every 10 parallel microarrays of slide point, using the plastic foil for sticking suitable size between each microarray It is separated, every point sample of each sample about 4nl, 2 parallel points.Promotion sample crosslinking in 4 hours is placed in moisturizing after point sample On slide, then contains the PBS of 20% fetal calf serum plus one layer in surface of glass slide, put for 4 DEG C after room temperature moisturizing is placed 1 hour Set overnight, on adequate closure slide non-specific protein crosslinking or binding site.Liquid is discarded, is air-dried, slide is put It is placed in the dry sealing of nitrogen charging in aluminium foil polybag, is placed at 4 DEG C and saves.Above procedure is successfully prepared the pre- egg for selecting antibody standard substance White chip.

3, sample diluting liquid: preparing the PBS (0.01mol/L, pH7.4) for containing 10% fetal calf serum, is added final concentration of 0.02% TweenC20, be sterile filtered packing, every bottle of 2ml.

4, it washs concentrated liquor: preparing the PBS (0.01mol/L, pH7.4) of 10 times of concentrations, be sterile filtered packing, and every bottle 20ml is diluted to 200ml using deionized water using preceding.

5, the concentrate of fluorescent marker anti-human antibody secondary antibody: using commercially available anti-human igg-Cy5 fluorescent marker secondary antibody and resist People's IgM-Cy3 fluorescent marker secondary antibody is sub-packed in brown vial after titrating suitable dilution respectively.

6, commercially available Streptavidin-Cy5 fluorescent marker secondary antibody and chain the concentrate of fluorescent marker Streptavidin: are used Mould Avidin-Cy3 fluorescent marker secondary antibody is sub-packed in brown vial after titrating suitable dilution respectively.

7, kit specification: specification should include the following contents, and the composition of kit, uses operation step at storage condition Suddenly, interpretation of result judgment method, points for attention etc..

The operating method and result of kit judge:

Operating method:

1, pre- chip of sealing is taken out at 4 DEG C, room temperature pre-temperature 15 minutes or more.

2,20ml is added in 180ml deionized water and washs concentrated liquor, prepare cleaning solution.

3, pre- point antigen and antibody standard substance chip hermetic bag are opened, chip is taken out and lays flat.Using sample diluting liquid by mark The working solution diluted is respectively taken 20 μ l by the concentrate for signing the dilution fluorescent marker Streptavidin of calibration, is added Each microarray sample cell, all surface of uniform fold sample cell, but not overflow sample cell.

4, moisturizing is incubated for 1 hour at 37 DEG C.

5, the liquid of coring on piece is quickly got rid of, surface of glass slide washing slide is flowed uniformly through with the cleaning solution of 20ml or more, altogether Washing slide 3 times.

6, using sample diluting liquid according to scheduled dilution proportion blood serum sample, recommend to dilute using 1:40.

7, the sample diluted is respectively taken into 20 μ l, the microarray sample cell of pre- point antigen chip, uniform fold sample is added The all surface in pond, but not overflow sample cell.Dilution is added in the chip microarray sample cell of point antibody standard substance in advance.

8, moisturizing is incubated for 1 hour at 37 DEG C.

9, the liquid of coring on piece is quickly got rid of, surface of glass slide washing slide is flowed uniformly through with the cleaning solution of 20ml or more, altogether Washing slide 3 times.

10, glimmering by what is diluted using sample diluting liquid by the dilution fluorescence secondary antibody working solution demarcated on label Light secondary antibody working solution respectively takes 20 μ l, each microarray sample cell, all surface of uniform fold sample cell is added, but not overflow Sample cell.

11, moisturizing is incubated for 1 hour at 37 DEG C.

12, the liquid of coring on piece is quickly got rid of, surface of glass slide washing slide is flowed uniformly through with the cleaning solution of 20ml or more, altogether Washing slide 3 times.

13, surface of glass slide washing slide is flowed uniformly through with the deionized water of 20ml or more, washed slide 1 time altogether.

14, it is air-dried, in reading each point on suitable instrument in the fluorescence intensity of 635nm, 530nm.

As a result judge:

1, for the single point of sample of single microarray, judge whether it is positive by the following method: calculating separately this The ratio and human IgG point 635nm fluorescence intensity of the 532nm fluorescence intensity of the 635nm fluorescence intensity and point of point and the point The ratio of 532nm fluorescence intensity takes the ratio of the two, then according to the public affairs of the antibody standard curve of the pre- spot film of human IgG standard items Formula calculates the corresponding antibody content of the antigen.Using the numerical value and scheduled standard ratio, judge whether this antigen I gG antibody is positive Property, positive then be counted as 1, feminine gender is counted as 0；Calculate separately the 532nm fluorescence intensity of the point and the 635nm fluorescence intensity of the point Ratio and people IgM point 635nm fluorescence intensity and the point 532nm fluorescence intensity ratio, the ratio both taken, then basis The formula of the antibody standard curve of the pre- spot film of people's IgM standard items calculates the corresponding antibody content of the antigen.Using the numerical value and in advance Fixed standard ratio judges whether this antigen I gM antibody is positive, positive then be counted as 1, and feminine gender is counted as 0.

2, for tested person corresponding for some microarray, positive antigen number calculates by the following method: described in 1. After method judges single-point number positive, the summation of 12 kinds of autoantibody single-point number positives is the positive antigen of tested person in array Number.

3, tested person's positive antigen number and preassigned are compared, when tested person's positive antigen number is greater than or equal to pre- calibration When quasi- value, that is, it is judged as the lung cancer positive.

Embodiment 4:

Using new early diagnosis lung cancer protein-chip and derivative reagent box diagnosis generaI investigation screening lung cancer suspected patient, High Risk of Lung Cancer crowd and healthy population

Using kit described in embodiment 3 (adding immunofluorescence technique using protein chip) detection patients with lung cancer 200, just Ordinary person compares 200, lung's benign lesion 200, High Risk of Lung Cancer crowd 2000, common community-based population 1000.Using just Ordinary person compares 200 delimitation pulmonary cancer diagnosis standards and carries out result judgement.As a result as shown in the table:

Patients with lung cancer 200, in normal controls 200, kit detects patients with lung cancer positive 164, normal 24 of people's positive, therefore the sensibility of the kit diagnosing is 82.00%, specificity is 88.00%, illustrates the reagent The Sensitivity Specificity of box diagnosing is higher.

In patients with lung cancer 200, I phase patients with lung cancer has 50, wherein 41 kit test results are the positive, early stage lung The sensibility of cancer (I phase) is 82%, is not remarkably decreased, and illustrates the Sensitivity Specificity of kit early diagnosis lung cancer It is higher.

35 of lung cancer are mistaken in 200 patients of lung's benign lesion, illustrate this method not only can with diagnosing, And antidiastole rate with higher for lung's benign lesion, the specificity of antidiastole are 82.50%.

282 are judged as lung cancer in High Risk of Lung Cancer crowd 2000, and people at highest risk's Positive rate is about 14.1%. 2000 people at highest risk are through follow-up, wherein 46 people are hereafter lung cancer by clinical definite, and 28 people's kits detect in this 46 people The positive illustrates that patients with lung cancer can be screened out by the kit with higher sensitivity from tested crowd and comes that sensibility is about 60.87%, so as to the screening for High Risk of Lung Cancer crowd.

103 are judged as lung cancer in detected common community-based population (healthy population) 1000.1000 common societies Area crowd is through follow-up, wherein 2 people are hereafter lung cancer by clinical definite, and all kit detections are positive in this 2 people, explanation Patients with lung cancer can be screened out by the kit with higher sensitivity from healthy population to be come, so as to be used for common community The screening of crowd.

(1) P53 antigen fragment, characteristic amino acid sequence is as shown in ID.1sequence:

NH3-

MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLS

PDDIEQWFTEDPGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWP

LSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLA

KTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCS

DSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDC

TTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCA

CPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKP

LDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSH

LKSKKGQSTSRHKKLMFKTEGPDSD-COOH

(2) SOX2 antigen fragment, characteristic amino acid sequence is as shown in ID.2sequence:

NH3-

MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRP

MNAFMVWSRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKR

PFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLA

PGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMMQ

DQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMN

GSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAP

CQAGDLRDMISMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAIN

GTLPLSHM-COOH

(3) COPB1 antigen fragment, characteristic amino acid sequence is as shown in ID.3sequence:

NH3-

MPLRLDIKRKLTARSDRVKSVDLHPTEPWMLASLYNGSVCVWNH

ETQTLVKTFEVCDLPVRAAKFVARKNWVVTGADDMQIRVFNYNT

LERVHMFEAHSDYIRCIAVHPTQPFILTSSDDMLIKLWDWDKKWSC

SQVFEGHTHYVMQIVINPKDNNQFASASLDRTIKVWQLGSSSPNFT

LEGHEKGVNCIDYYSGGDKPYLISGADDRLVKIWDYQNKTCVQTL

EGHAQNVSCASFHPELPIIITGSEDGTVRIWHSSTYRLESTLNYGME

RVWCVASLRGSNNVALGYDEGSIIVKLGREEPAMSMDANGKIIWA

KHSEVQQANLKAMGDAEIKDGERLPLAVKDMGSCEIYPQTIQHNP

NGRFVVVCGDGEYIIYTAMALRNKSFGSAQEFAWAHDSSEYAIRES

NSIVKIFKNFKEKKSFKPDFGAESIYGGFLLGVRSVNGLAFYDWDN

TELIRRIEIQPKHIFWSDSGELVCIATEESFFILKYLSEKVLAAQETHE

GVTEDGIEDAFEVLGEIQEIVKTGLWVGDCFIYTSSVNRLNYYVGG

EIVTIAHLDRTMYLLGYIPKDNRLYLGDKELNIISYSLLVSVLEYQT

AVMRRDFSMADKVLPTIPKEQRTRVAHFLEKQGFKQQALTVSTDP

EHRFELALQLGELKIAYQLAVEAESEQKWKQLAELAISKCQFGLAQ

ECLHHAQDYGGLLLLATASGNANMVNKLAEGAERDGKNNVAFM

SYFLQGKVDACLELLIRTGRLPEAAFLARTYLPSQVSRVVKLWREN

LSKVNQKAAESLADPTEYENLFPGLKEAFVVEEWVKETHADLWPA

KQYPLVTPNEERNVMEEGKDFQPSRSTAQQELDGKPASPTPVIVAS

HTANKEEKSLLELEVDLDNLELEDIDTTDINLDEDILDD-COOH

(4) EFHD2 antigen fragment, characteristic amino acid sequence is as shown in ID.4sequence:

NH3-

MATDELATKLSRRLQMEGEGGGETPEQPGLNGAAAAAAGAPDEA

AEALGSADCELSAKLLRRADLNQGIGEPQSPSRRVFNPYTEFKEFSR

KQIKDMEKMFKQYDAGRDGFIDLMELKLMMEKLGAPQTHLGLKN

MIKEVDEDFDSKLSFREFLLIFRKAAAGELQEDSGLCVLARLSEIDV

SSEGVKGAKSFFEAKVQAINVSSRFEEEIKAEQEERKKQAEEMKQR

KAAFKELQSTFK-COOH

(5) EIF4G3 antigen fragment, characteristic amino acid sequence is as shown in ID.5sequence:

NH3-

MNSQPQTRSPGGFRPIQFFQRPQIQPPRATIPNSSPSIRPGAQTPTAV

YQANQHIMMVNHLPMPYPVPQGPQYCIPQYRHSGPPYVGPPQQYP

VQPPGPGPFYPGPGPGDFPNAYGTPFYPSQPVYQSAPIIVPTQQQPPP

AKREKKTIRIRDPNQGGKDITEEIMSGGGSRNPTPPIGRPTSTPTPPQ

LPSQVPEHSPVVYGTVESAHLAASTPVTAASDQKQEEKPKPDPVLK

SPSPVLRLVLSGEKKEQEGQTSETTAIVSIAELPLPPSPTTVSSVARST

IAAPTSSALSSQPIFTTAIDDRCELSSPREDTIPIPSLTSCTETSDPLPTN

ENDDDICKKPCSVAPNDIPLVSSTNLINEINGVSEKLSATESIVEIVK

QEVLPLTLELEILENPPEEMKLECIPAPITPSTVPSFPPTPPTPPASPPH

TPVIVPAAATTVSSPSAAITVQRVLEEDESIRTCLSEDAKEIQNKIEV

EADGQTEEILDSQNLNSRRSPVPAQIAITVPKTWKKPKDRTRTTEE

MLEAELELKAEEELSIDKVLESEQDKMSQGFHPERDPSDLKKVKA

VEENGEEAEPVRNGAESVSEGEGIDANSGSTDSSGDGVTFPFKPES

WKPTDTEGKKQYDREFLLDFQFMPACIQKPEGLPPISDVVLDKINQ

PKLPMRTLDPRILPRGPDFTPAFADFGRQTPGGRGVPLLNVGSRRS

QPGQRREPRKIITVSVKEDVHLKKAENAWKPSQKRDSQADDPENIK

TQELFRKVRSILNKLTPQMFNQLMKQVSGLTVDTEERLKGVIDLVF

EKAIDEPSFSVAYANMCRCLVTLKVPMADKPGNTVNFRKLLLNRC

QKEFEKDKADDDVFEKKQKELEAASAPEERTRLHDELEEAKDKAR

RRSIGNIKFIGELFKLKMLTEAIMHDCVVKLLKNHDEESLECLCRLL

TTIGKDLDFEKAKPRMDQYFNQMEKIVKERKTSSRIRFMLQDVIDL

RLCNWVSRRADQGPKTIEQIHKEAKIEEQEEQRKVQQLMTKEKRR

PGVQRVDEGGWNTVQGAKNSRVLDPSKFLKITKPTIDEKIQLVPKA

QLGSWGKGSSGGAKASETDALRSSASSLNRFSALQPPAPSGSTPSTP

VEFDSRRTLTSRGSMGREKNDKPLPSATARPNTFMRGGSSKDLLD

NQSQEEQRREMLETVKQLTGGVDVERNSTEAERNKTRESAKPEISA

MSAHDKAALSEEELERKSKSIIDEFLHINDFKEAMQCVEELNAQGL

LHVFVRVGVESTLERSQITRDHMGQLLYQLVQSEKLSKQDFFKGFS

ETLELADDMAIDIPHIWLYLAELVTPMLKEGGISMRELTIEFSKPLLP

VGRAGVLLSEILHLLCKQMSHKKVGALWREADLSWKDFLPEGED

VHNFLLEQKLDFIESDSPCSSEALSKKELSAEELYKRLEKLIIEDKAN

DEQIFDWVEANLDEIQMSSPTFLRALMTAVCKAAIIADSSTFRVDT

AVIKQRVPILLKYLDSDTEKELQALYALQASIVKLDQPANLLRMFF

DCLYDEEVISEDAFYKWESSKDPAEQNGKGVALKSVTAFFTWLRE

AEEESEDN-COOH

(6) PCNA antigen fragment, characteristic amino acid sequence is as shown in ID.6sequence:

NH3-

MFEARLVQGSILKKVLEALKDLINEACWDISSSGVNLQSMDSSHVS

LVQLTLRSEGFDTYRCDRNLAMGVNLTSMSKILKCAGNEDIITLRA

EDNADTLALVFEAPNQEKVSDYEMKLMDLDVEQLGIPEQEYSCVV

KMPSGEFACICRDLSHIGDAVVISCAKDGVKFSASGELGNGNI-

COOH

Claims (4)

1. a kind of purposes of protein-chip in the diagnostic product of preparation early diagnosis or generaI investigation screening lung cancer, feature exist In:

The protein-chip includes but not only comprising the protein marker of following 6 kinds of lung cancer autoantigen proteins: (1) P53 Antigen fragment, characteristic amino acid sequence is as shown in ID.1sequence；(2) SOX2 antigen fragment, characteristic amino acid sequence As shown in ID.2sequence；(3) COPB1 antigen fragment, characteristic amino acid sequence is as shown in ID.3sequence；(4) EFHD2 antigen fragment, characteristic amino acid sequence is as shown in ID.4sequence；(5) EIF4G3 antigen fragment, feature amino Acid sequence is as shown in ID.5sequence；(6) PCNA antigen fragment, characteristic amino acid sequence is as shown in ID.6sequence.

2. purposes as described in claim 1, the protein marker of the autoantigen protein matter is recombination expression and purification Protein.

3. a kind of purposes of protein-chip diagnostic kit in the diagnostic product of preparation early diagnosis or generaI investigation screening lung cancer, It is characterized in that, it uses protein-chip test sample as described in claim 1, the protein-chip diagnostic kit It include: detection slide, detection correction slide, sample diluting liquid, cleaning solution, fluorescence secondary antibody；

In the recombinant protein that expression in escherichia coli includes 6 kinds of antigen property amino acid sequences as described in claim 1 Or recombination fusion protein, these target proteins of purifies and separates make its purity reach 80% or more, in each antigen and human IgG, people Suitable biotinylated BSA is added in IgM, with suitable concentration point sample in the glass substrate handled through aldehyde radical, antigen By covalent cross-linking on substrate, remaining crosslink sites are closed using cow's serum, 4 DEG C of kept dries after drying are detected Slide；

Human IgG and human IgM antibody standard items are subjected to doubling dilution, suitable concentration gradient point sample is selected to handle in through aldehyde radical Glass chip on, covalent cross-linking on substrate, is closed remaining crosslink sites using cow's serum by antibody standard substance, dry 4 DEG C of kept dries afterwards obtain detection correction slide；

PBS containing 10% cow's serum is sterile filtered and is bottled, sample diluting liquid is obtained；

PBS containing 0.02%Tween20 is sterile filtered and is bottled, cleaning solution is obtained；

By the Streptavidin-Cy5 fluorescent marker secondary antibody containing acceptable diluent degree, Streptavidin-Cy3 fluorescent marker secondary antibody, Goat anti-human igg's-Cy5 fluorescent marker secondary antibody and the bottling of goat-anti people's IgM-Cy3 fluorescent marker secondary antibody, obtain fluorescence secondary antibody.

4. purposes as claimed in claim 3, it is characterised in that also put carcinomebryonic antigen on the detection slide.