CN110333352A

CN110333352A - Tumour related auto-antibodies and tumor markers combined detection kit

Info

Publication number: CN110333352A
Application number: CN201910725141.2A
Authority: CN
Inventors: 刘浩; 李宾; 王东; 胡欣; 张涛
Original assignee: Gene Technology (wuhan) Co Ltd; Gene Technology (shanghai) Ltd By Share Ltd
Current assignee: Gene Technology (wuhan) Co Ltd; Gene Technology (shanghai) Ltd By Share Ltd
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2019-10-15

Abstract

The present invention relates to method, reagent and kits based on tumour related auto-antibodies and the joint-detection of tumor markers.In the present invention, the combination of antigen protein and one or more tumor markers antibody is corresponded to using three or more tumour related auto-antibodies, optimization design detection reagent and kit, substantially increase the sensitivity and accuracy of diagnosing tumor, the early diagnosis particularly suitable for tumour.

Description

Tumour related auto-antibodies and tumor markers combined detection kit

Technical field

The invention belongs to the diagnosis of tumor related marker object or detection technique fields, specifically, the present invention relates to one kind to swell Tumor related auto-antibodies and tumor markers combined detection kit.

Background technique

" the Global Cancer Statistics 2018 " issued according to World Cancer Research Foundation, it is contemplated that 2018 Year, total new cancer cases were up to 18,000,000, and the whole world is in advance in respect of 9,600,000 cancer mortality cases, wherein the newly-increased disease of China Number of cases account for 380.4 ten thousand, death number account for 229.6 ten thousand.

Although with the continuous appearance of new drug, new treatment, new technology, and country adds in the continuous of medical field investment Greatly, 5 years survival rates of cancer have been promoted to the 37.2% of 2012-2015 from 30.9% in 2003~2005 years, but due to hair The raising of sick rate, malignant tumour are still in the second disease death reason that China is only second to cardiovascular disease, are caused every year The death of nearly three million people accounts for disease dies population 27%.The case where high-incidence cancer kind such as lung cancer, liver cancer, much allows of no optimist --- hair The sick highest lung cancer five year survival rate of rate only 19.5%, morbidity number of cases account for more than half liver cancer of the whole world and more there was only 12.15%.And And due to the accessibility of medical resource and economy etc., rural five year survival rate (33.6%) and city (46.7%) are still There is obvious gap.

Cancer burden still rapid increase year by year personal causes to country, society, family and all very big influence.With hair 60~70% whole 5 years survival rates up to country are compared, and Chinese gap more comes from other than treatment means and investment Screening and ability of discovery in infantile tumour.China 5 years survival rates have reached 82% breast cancer, if it find that when occurred Far-end transfer, survival rate is also only less than 30%；And if lung cancer early stage i.e. be diagnosed, survival rate is still reachable within 5 years To 50~60%.The more cost of medical service of some precancerous lesions of leisure opinion and early-stage cancer, the only half in advanced stage even 1/5th. The cancer early diagnosis that China successively starts rural area and city thus early controls project, but due to huge population bring to finance and The reasons such as the great demand of medical and technical staff, the accessibility of related Clinics and compliance, Popular Science Education, still much fail to reach It is universal to the whole nation.Due to cancer early stage patient without apparent discomfort, carcinoma small volume, cancer location be concealed and some patientss Tumor Marker Levels do not increase significantly, and the screening method for being based primarily upon Imaging Method and specific tumors marker at present is sensitive Spend relatively low, it is higher that there is also false positives, the inadequate problem of accuracy, so that some patientss is missed early detection and treat Chance, it is also possible to will increase the workload and financial burden of later period follow-up visit monitoring.

To sum up, exploitation it is more economical, effectively, can and cancer early detection and diagnostic method, it is imperative.

Summary of the invention

The purpose of the present invention is to provide a kind of tumour related auto-antibodies and tumor markers combined detection kits.

In the first aspect of the present invention, the purposes of tumour related auto-antibodies and tumor markers is provided, for as examining Target break to prepare the diagnostic reagent or kit of diagnosing tumour, in which:

(1) the tumour related auto-antibodies correspond respectively to antigen P53, SOX2 and CK8；

(2) tumor markers include selected from the group below 1,2 or 3 kind: CEA, NSE, SCC.

In the second aspect of the present invention, the reagent of specific detection tumour related auto-antibodies or tumor markers is provided Purposes is used to prepare the kit of diagnosing tumour；Wherein,

(1) reagent of the specific detection tumour related auto-antibodies includes: that tumour related auto-antibodies are corresponding anti- Former P53, SOX2 and CK8；

(2) reagent of the specific detection tumor markers includes: that specific detection selected from the group below 1,2 or 3 kind are swollen The reagent of tumor markers: CEA, NSE, SCC.

In another preferred example, the corresponding antigen of the tumour related auto-antibodies further includes selected from the group below 1,2 or more Kind (such as 3,4,5,6,7 kind): HuD, CAGE, NY-ESO-1, GBU4-5, P62, KRAS and MAGE A4.

In another preferred example, the corresponding antigen of the tumour related auto-antibodies includes: P53, SOX2, CK8 and HuD.

In another preferred example, the corresponding antigen of the tumour related auto-antibodies includes: P53, SOX2, CK8, HuD and CAGE。

In another preferred example, the corresponding antigen of the tumour related auto-antibodies includes: P53, SOX2, CK8, HuD, CAGE and NY-ESO-1.

In another preferred example, the tumor markers include: CEA；Or CEA and NSE；Or CEA and SCC；Or CEA, NSE and SCC.

In another preferred example, in (1), the diagnostic reagent is for detecting tumour correlation described in sample to be tested certainly There are situation or the reagents of amount for body antibody；Preferably, the diagnostic reagent is suitable for chemoluminescence method or fluorescence method The reagent of detection.

In another preferred example, in (2), the diagnostic reagent is for detecting tumor markers described in sample to be tested There are situation or the reagents of amount；Preferably, the diagnostic reagent is the examination containing the antibody for resisting the tumor markers Agent；Preferably, the diagnostic reagent is the reagent suitable for chemoluminescence method or Fluorometric assay；

In another preferred example, in (1) and (2), the diagnostic reagent is all based on the detection reagent of chemoluminescence method；Or (1) and in (2), the diagnostic reagent is all based on the detection reagent of fluorescence method.

In another preferred example, the tumour related auto-antibodies correspond to antigen and are attached on solid phase carrier；Preferably, institute Stating solid phase carrier includes but is not limited to: particle, microballoon, slide, test strips；Preferably, the tumour related auto-antibodies Corresponding antigen is expressed by expression vector；More preferably, the tumour related auto-antibodies are corresponded into the cDNA of antigen, biotin acceptor Polypeptid DNA and biotin ligase are gene constructed to an expression vector, prepare biotinylated protein；Utilize biotin and strepto- Tumour related auto-antibodies are corresponded to antigen protein and are indirectly secured to solid phase carrier by the specific reaction between Avidin.

In another preferred example, the antibody of the tumor markers is resisted to be attached on solid phase carrier；Preferably, the solid phase Carrier includes but is not limited to: particle (such as magnetic particle), microballoon, slide, test strips, plastic bead, liquid-phase chip, microwell plate or parent And film.

In another preferred example, the diagnostic reagent is the diagnostic reagent using serum as sample to be tested, with blood plasma be to The diagnostic reagent of sample or using tissue or cell as the diagnostic reagent of sample to be tested.

In another preferred example, the tumour related auto-antibodies are carried out using escherichia expression system and corresponds to antigen Expression.

In the third aspect of the present invention, a kind of kit for diagnosing tumour is provided, includes: in the kit

(1) reagent of specific detection tumour related auto-antibodies, the tumour related auto-antibodies correspond respectively to (such as It is incorporated into) antigen P53, SOX2 and CK8；

(2) reagent of specific detection selected from the group below 1,2 or 3 kind of tumor markers: CEA, NSE, SCC.

In another preferred example, in (1), the diagnostic reagent is that tumour in sample to be tested is related itself to be resisted for detecting There are situation or the reagents of amount for body；Preferably, the diagnostic reagent is corresponding containing the tumour related auto-antibodies The reagent of antigen；Preferably, the diagnostic reagent is the reagent suitable for chemoluminescence method or Fluorometric assay.

It in another preferred example, further include (but being not limited to) ingredient selected from the group below: solid phase carrier in the kit, it is dilute Release liquid, reference substance, standard items, quality-control product, anti-human IgG antibodies, detection antibody, luminescence reagent, cleaning solution；Preferably, the mark Quasi- product include: tumor markers standard items, humanization tag antibody standard items；Preferably, the quality-control product includes: tumor-marker Object quality-control product, humanization tag antibody quality-control product；Preferably, the solid phase carrier includes but is not limited to: particle, microballoon, glass Piece, test strips.

In another preferred example, the tumour includes: infantile tumour, is preferably included in front of the IV phase or before the III phase Tumour more preferably includes in incubation period, precancerous lesion, the tumour of I phase or II phase；In without the apparent uncomfortable stage Tumour；Tumour small in size (such as diameter of tumor is less than 30mm or smaller, preferably less than 20mm)；Cancer location is concealed to swell Tumor；

In another preferred example, the tumour is lung cancer.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.

Detailed description of the invention

Fig. 1 difference tumour related auto-antibodies identify the result figure of Chinese Healthy, patients with lung cancer, and ordinate is kidney-Yang Property rate, abscissa is false positive rate.

Figure 1A, P53 autoantibody detect ROC curve (Receiver operating curve).

Figure 1B, SOX2 autoantibody detect ROC curve.

Fig. 1 C, CK8 autoantibody detect ROC curve.

Fig. 1 D, HuD autoantibody detect ROC curve.

Fig. 1 E, CAGE autoantibody joint-detection ROC curve.

Fig. 1 F, NY-ESO-1 autoantibody detect ROC curve.

Fig. 1 G, GBU4-5 autoantibody detect ROC curve.

Fig. 1 H, MAGE A4 autoantibody detect ROC curve.

The combination of three or more tumour related auto-antibodies of Fig. 2 identifies the result figure of Chinese Healthy, patients with lung cancer.

Fig. 2A, P53, SOX2, CK8 autoantibody joint-detection ROC curve.

Fig. 2 B, P53, SOX2, CK8, HuD autoantibody joint-detection ROC curve.

Fig. 2 C, P53, SOX2, CK8, HuD, CAGE autoantibody joint-detection ROC curve.

Fig. 2 D, P53, SOX2, CK8, HuD, CAGE, NY-ESO-1 autoantibody joint-detection ROC curve.

Fig. 3, three or more tumour related auto-antibodies and one or more of tumor markers joint-detection China's Healthies People, patients with lung cancer result figure.

Fig. 3 A, P53, SOX2, CK8 autoantibody and CEA joint-detection ROC curve.

Fig. 3 B, P53, SOX2, CK8, HuD autoantibody and CEA, NSE joint-detection ROC curve.

Specific embodiment

The present inventor passes through in-depth study, discloses and examines for the joint of tumour related auto-antibodies and tumor markers Method, reagent and the kit of survey.It is corresponding using P53, SOX2 and CK8 tumour related auto-antibodies are included at least in the present invention The combination of antigen protein and one or more tumor markers (CEA, NSE or SCC) antibody, optimization design detection reagent and Kit substantially increases the sensitivity and accuracy of diagnosing tumor.

As used herein, " sample to be tested " or " sample to be tested " refers to sample to be detected.In the present invention, described is to be measured Sample may is that serum, blood plasma are derived from the tissue of diseased region, bronchoalveolar lavage fluid, hydrothorax, sputum etc.；Preferably serum or Blood plasma.

As used herein, " diagnosis " word refers to those skilled in the art to assess and/or determine that an individual suffers from one The method for a possibility that specified disease, illness or disease are levied." diagnosis " word also includes that detecting suffers from what a disease, illness or disease were levied Tendency determines the therapeutic efficiency of a drug therapy, or predicts the reaction to drug therapy.The diagnostic method of this disclosure can Individually carry out, or and other to analyze the diagnosis of disease, illness or disease sign and/or by stages method carries out simultaneously.

The present inventor is by being extensively studied screening and repetition test, and discovery is in clinical tumor diagnosis, with tumour correlation As target, conjunctive use detects the technology and detection tumour of tumour related auto-antibodies for autoantibody and tumor markers The technology of marker can extremely efficient improve accuracy, the sensibility of clinical diagnosis.

Early stage tumor development, tumour cell generates gene mutation, dystopy or recombination, is immunized after GAP-associated protein GAP release System identification is " non-self albumen " as antigen and immune response occurs, and generates the antibody for being directed to the antigen, i.e. tumour Autoantibody.Autoantibody is present in body-internal-circulation in the form of dissociating or be combined into antigen-antibody complex with related antigen. But difference, single autoantibody are difficult to accurately speculate the incidence of all lung cancer for the reaction of Different Individual immune system. In the present invention, it is determined that the several autoantibody for tumour antigen for being suitable for detection, by the autoantibody of tumour antigen As tumor related marker object, tumour autoantibody is detected using the corresponding antigen of tumour related auto-antibodies.The present inventor It was found that this detection method has relatively high specificity and sensibility, facilitate the tumour for clinically realizing more early stage Screening.There is sensibility and stability in view of immune system, humoral immune reaction has the function of amplifying tumor signal, can compare Imaging diagnosis goes out cancer cell and shifts to an earlier date the raising for detecting cancer autoantibody in blood in 5 years.

Tumor markers, which refer to, is present in tumor tissues itself by what tumor tissues generated, or secretes to other bodies such as blood Liquid, or stimulated by tumor tissues, by host cell generation, content is apparently higher than a substance of normal reference value.In recent years, Protide tumor markers are widely used to antidiastole, observation of curative effect and the Index for diagnosis of tumour, but clinic is examined at present The disconnected tumor markers used are difficult to be individually used for the screening to infantile tumour, generally require in conjunction with other diagnostic modes to improve Diagnostic accuracy.In addition, having differences in the selection of tumor markers for different tumor types, its diagnosis is also improved Difficulty.And in the present invention, the diagnostic techniques of the autoantibody for tumour antigen is combined using tumor markers, unexpectedly Improve diagnostic accuracy.

In the present invention, also directed to property conjunctive use is based on chemiluminescent detection technique or detection technique of fluorescence. In the art for the detection of antibody generally using classical ELISA method, this may be those skilled in the art for antibody The conventional thinking of detection.And tumour related auto-antibodies are detected simultaneously with chemiluminescence or fluorescent technique in the present invention It is had not been reported with the combined method of tumor markers.By appropriate selection, using chemiluminescence or fluorescent technique platform The method for detecting tumour related auto-antibodies and tumor markers combination simultaneously helps to improve the recall rate of malignant tumour and examines Disconnected accuracy, has both higher sensibility and high specificity.In addition, being also beneficial to simplify the design of reagent, and simplify behaviour Make process.

Above-mentioned new discovery based on the present inventor provides tumour related auto-antibodies and tumor markers as diagnosis Purposes of the target in the diagnostic reagent or kit for preparing diagnosing tumour, the tumour related auto-antibodies correspond respectively to (being such as incorporated into) antigen P53, SOX2 and CK8；The tumor markers include selected from the group below 1,2 or 3 kind: CEA, NSE, SCC.

Alternatively, the corresponding antigen of tumour related auto-antibodies further includes selected from the group below 1,2 Or a variety of (such as 3,4,5,6,7 kind): HuD, CAGE, NY-ESO-1, GBU4-5, P62, KRAS and MAGE A4.

Based on new discovery of the invention, the examination of specific detection tumour related auto-antibodies or tumor markers is also provided The purposes of agent is used to prepare the kit of diagnosing tumour.

The P53, amino acid sequence can be as shown in SEQ ID NO:1；The SOX2, amino acid sequence can As shown in SEQ ID NO:2；The CK8, amino acid sequence can be as shown in SEQ ID NO:3；The HuD, Amino acid sequence can be as shown in SEQ ID NO:4；The CAGE, amino acid sequence can be as shown in SEQ ID NO:5； The NY-ESO-1, amino acid sequence can be as shown in SEQ ID NO:6；The GBU4-5, amino acid sequence can As shown in SEQ ID NO:7；The P62, amino acid sequence can be as shown in SEQ ID NO:8；The KRAS, Amino acid sequence can be as shown in SEQ ID NO:9；The MAGE A4, amino acid sequence can be such as SEQ ID NO:10 It is shown.

The invention also includes the conservative sequence variant forms that above-mentioned tumour related auto-antibodies correspond to antigen.The change Special-shaped formula includes (but being not limited to): several (such as 1-20, most preferably 1-10, also more preferably such as 1-8,1-5) amino Acid missing, insertion and/or substitution, and C-terminal and/or N-terminal addition or lack it is one or several (within such as 20, More preferably it is within 5 within preferably 10) amino acid.Antigen corresponding with the tumour related auto-antibodies is homologous Property it is high (for example homology is 90% or higher, such as homology 95%, 98% or 99%) and it is more with wild type identical function Peptide is also included in the present invention.Above-mentioned tumour related auto-antibodies correspond to the segment of antigen or determinant is also contained in the present invention It is interior.

Based on new discovery of the invention, also provide it is a kind of have both it is related to the tumour of high specific compared with high sensitivity itself Antibody and tumor markers combined detection kit, to improve the early diagnostic rate of tumour.

In a preferred embodiment of the invention, described to be used for joint-detection tumour related auto-antibodies and tumor-marker The detection kit of object corresponds to the solid phase of antigen protein and tumor markers antibody including being coated with tumour related auto-antibodies respectively Carrier.The detection kit corresponds to antigen protein and one or more tumour mark using three or more tumour related auto-antibodies The combination of will object antibody.The detection kit detects tumour correlation using chemiluminescence or fluorescent technique platform simultaneously and itself resists Body and tumor markers.

As preferred embodiment of the invention, it includes: P53 that the tumour related auto-antibodies, which correspond to antigen, SOX2 and CK8；Or P53, SOX2, CK8 and HuD；Or P53, SOX2, CK8, HuD and CAGE；Or P53, SOX2, CK8, HuD, CAGE and NY- ESO-1.As preferred embodiment of the invention, the tumor markers include: CEA；Or CEA and NSE；Or CEA and SCC；Or CEA, NSE and SCC.The demonstration of embodiment according to the present invention, currently preferred above-mentioned antigen and tumor markers, for The specificity and sensibility of lung cancer are especially excellent.

According to the present invention, the tumour related auto-antibodies correspond to antigen or antitumor marker antibody is fixed on solid phase load Body, the solid phase carrier can be but not limited to: magnetic particle, microballoon, plastic bead, liquid-phase chip, microwell plate, affinity membrane, slide Or test paper.

According to the present invention, the fixing means of the antigen protein or antibody includes direct coating and indirect coating method, is adopted It is by the specific reaction between biotin and Streptavidin that antigen protein is indirect with direct coating or indirect coating method It is fixed on solid phase carrier, antibody is directly fixed on solid phase carrier using direct coating.

According to the present invention, the kit further includes Sample dilution, anti-human IgG antibodies, detection antibody, antibody dilution Liquid, cleaning solution, tumor markers standard items, humanization tag antibody standard items, tumor mark quality control product, humanization label are anti- Body quality-control product, luminescent solution.

According to the present invention, signal detecting method used is chemoluminescence method, it is preferable that luminous agent used is AMPPD, 4- MUP or acridinium ester.

According to the present invention, the detection antibody or anti-human IgG antibodies have detectable marker；The detectable mark Remember that object is preferably alkaline phosphatase.

In a preferred embodiment of the invention, it is anti-that tumour correlation itself is detected according to the principle of Dot-ELISA Body.Including the corresponding antigen protein of tumour related auto-antibodies is connected on solid phase carrier, in sample autoantibody to be measured with Be combined into solid phase antigen-by inspection auto-antibody complex, then it is anti-by itself is examined with enzyme mark anti-human IgG antibodies and solid phase antigen- Autoantibody in nanocrystal composition combines, and forms solid phase antigen-by inspection autoantibody-enzyme mark anti-human IgG antibodies' compound, then Luminous intensity after measurement plus shiner, determines autoantibody to be measured.

In a preferred embodiment of the invention, the tumour is detected according to the principle of double antibody sandwich enzyme immunization Marker.Including tumor markers antibody to be fixed on solid phase carrier, tumor markers antigen to be measured is in combination in sample At solid phase antibody-by inspection antigenic compound, then it is multiple by inspection antigen with another enzyme mark tumor-marker analyte detection antibody and solid phase antibody- Antigen binding in conjunction object forms solid phase antibody-by inspection antigen-enzyme mark and detects antibody complex, after then measurement adds shiner Luminous intensity, determine tumor markers content to be measured.

As preferred embodiment of the invention, antigen detection autoantibody and utilization are corresponded to using tumour related auto-antibodies In detecting step of the antitumor marker antibody to detect tumor markers, it is all made of the detection based on chemoluminescence method；Alternatively, It is all made of the detection based on fluorescence method.

As preferred embodiment of the invention, the test object is human serum or blood plasma.Serum (or blood plasma) dilution Or stoste is for detecting autoantibody and Tumor Marker Levels.

Of the invention mainly has the beneficial effect that:

1, using tumour related auto-antibodies as the marker of diagnosing tumor, the multiple tumour related auto-antibodies of joint-detection Horizontal and Tumor Marker Levels, can obtain relatively high sensibility and high specificity, for clinically early screening Malignant tumour provides possibility.

2, joint-detection tumor markers improve the sensitivity and specificity of detection.The relevant detection kit packet of the present invention It includes three or more tumour related auto-antibodies and corresponds to antigen combination, the detection of tumour related auto-antibodies can be obtained high special Property, joint-detection tumor markers will obtain higher detection sensitivity.

3, chemiluminescence or fluorescent technique detection are carried out personalizedly, further increase detection sensitivity and quantitative stabilization Property.Make to operate simultaneously more convenient.

4, magnetic particle, microballoon or plastic bead increase surface of solid phase carriers product, and amplified signal simultaneously shortens detection time.Using magnetic Particle makes immune response be converted to reactive liquid solution, not only increases response area as solid phase carrier, at the same also accelerate reaction into Row.

5, method of the invention and kit are especially early diagnosed particularly suitable for pulmonary cancer diagnosis, have it is highly sensitive, High specific, it is easy to operate, detection cycle is short the features such as.Detection accuracy can be effectively improved, and reduces the false negative of result.This In the embodiment of invention, presents the method for the present invention and kit is that the diagnosis of early stage, volume small tumour has well Sensitivity and accuracy.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.

Embodiment 1, tumour related auto-antibodies correspond to antigen protein and antitumor marker antibody is coated with magnetic particle respectively

The method of direct immobilized antigen albumen to magnetic particle is as follows: from Bacillus coli expression and purifying acquisition with His mark The tumour related auto-antibodies of the recombination of label correspond to antigen protein P53, SOX2, CK8, HuD, CAGE, NY-ESO-1, GBU4-5, P62, KRAS and MAGE A4.Add 2.5 μ g recombinant proteins to 200 μ L magnetic particle suspensions (0.05M MES, pH 5.0, containing 1.25 × 10⁶Magnetic particle), dark in be incubated at room temperature 2 hours.20 μ L 1mg/mL EDC are added then to mixture, are incubated at room temperature in dark Overnight.It is cleaned 4 times using 1mL PBS (containing 1%BSA, 0.2%Tween-20 and 0.05% Sodium azide, pH 7.4), 500 μ L Closing/preservation buffer (PBS-TBN contains 0.1%BSA, 0.02%Tween-20 and 0.05% Sodium azide, pH 7.4) is resuspended Magnetic particle and 2~8 DEG C of preservations in dark.

The method of indirect immobilized antigen albumen to magnetic particle is as follows: tumour related auto-antibodies are corresponded to antigen protein CDNA, biotin acceptor polypeptid DNA and gene constructed to the same expression vector of biotin ligase, utilize Bacillus coli expression system Controlling is for biotinylated protein.Using the specific reaction between biotin and Streptavidin by tumour related auto-antibodies Corresponding antigen protein is indirectly secured to respectively on the magnetic particle with Streptavidin, and it is micro- that each antigen protein corresponds to a kind of magnetic Grain.

The method for directly fixing antitumor marker antibody to magnetic particle is as follows: taking appropriate magnetic particle (5 × 10⁵) extremely micro In buret, the cleaning of 100 μ L deionized waters is primary, and 80 μ L activation buffer (100mM NaH are added₂PO₄, pH 6.2) and magnetic is resuspended 10 μ L 50mg/mL sulfo-NHS (N- hydroxy thiosuccinimide) and 10 μ L 50mg/mL EDC (1- (3- are added in particle Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) for activating magnetic particle, magnetic particle suspension is put in room in dark Temperature, which is incubated for 20 minutes, activates it sufficiently.It is used in combination twice using coupling buffer (0.05M MES, pH 5.0) washing magnetic particle Magnetic particle is resuspended in 100 μ L coupling buffers, and 5 μ g Anti-CEA, Anti-NSE or Anti-SCC Dan Ke then are added to suspension Mixture is put in and is incubated at room temperature 2 hours in dark and ceaselessly shakes by grand antibody.Finally using PBS (containing 0.1%BSA, 0.02%Tween-20 and 0.05% Sodium azide, pH 7.4) cleaning is put in dark 4 DEG C of preservations twice, after resuspension.Every primary antibody is swollen Tumor markers antibody corresponds to a kind of magnetic particle.

Embodiment 2, the preparation for detecting tumour related auto-antibodies and tumor markers kit

The present invention, which is prepared for one kind according to the principle of Dot-ELISA and double antibody sandwich method, can be used for lung cancer early stage The a variety of autoantibodies and tumor markers combined detection kit of screening and diagnosis.The principle of Dot-ELISA is will to swell The corresponding antigen protein of tumor related auto-antibodies is connected on solid phase carrier, and autoantibody to be measured is in combination at solid phase in sample Antigen-is by inspection auto-antibody complex, then with enzyme mark anti-human IgG antibodies and solid phase antigen-by inspection auto-antibody complex Autoantibody combines, and forms solid phase antigen-by inspection autoantibody-enzyme mark anti-human IgG antibodies' compound, then measurement adds shiner Luminous intensity afterwards determines autoantibody to be measured.

It is that tumor markers antibody is fixed on solid phase carrier between the principle of double antibody sandwich enzyme immunization, in sample Tumor markers antigen to be measured it is in combination at solid phase antibody-by inspection antigenic compound, then with another enzyme mark tumor-marker quality testing Antibody and solid phase antibody-are surveyed by the antigen binding in inspection antigenic compound, formation solid phase antibody-is detected anti-by inspection antigen-enzyme mark Nanocrystal composition, then measurement adds the luminous intensity after shiner, determines tumor markers content to be measured.

In the present embodiment, the kit forms are as follows:

A, for the reagent of tumour related auto-antibodies detection

1, different tumour related auto-antibodies correspond to antigen protein (P53, SOX2, CK8, HuD, CAGE, NY-ESO-1, GBU4-5, P62, KRAS and MAGE A4) coated magnetic particle and unloaded body protein (Vector) coated magnetic particle；

2, the humanization Anti-His antibody of certain concentration is as standard items；

3, the humanization Anti-His antibody of high concentration and low concentration is as quality-control product；

4, the anti-human IgG antibodies of alkaline phosphatase (ALP) label.

B, for the reagent of the detection of tumor markers:

1, tumor markers antibody (Anti-CEA, Anti-NSE, Anti-SCC) coated magnetic particle；

2, multi-tumor marker (CEA, NSE, SCC) definite value quality-control product of high concentration and low concentration；

3, CEA, NSE, SCC of alkali phosphatase enzyme mark detect antibody；

C, other solution or apparatus

1, Sample dilution: contain the PBST buffer of 1% (W/V) BSA；

2, antibody diluent: contain the PBST buffer of 1% (W/V) BSA；

3, concentrated cleaning solution: 10 × PBST buffer containing 0.5% Tween-20；

4, reaction vessel: reaction cup；

5, luminescent solution: 100 μ g/mL AMPPD, 0.1M Tris-HCl, 0.1M NaCl, 50mM MgCl₂(pH 9.5)。

Embodiment 3, detection method

1. sample and standard items prepare

It takes humanization Anti-His antibody standard substance 1 to dilute twice and obtains standard items 2, then gradient dilution obtains standard items 3 ~6；

It takes CEA standard items 1, NSE standard items 1, SCC standard items 1 to dilute 5 times respectively and obtains CEA standard items 2, NSE standard items 2, SCC standard items 2, then gradient dilution obtain CEA standard items 3~6, NSE standard items 3~6 and SCC standard items 3~6；

Part serum is taken to dilute 100 times for detecting autoantibody using Sample dilution.

2. detecting autoantibody

2.1 production standard curves: being that X-axis draws standard song using luminous intensity as Y-axis, antibody concentration denary logarithm Line calculates standard curve regression equation.

2.1.1 standard items 1~6 50 μ L are added in 12 reaction cups respectively, are divided into 6 groups；

2.1.2 20 μ L Vector-His magnetic particle suspensions is taken to be added in each reaction cup；

2.1.3 50 μ L of Sample dilution is added, incubates 15 minutes for 37 DEG C, is washed out 5 times after mixing；

2.1.4 100 μ L of anti-human IgG antibodies' dilution is added；

2.1.5 it incubates 15 minutes for 37 DEG C, is washed out 5 times after mixing；

2.1.6 disperse magnetic particle, luminescent solution (containing AMPPD) 100 μ L is added；

2.1.7 it is protected from light 1~5 minute detection luminous intensity after mixing.

2.2 detection sample autoantibodies

2.2.1 it is separately added into 50 μ L of sample to be tested after diluting in 22 reaction cups, is divided into 11 groups, in 2 groups of 4 reaction cups In be separately added into quality-control product 1 and quality-control product 2, each 50 μ L；

2.2.2 sample to be tested reaction cup is separately added into 20 μ L P53-His magnetic particles, SOX2-His magnetic particle, CK8-His Magnetic particle, HuD-His magnetic particle, CAGE-His magnetic particle, NY-ESO-1-His magnetic particle, GBU4-5-His magnetic particle, P62- His magnetic particle, KRAS-His magnetic particle, MAGE A4-His magnetic particle or Vector-His magnetic particle suspension, 1 He of quality-control product 2 reaction cup of quality-control product is separately added into 20 μ L Vector-His magnetic particle suspensions；

2.2.3 50 μ L of Sample dilution is added, incubates 15 minutes for 37 DEG C, is washed out 5 times after mixing；

2.2.4 100 μ L of anti-human IgG antibodies' dilution is added；

2.2.5 it incubates 15 minutes for 37 DEG C, is washed out 5 times after mixing；

2.2.6 disperse magnetic particle, luminescent solution (containing AMPPD) 100 μ L is added；

2.2.7 it is protected from light 1~5 minute detection luminous intensity after mixing.

2.3 calculate sample autoantibody

Corresponding background luminance value is individually subtracted in the luminous intensity values that antigen detects in same serum sample (Vector-His magnetic particle group luminous intensity values are multiplied by transformation ratio), obtains the absolute luminescence intensity value of antigen, will absolutely send out Light intensity value substitutes into standard curve regression equation calculation sample the autoantibody concentrations value for being directed to this antigen.

3. detecting tumor markers (TM) CEA, NSE, SCC

3.1 are added Sample dilution, standard items 1~6, tumor mark quality control product 1, TM quality-control product in reaction cup respectively 2 or 25 μ L of sample to be tested does two in parallel；

3.2 are added 20 μ L of magnetic particle (attachment Anti-CEA or Anti-NSE or Anti-SCC) suspension, add sample 50 μ L of dilution；

It incubates 15 minutes for 37 DEG C, is washed out 5 times after 3.3 mixings；

3.4 are added the 100 μ L of detection antibody diluent of corresponding alkali phosphatase enzyme mark；

It incubates 17 minutes for 37 DEG C, is washed out 5 times after 3.5 mixings；

3.6 dispersion magnetic particles, are added luminescent solution (containing AMPPD) 100 μ L；

3.7 are protected from light 1~5 minute detection luminous intensity after mixing；

3.8 using luminous intensity as Y-axis, tumor markers concentration denary logarithm be X-axis draw standard curve, pass through Four parameter logistic curves drawn calculate test sample TM concentration.

4. result judges

4.1 provide each autoantibody detection level and tumor markers concentration level respectively, so as to more multiple detections As a result, judging autoantibodies in serum and Tumor Marker Levels variation.

4.2 provide the boundary value suggested and judgment method:

If any tumor markers (CEA, NSE or SCC) or autoantibody are more than the high boundary value of setting, determine The index result is " high level "；

If any tumor markers (CEA, NSE or SCC) or autoantibody are more than the middle boundary value of setting, but are lower than High boundary value then determines that the index result is " middle level "；

If tumor markers (CEA, NSE or SCC) and autoantibody determine that this refers to lower than the middle boundary value of setting Marking result is " no significant tumor markers or autoantibody are measured ".

4.3 comprehensive 4.1 and 4.2 as a result, providing the testing result of sample.

Embodiment 4, single tumour related auto-antibodies detection patients with lung cancer combine inspection with multiple tumour related auto-antibodies The effect for surveying patients with lung cancer compares

Sample prepares: having collected the serum (lung cancer group) of 100 early stage of lung cancer patients and the serum of 100 Healthy Peoples (control group).All patients with lung cancer through CT detect display tubercle be less than 20mm, through pathological diagnosis be diagnosed as the lung cancer I phase (57) or II phase (43), Serum of Patients with Lung Cancer acquire before patient receives any chemicotherapy.Healthy Human Serum comes from health examination people Group, without any malignant tumour related disease.

Using kit of the invention and detection method above-mentioned respectively to 100 Serum of Patients with Lung Cancer and 100 health The level of tumour related auto-antibodies or Tumor Marker Levels are detected in human serum.

As Figure 1A~H carries out autoantibody inspection to the serum of patients with lung cancer using the magnetic particle for being attached with single antigen It surveys.The results show that area (AUC) is no more than 0.66 under the performance curve (ROC curve) drawn, diagnosis only has lower standard True property.In order to improve diagnostic accuracy, present inventor has performed extensive researchs, determine Combining diagnosis scheme.

Using three kinds of magnetic particles for being attached with antigen " P53, SOX2, CK8 " respectively, itself is carried out to the serum of patients with lung cancer Antibody combined detection.The results show that area can be more than 0.7 under the ROC curve drawn, numerical value 0.716, it is seen that diagnosis is accurate Property is improved, such as Fig. 2A.

Using four kinds of magnetic particles for being attached with antigen " P53, SOX2, CK8, HuD " respectively, the serum of patients with lung cancer is carried out Autoantibody joint-detection.The results show that area is more than 0.7 under the ROC curve drawn, numerical value 0.733, it is seen that diagnosis is accurate Property, which has, more to be significantly improved, such as Fig. 2 B.

Using four kinds of magnetic particles for being attached with antigen " P53, SOX2, CK8, HuD, CAGE " respectively, to the blood of patients with lung cancer It is clear to carry out autoantibody joint-detection.The results show that area figures are 0.76 under the ROC curve drawn, it is seen that diagnostic accuracy It further improves, such as Fig. 2 C.

Using six kinds of magnetic particles for being attached with antigen " P53, SOX2, CK8, HuD, CAGE, NY-ESO-1 " respectively, to lung cancer The serum of patient carries out autoantibody joint-detection.The results show that area figures are 0.778 under the ROC curve drawn, it is seen that examine Disconnected accuracy further improves, such as Fig. 2 D.

Embodiment 5, multiple tumour related auto-antibodies joint-detections merge one or more of tumor markers joint-detections

In order to further improve diagnostic accuracy, present inventor has performed further furtheing investigate, including diagnosis mark The research of will object and the research of methodology.Having determined will be one or more of the merging of multiple tumour related auto-antibodies joint-detections Tumor markers joint-detection advanced optimizes method.

It is micro- using the three kinds of magnetic particles and the coated magnetic of Anti-CEA that are attached with antigen " P53, SOX2, CK8 " respectively Grain carries out for " P53, SOX2, CK8 " autoantibody and for the joint-detection of CEA antigen the serum of patients with lung cancer.Connection It closes testing result to show, area figures are 0.819 under ROC curve, it is seen that diagnostic accuracy is significantly higher than the feelings in embodiment 4 Shape, such as Fig. 3 A.

Utilize the four kinds of magnetic particles and Anti-CEA, Anti-NSE packet for being attached with antigen " P53, SOX2, CK8, HuD " Two kinds of magnetic particles of quilt, to the serum of patients with lung cancer carry out for " P53, SOX2, CK8, HuD " autoantibody and for CEA, The joint-detection of NSE antigen.Joint detection results show that area figures are 0.914 under ROC curve, it is seen that diagnostic accuracy is non- Chang Gao, such as Fig. 3 B.

When therefore, by three or more autoantibody magnetic bead and one or more of antibody magnetic bead joint-detections, AUC will surpass 0.9 is crossed, shows that diagnosis has very high accuracy.

To sum up, joint-detection tumour related auto-antibodies and the method for tumor markers combination help to improve pernicious swollen The recall rate and diagnostic accuracy of tumor.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

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Ser Glu Glu Gln Gly Gly Gln Gly Thr Pro Pro Ala Glu Asp Ala Ala

1125 1130 1135

Cys Leu Gln Ser Pro Gln Pro Glu Asp Thr Gly Ala Glu Gly Gly Ala

1140 1145 1150

Glu Ser Lys Thr Ser Ser Glu Asn Gln Lys Pro Gly Gly Tyr Leu Val

1155 1160 1165

Phe Lys Arg Trp Leu Ser Ser Asn Arg

1170 1175

<210> 8

<211> 443

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 8

Met Gln Arg Arg Asp Asp Pro Ala Ala Arg Met Ser Arg Ser Ser Gly

1 5 10 15

Arg Ser Gly Ser Met Asp Pro Ser Gly Ala His Pro Ser Val Arg Gln

20 25 30

Thr Pro Ser Arg Gln Pro Pro Leu Pro His Arg Ser Arg Gly Gly Gly

35 40 45

Gly Gly Ser Arg Gly Gly Ala Arg Ala Ser Pro Ala Thr Gln Pro Pro

50 55 60

Pro Leu Leu Pro Pro Ser Ala Thr Gly Pro Asp Ala Thr Val Gly Gly

65 70 75 80

Pro Ala Pro Thr Pro Leu Leu Pro Pro Ser Ala Thr Ala Ser Val Lys

85 90 95

Met Glu Pro Glu Asn Lys Tyr Leu Pro Glu Leu Met Ala Glu Lys Asp

100 105 110

Ser Leu Asp Pro Ser Phe Thr His Ala Met Gln Leu Leu Thr Ala Glu

115 120 125

Ile Glu Lys Ile Gln Lys Gly Asp Ser Lys Lys Asp Asp Glu Glu Asn

130 135 140

Tyr Leu Asp Leu Phe Ser His Lys Asn Met Lys Leu Lys Glu Arg Val

145 150 155 160

Leu Ile Pro Val Lys Gln Tyr Pro Lys Phe Asn Phe Val Gly Lys Ile

165 170 175

Leu Gly Pro Gln Gly Asn Thr Ile Lys Arg Leu Gln Glu Glu Thr Gly

180 185 190

Ala Lys Ile Ser Val Leu Gly Lys Gly Ser Met Arg Asp Lys Ala Lys

195 200 205

Glu Glu Glu Leu Arg Lys Gly Gly Asp Pro Lys Tyr Ala His Leu Asn

210 215 220

Met Asp Leu His Val Phe Ile Glu Val Phe Gly Pro Pro Cys Glu Ala

225 230 235 240

Tyr Ala Leu Met Ala His Ala Met Glu Glu Val Lys Lys Phe Leu Val

245 250 255

Pro Asp Met Met Asp Asp Ile Cys Gln Glu Gln Phe Leu Glu Leu Ser

260 265 270

Tyr Leu Asn Gly Val Pro Glu Pro Ser Arg Gly Arg Gly Val Pro Val

275 280 285

Arg Gly Arg Gly Ala Ala Pro Pro Pro Pro Pro Val Pro Arg Gly Arg

290 295 300

Gly Val Gly Pro Pro Arg Gly Ala Leu Val Arg Gly Thr Pro Val Arg

305 310 315 320

Gly Ala Ile Thr Arg Gly Ala Thr Val Thr Arg Gly Val Pro Pro Pro

325 330 335

Pro Thr Val Arg Gly Ala Pro Ala Pro Arg Ala Arg Thr Ala Gly Ile

340 345 350

Gln Arg Ile Pro Leu Pro Pro Pro Pro Ala Pro Glu Thr Tyr Glu Glu

355 360 365

Tyr Gly Tyr Asp Asp Thr Tyr Ala Glu Gln Ser Tyr Glu Gly Tyr Glu

370 375 380

Gly Tyr Tyr Ser Gln Ser Gln Gly Asp Ser Glu Tyr Tyr Asp Tyr Gly

385 390 395 400

His Gly Glu Val Gln Asp Ser Tyr Glu Ala Tyr Gly Gln Asp Asp Trp

405 410 415

Asn Gly Thr Arg Pro Ser Leu Lys Ala Pro Pro Ala Arg Pro Val Lys

420 425 430

Gly Ala Tyr Arg Glu His Pro Tyr Gly Arg Tyr

435 440

<210> 9

<211> 189

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 9

Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys

1 5 10 15

Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr

20 25 30

Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly

35 40 45

Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr

50 55 60

Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys

65 70 75 80

Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr

85 90 95

Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val

100 105 110

Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys

115 120 125

Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr

130 135 140

Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val

145 150 155 160

Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys

165 170 175

Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met

180 185

<210> 10

<211> 317

<212> PRT

<213>homo sapiens (Homo sapiens)

<400> 10

Met Ser Ser Glu Gln Lys Ser Gln His Cys Lys Pro Glu Glu Gly Val

1 5 10 15

Glu Ala Gln Glu Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Thr

20 25 30

Thr Glu Glu Gln Glu Ala Ala Val Ser Ser Ser Ser Pro Leu Val Pro

35 40 45

Gly Thr Leu Glu Glu Val Pro Ala Ala Glu Ser Ala Gly Pro Pro Gln

50 55 60

Ser Pro Gln Gly Ala Ser Ala Leu Pro Thr Thr Ile Ser Phe Thr Cys

65 70 75 80

Trp Arg Gln Pro Asn Glu Gly Ser Ser Ser Gln Glu Glu Glu Gly Pro

85 90 95

Ser Thr Ser Pro Asp Ala Glu Ser Leu Phe Arg Glu Ala Leu Ser Asn

100 105 110

Lys Val Asp Glu Leu Ala His Phe Leu Leu Arg Lys Tyr Arg Ala Lys

115 120 125

Glu Leu Val Thr Lys Ala Glu Met Leu Glu Arg Val Ile Lys Asn Tyr

130 135 140

Lys Arg Cys Phe Pro Val Ile Phe Gly Lys Ala Ser Glu Ser Leu Lys

145 150 155 160

Met Ile Phe Gly Ile Asp Val Lys Glu Val Asp Pro Ala Ser Asn Thr

165 170 175

Tyr Thr Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly

180 185 190

Asn Asn Gln Ile Phe Pro Lys Thr Gly Leu Leu Ile Ile Val Leu Gly

195 200 205

Thr Ile Ala Met Glu Gly Asp Ser Ala Ser Glu Glu Glu Ile Trp Glu

210 215 220

Glu Leu Gly Val Met Gly Val Tyr Asp Gly Arg Glu His Thr Val Tyr

225 230 235 240

Gly Glu Pro Arg Lys Leu Leu Thr Gln Asp Trp Val Gln Glu Asn Tyr

245 250 255

Leu Glu Tyr Arg Gln Val Pro Gly Ser Asn Pro Ala Arg Tyr Glu Phe

260 265 270

Leu Trp Gly Pro Arg Ala Leu Ala Glu Thr Ser Tyr Val Lys Val Leu

275 280 285

Glu His Val Val Arg Val Asn Ala Arg Val Arg Ile Ala Tyr Pro Ser

290 295 300

Leu Arg Glu Ala Ala Leu Leu Glu Glu Glu Glu Gly Val

305 310 315

Claims

1. the purposes of tumour related auto-antibodies and tumor markers, for being marked with the diagnosis for preparing diagnosing tumour as diagnosis target Reagent or kit, in which:

2. the purposes of the reagent of specific detection tumour related auto-antibodies or tumor markers, is used to prepare the examination of diagnosing tumour Agent box；Wherein,

(1) reagent of the specific detection tumour related auto-antibodies includes: the corresponding antigen of tumour related auto-antibodies P53, SOX2 and CK8；

(2) reagent of the specific detection tumor markers includes: specific detection selected from the group below 1,2 or 3 kind of tumour mark The reagent of will object: CEA, NSE, SCC.

3. the purposes as described in claim 1~2 is any, which is characterized in that the corresponding antigen of the tumour related auto-antibodies It further include selected from the group below 1,2 or a variety of: HuD, CAGE, NY-ESO-1, GBU4-5, P62, KRAS and MAGE A4；Or

The tumor markers include: CEA；Or CEA and NSE；Or CEA and SCC；Or CEA, NSE and SCC.

4. the purposes as described in claim 1~2 is any, which is characterized in that (1) in, the diagnostic reagent is for detecting There are situation or the reagents of amount for tumour related auto-antibodies described in sample to be tested；Preferably, the diagnostic reagent is Suitable for chemoluminescence method or the reagent of Fluorometric assay；Or

(2) in, the diagnostic reagent is that there are situation or amounts for detecting tumor markers described in sample to be tested Reagent；Preferably, the diagnostic reagent is the reagent containing the antibody for resisting the tumor markers；Preferably, described examines Disconnected reagent is the reagent suitable for chemoluminescence method or Fluorometric assay；

Preferably, the diagnostic reagent is all based on the detection reagent of chemoluminescence method in (1) and (2)；Or in (1) and (2), The diagnostic reagent is all based on the detection reagent of fluorescence method.

5. purposes as claimed in claim 4, which is characterized in that the tumour related auto-antibodies correspond to antigen and are attached to solid phase On carrier；Preferably, the solid phase carrier includes: particle, microballoon, slide, test strips；Preferably, the tumour correlation is certainly Body antibody corresponds to antigen and is expressed by expression vector；More preferably, the tumour related auto-antibodies are corresponded into the cDNA of antigen, biology Plain receptor polypeptides DNA and biotin ligase are gene constructed to an expression vector, prepare biotinylated protein；Utilize biotin The corresponding antigen protein of tumour related auto-antibodies is indirectly secured to solid phase carrier by specific reaction between Streptavidin； And/or

The antibody of the tumor markers is resisted to be attached on solid phase carrier；Preferably, the solid phase carrier include: particle, microballoon, Slide, test strips, plastic bead, liquid-phase chip, microwell plate or affinity membrane；And/or

The diagnostic reagent is the diagnostic reagent using serum as sample to be tested, using blood plasma as the diagnostic reagent of sample to be tested or with Tissue or cell are the diagnostic reagent of sample to be tested.

6. a kind of kit for diagnosing tumour, which is characterized in that include: in the kit

(1) reagent of specific detection tumour related auto-antibodies, the tumour related auto-antibodies correspond respectively to antigen P53, SOX2 and CK8；

7. kit as claimed in claim 6, which is characterized in that (1) in, the diagnostic reagent is for detecting to test sample There are situation or the reagents of amount for tumour related auto-antibodies in this；Preferably, the diagnostic reagent is containing described swollen Tumor related auto-antibodies correspond to the reagent of antigen；Preferably, the diagnostic reagent is suitable for chemoluminescence method or fluorescence method The reagent of detection；Or

8. kit as claimed in claims 6 or 7, which is characterized in that preferably, the tumour related auto-antibodies are corresponding Antigen further includes selected from the group below 1,2 or a variety of: HuD, CAGE, NY-ESO-1, GBU4-5, P62, KRAS and MAGE A4；Or

9. kit as claimed in claims 6 or 7, which is characterized in that further include ingredient selected from the group below in the kit: Solid phase carrier, dilution, reference substance, standard items, quality-control product, anti-human IgG antibodies detect antibody, luminescence reagent, cleaning solution；Compared with Goodly, the standard items include: tumor markers standard items, humanization tag antibody standard items；Preferably, the quality-control product packet It includes: tumor mark quality control product, humanization tag antibody quality-control product；Preferably, the solid phase carrier includes: particle, microballoon, glass Piece, test strips.

The kit as described in 10. purposes or claim 6~9 as described in Claims 1 to 5 is any are any, which is characterized in that The tumour includes: infantile tumour, is preferably included in the tumour before the IV phase or before the III phase, more preferably includes in latent Phase, precancerous lesion, the tumour of I phase or II phase；In the tumour without the apparent uncomfortable stage；Tumour small in size；Cancer The tumour of position secret；Preferably, the tumour is lung cancer.