CN110196328A - Application and its kit of one group of tumor associated antigen in cancer of the esophagus early screening kit - Google Patents

Abstract

The invention belongs to oncomolecularbiology fields, and in particular to application and its kit of one group of tumor associated antigen in cancer of the esophagus early screening kit.Kit of the present invention includes solid phase carrier and the cancer of the esophagus tumor associated antigen that is coated on solid phase carrier, and the cancer of the esophagus tumor associated antigen is CP96 gene expression object, CRP78 gene expression object, MMP-10 gene expression object, cav-1 gene expression object, CDC25B gene expression object, PGK1 gene expression object and HSP105 gene expression object；The kit further includes the secondary antibody of phycoerythrin conjugation.Kit of the present invention is up to 96.1% and 95.1% to the detection specificity and sensibility of the cancer of the esophagus respectively；Also, compared with organizing biopsy, kit of the present invention need to only acquire the blood of crowd to be checked, so that it may which its risk for suffering from the cancer of the esophagus of screening, wound is small, Small side effects, and easy to use, has good potential applicability in clinical practice.

Description

Application and its examination of one group of tumor associated antigen in cancer of the esophagus early screening kit Agent box

Technical field

The invention belongs to oncomolecularbiology fields, and in particular to one group of tumor associated antigen is in cancer of the esophagus early screening Application and its kit in kit.

Background technique

The cancer of the esophagus is that one of most common six big malignant tumours, morbidity and mortality account for the 6th in the world；China It is Incidence of esophageal cancer and the highest country of the death rate, national disease incidence occupies the 6th, and the death rate occupies the 4th.It is global annual new Increase in 500,000 patient with esophageal carcinoma, more than half occurs in China.Crowd is dramatically different with west, and Chinese human esophagus cancer is outstanding Epidemiologic feature is significant regionality distribution difference and apparent familial aggregation.Henan, Hebei and Shanxi San Sheng have a common boundary Wild jujube in Taihang Mountain Area, it is also Incidence of esophageal cancer and the highest area of the death rate in the world that Henan Linzhou City, which is domestic,.Chinese and western oesophagus The histological type of cancer and the significantly different problem in science for leading to Chinese and western focus of attention of popular feature and Research Thinking are obvious Difference, research achievement is mutually used for reference and shared difficulty.

Cancer of the esophagus poor prognosis, 5 years survival rates of advanced esophagus cancer patients only 15% or so, and the 5 of early stage patient with esophageal carcinoma Year survival rate is 90% or so.Although 5 years survival rates of the early stage cancer of the esophagus are apparently higher than middle and advanced stage, the trouble clinically made a definite diagnosis for the first time In person, early carcinoma only accounts for 5% or so.Therefore the diagnosis for improving the early stage cancer of the esophagus is of crucial importance.Current clinically normal scope skill Art.However traditional Endoscopy will use idodine, however the use of idodine may cause certain adverse reactions to human body；Scope Narrow band imaging uses electron stain, the side effect without chemical stain, but expensive and endoscopy has wound, efficiency It is low, therefore limit the popularization of scope Narrow-Band Imaging inspection.

Oesophagus cancer susceptibility gene is screened using molecular mechanism, positioning esophageal canceration key drives gene, establishes people at highest risk With early detection molecular marker screening system, early carcinoma discovery rate is improved, is the key that reduce mortality rate of esophageal cancer.By molecule Kit is made in marker, is applied to clinical detection, and the discovery rate of the early stage cancer of the esophagus can be improved, and reduces the wound to patient. Therefore, a kind of kit convenient for carrying out suffering from cancer of the esophagus risk supervision to people at highest risk is developed to be of great significance.

Summary of the invention

The purpose of the present invention is to provide application of one group of tumor associated antigen in cancer of the esophagus early screening kit, originally Another of invention is designed to provide a kind of cancer of the esophagus early screening kit.

Based on above-mentioned purpose, the present invention adopts the following technical scheme:

Application of one group of tumor associated antigen in cancer of the esophagus early screening kit, one group of tumor associated antigen are CP96 gene expression object, CRP78 gene expression object, MMP-10 gene expression object, cav-1 gene expression object, CDC25B gene table Up to object, PGK1 gene expression object and HSP105 gene expression object.

It is another object of the present invention to provide a kind of cancer of the esophagus early screening kits, for assessing esophageal patient's cancer Occurrence risk, and indicate the cancer of the esophagus by stages.Based on the purpose, the technical scheme adopted by the invention is as follows: a kind of cancer of the esophagus Early screening kit, including solid phase carrier and the cancer of the esophagus tumor associated antigen being coated on solid phase carrier, the cancer of the esophagus Tumor associated antigen be CP96 gene expression object (designated hereinafter simply as CP96), CRP78 gene expression object (designated hereinafter simply as CRP78), MMP-10 gene expression object (designated hereinafter simply as MMP-10), cav-1 gene expression object (designated hereinafter simply as cav- 1), CDC25B gene expression object (designated hereinafter simply as CDC25B), PGK1 gene expression object (designated hereinafter simply as PGK1) and HSP105 gene expression object (designated hereinafter simply as HSP105)；The kit further includes the secondary antibody of phycoerythrin conjugation, algae red egg The secondary antibody of white conjugation is in the following secondary antibody for being referred to as PE conjugation.

Preferably, BioPlex pearl is conjugated on cancer of the esophagus tumor associated antigen.

Preferably, which further includes Sample dilution, developing solution, terminate liquid and cleaning solution.

Preferably, Sample dilution is to contain 1%(w/v) the PBST buffer of BSA.

Preferably, developing solution is mixed in equal volume by developing solution A and developing solution B, and the developing solution A is 0.02%(w/v) TMB, developing solution B are 0.006%(w/v) urea peroxide element.

Preferably, terminate liquid 10%(v/v) sulfuric acid solution.

Preferably, cleaning solution is containing 0.05%(v/v) the PBST buffer of Tween-20；The concentration of the PBST buffer is 0.01mol/L；The pH of the cleaning solution is 7.4.

Compared with prior art, the invention has the benefit that

1. the cell compared with the normal esophageal tissue epithelial cell for adjoining esophageal neoplasm in esophageal neoplasm block includes unique one Histone/tumour antigen.The presence of tumour antigen is related with for the generation of autoantibody of these tumour antigens.In the present invention Tumor associated antigen is CP96 gene expression object, CRP78 gene expression object, MMP-10 gene expression object, cav-1 gene expression Object, CDC25B gene expression object, PGK1 gene expression object and HSP105 gene expression object can serve as cancer of the esophagus screening and One group of new marker of diagnosis.Kit provided by the invention detects people to be checked using one group of cancer of the esophagus tumor associated antigen The content of corresponding antibody, assesses the risk that individual to be checked suffers from the cancer of the esophagus with this, can be used for facing for the early stage cancer of the esophagus in body serum Bed auxiliary diagnosis, according to gene expression product standard listed by table 4, which meets lowest bid listed by the phase Standard can be diagnosed as the current period cancer of the esophagus.Kit of the present invention is up to 96.1% to the detection specificity and sensibility of the cancer of the esophagus respectively With 95.1%.

2. kit of the present invention need to only acquire the blood of crowd to be checked compared with organizing biopsy, so that it may its trouble food of screening The risk of pipe cancer, wound is small, Small side effects, and easy to use, has good potential applicability in clinical practice.

Detailed description of the invention

Fig. 1 is the knot based on protein expression profiles between two-dimentional mass spectrography cancer of the esophagus tumor biopsy and the normal tissue adjoined Fruit；

Fig. 2 is the vivoexpression flow chart of biomarker；

Fig. 3 is the purifying flow chart of biomarker；

Fig. 4 is the detection schematic diagram of kit of the present invention；

Fig. 5 is that the compound for the secondary antibody immune response that biomarker-BioPlex pearl conjugate and primary antibody and PE are conjugated is illustrated Figure；

Fig. 6 is the workflow using BioPlex systematic survey autoantibody；

Fig. 7 is the standard curve in the concentration and BioPlex pearl and secondary antibody of primary antibody between the signal of both PE for being conjugated.

Specific embodiment

Embodiment 1: the discovery and identification of biomarker in oesophagus tumor

(1) albumen is extracted from the biopsy of patient

It collects the pairing biopsy (tumor biopsy is relative to the normal tissue adjoined) of 500mg patient and is washed with PBS, by it It is immersed in quick-frozen in liquid nitrogen, then takes out the freezing tissue, be homogenized the freezing tissue using pestle and mortar, to the sample of homogenate 5 μ L cracking aqueous solution is added in product, cracking aqueous solution includes 8mol/L urea, 4%(v/v) CHAPS, 2%(v/v) IPG buffer With the PMSF of 0.2mg/mL, then it is vortexed and mixes at least 5min until tissue is completely dispersed；Then at 4 DEG C, with 14000rpm/ Pyrolysis product centrifugal clarification is taken supernatant spare by the revolving speed centrifugation 10min of min；Utilize cleaning reagent box, that is, 2D Clean Up kit cleans supernatant further to remove desalination and impurity；With the rehydration solution of 1 μ L (DTT&IPG buffer is not added Cracking aqueous solution) pellet is resuspended；Antigen protein concentration is measured followed by Bio-Rad albuminometry, and will be resisted Former albumen is saved according to 200g/ pipe aliquot in -70 DEG C.

(2) albumen is parsed using two dimensional electrophoresis

2.8mg DTT, 5 μ L pharmalyte or IPG buffers and 2 μ L bromophenol blues are added into 1mL rehydration solution；It will 75pg antigen protein sample is added to the Immobiline DryStrip(i.e. IPG of the 13cm containing 250 μ L rehydration solutions Item), after removing protection cap, ipg strip is placed in item frame, under the gel of ipg strip is lateral, and is covered with cover Fluid To prevent ipg strip to be dehydrated during electrophoresis, ipg strip is then placed on Ettan IPGphor(Amersham) on to carry out etc. Electric point focusing, this is the first dimension electrophoresis.

After the first dimension electrophoresis, ipg strip is balanced with balance solution, balance solution (urea, the 2%(v/ of 6mol/L V) SDS, 50mM Tris HCI pH 6.8,30%(v/v) glycerol, 0.002%(v/v) bromophenol blue, 10mg/mL DTT and 25mg/ The PBS buffer solution of mL IAA)；Then ipg strip is washed 4-5 times with Ix SDS electrophoretic buffer；Ipg strip is placed on the second dimension to coagulate Simultaneously with sealing solution covering, sealing solution is added with 0.5%(v/v at the top of glue) low melting-point agarose and 0.002%(v/v) bromine The Ix SDS electrophoretic buffer of phenol indigo plant；Then it carries out two dimensional electrophoresis: being first that 30mA carries out electrophoresis 15min with size of current, connect With size of current be 60mA carry out electrophoresis 3-4h.

After completing two dimensional electrophoresis, ipg strip is taken out from box, is fixed and is used cma staining.It identifies and represents 7 7 points of the albumen of kind up-regulation.In order to identify albumen, the gel slice of Silver stain is decolourized and use trypsin digestion with from Albumen is discharged in ipg strip makes peptide mass fingerprinting spectrum for MALDI-TOF analysis, to detect the kind for discharging albumen in ipg strip Class and its content.

(3) identification of biomarker

Based on Two way chromatograms-mass spectrography to cancer of the esophagus tumour cell compared with normal esophageal epithelial cell, find oesophagus cancerous swelling There are one group of unique albumen in oncocyte, and are identified using two dimension/mass spectrography the unique albumen of the group, share seven kinds Albumen in oesophagus tumor there are specific expressed, this seven kinds of albumen be respectively CP96, CRP78, MMP-10, cav-1, CDC25B, PGK1 and HSP105, as a result as shown in Figure 1.This seven kinds of albumen can be used as biomarker group, biomarker group egg White molecular weight is as shown in table 1；It moreover has been found that the life in the antibody and cancer of the esophagus tumour cell in the serum of patient with esophageal carcinoma The abundance of object marker is positively correlated, so the diagnosis to the cancer of the esophagus can be used to by the detection to Serum Antibody.

The molecular weight of 1 biomarker histone of table

Embodiment 2: the vivoexpression and protein purification of biomarker group

(1) vivoexpression of biomarker group

Based on the amino acid sequence for the biomarker being targeted, biomarker is expressed using the C DNA cloning being commercially synthesized Group, the vivoexpression process of biomarker group is as shown in Fig. 2, detailed process is as follows:

The plasmid with HIS label of CDNA insert containing encoding human marker group is converted into DH5 competent cell, Choose single colony and grow it in bacterial cultures, expands the number of plasmid, and mention from bacterium by miniprep Plasmid is taken, plasmid is further converted into BL21DE3 or BL21DE3pLysS competent cell, select the bacterium of conversion and is made It grows in 2X 100mL LB culture medium, when it is 0.06 that bacterial cultures, which reaches OD value, adds to 100mL bacterial cultures Enter 200 μM of IPTG, the bacterial cultures using 100mL without IPTG shakes bacterial cultures as negative control at 30 DEG C It is incubated for, after being incubated at least 3h or more or being incubated overnight, takes 500 μ L bacterial cultures to store at -20 DEG C spare.

500 μ L are mixed in 500mL centrifugal bottle by IPTG induction and 500 μ L without the bacterial cultures that IPTG is induced It closes uniformly, at 4 DEG C, collects bacterial cell after being centrifuged 20min with the revolving speed of 9000rpm/min, take 500 μ L supernatants as another One negative control, and discard remaining supernatant, to different point (bacterium pellet) collections bacterial cultures and Negative control carries out SDS-PAGE electrophoresis to parse albumen, then stays overnight ipg strip with Coomassie blue stain, takes off by ipg strip After color, it can confirm by inspection size and compared with negative control protein induced.

(2) purifying of biomarker group

Detailed process to the protein purification of biomarker group is as shown in figure 3, detailed process is as follows:

Bacterial cell pellet will be collected after centrifugation to be resuspended in 10mL solubilization buffer by being vortexed at room temperature, it will The cell of resuspension is put into 50mL centrifuge tube, centrifuge tube is placed in always to keep low temperature on trash ice, by with 70% amplitude 10 wheel ultrasounds are carried out, crack cell completely, every wheel ultrasound 30 seconds is spaced 30 seconds, and the cell of cracking is at 4 DEG C, with 10, The revolving speed of 000rpm/min is centrifuged lh, and supernatant is transferred in dialysis tubing to after centrifugation and is immersed in 4 DEG C of the unfiltered PBS of 1L In buffer, and continue to stir 4-6h, then with another 1L start buffer continuation dialysed overnight, and with 0.22 μm of aperture Filter disc and syringe further filter the supernatant being dialyzed overnight, and have loaded 0.1M nickel sulfate then to being equipped with Filtered sample is loaded in the AKTA machine of HiTrap chelate column, the setting program on AKTA machine, automatically by eluent With multiple fraction collectors, the albumen purified from different fractions carries out SDS-PAGE analysis and checks to determine biomarker histone The degree of purifying.

Embodiment 3: the preparation of biomarker-BioPlex pearl conjugate

The biomarker histone that is prepared referring to method as described in example 2 or using purchase purity up to 90% or more Biomarker histone, carries out biomarker histone and BioPlex pearl is coupled, and detailed process is, by what is be not coupled BioPlex pearl, which is vortexed, mixes 30s, then ultrasonication 15s, by with the revolving speed of 10000rpm/min by 100 μ L BioPlex Pearl is centrifuged 4min, and 1,250,000 BioPlex pearl is collected in reaction tube, and with 100 μ L BioPlex pearl washing buffers Carry out centrifuge washing after, BioPlex pearl is resuspended in 80 μ L BioPlex pearl activation buffers, activation buffer be comprising The MES buffer of EDC/NHS activation system adds the 10 freshly prepared N- hydroxyl sulphurs of μ L 50mg/mL then to BioPlex pearl Sour succinimide (S-NHS) aqueous solution and the 10 freshly prepared 1- ethyl -3-(3- dimethylamino-propyls of μ L 50mg/mL) carbon Change diimine (EDC) aqueous solution, is incubated for 20min in the dark at room temperature, washs BioPlex pearl two with the PBS buffer solution of 150 μ L It is secondary.

7 kinds of biomarker histones, each 10 μ of 7 kinds of biomarker histones are added into washed BioPlex pearl G, and being filled it up with total volume to 500 μ L with PBS buffer solution, shakes be incubated for 2h in the dark, then with the revolving speed of 6000rpm/min from Supernatant is removed after heart 4min, 250 μ L Block buffers are added into sediment and shakes 30min in the dark, then with identical Revolving speed be centrifuged 4min, and remove supernatant, be resuspended in store buffer after BioPlex pearl is washed with the PBS buffer solution of 150 μ L In liquid, store buffer liquid is the Tris buffer comprising deproteinized agent, preservative and Sodium azide, and is stored at 4 DEG C, utilizes blood Cell counter counts the number of BioPlex pearl.

Embodiment 4: the composition of kit

A kind of kit for screening of the early stage cancer of the esophagus, is made of the following contents:

1, biomarker group-BioPlex pearl conjugate is conjugated by CP96-BioPlex pearl conjugate, CRP78-BioPlex pearl Object, MMP-10-BioPlex pearl conjugate, cav-1-BioPlex pearl conjugate, CDC25B-BioPlex pearl conjugate, PGK1- BioPlex pearl conjugate and HSP105-BioPlex pearl conjugate form, and a kind of antigen egg is conjugated on each BioPlex pearl White, above-mentioned biomarker group-BioPlex pearl conjugate is prepared referring to method described in embodiment 3.

2, the solid phase carrier of biomarker group-BioPlex pearl conjugate: 96 hole elisa Plates, biomarker group- Layout of the BioPlex pearl conjugate in 96 hole elisa Plates ELISA Plates is as shown in table 2.

Layout of the 2 biomarker group-BioPlex pearl conjugate of table in 96 hole elisa Plates ELISA Plates

*: the secondary antibody of antigen and PE conjugation is added without in the hole

*: patients serum is added without in the hole

3, the secondary antibody of PE conjugation

Preparation method:

(1) 4.08mg PE is taken to be dissolved in the phosphate buffer (containing 0.1mol/LNaCl) that 1mL 0.1mol/L pH is 7.4, it is molten After solution mixes, solution after 0.5mL mixing is taken out, 10 μ L SPDP anhydrous methanol liquid (2.65mg/mL), SPDP/ albumen mole is added Than being 10,5min is reacted at 22 DEG C, carries out column chromatography using Sephadex G-50 (1 × 17cm), and use 100mmol/L PH7.4 PBS(NaCl containing 0.1mol/L) it is balanced and elutes.

(2) taking concentration after elution is PE the and 0.27mg/mL secondary antibody mixed in equal amounts of 0.77mg/mL, reacts 6h at 22 DEG C It is saved backup at 4 DEG C afterwards.

(3) secondary antibody that the PE of above-mentioned preparation is conjugated, is dissolved in 0.01mol/L pH7.4 PB buffer and (containing 0.1mol/L DETA, 1mol/L iodoacetamide, 1%(w/v) BSA and 0.1%(w/v) NaN3), saved at 0~5 DEG C.

4, Sample dilution；Sample dilution is to contain 1%(w/v) the PBST buffer of BSA.

5, developing solution；Developing solution is made of developing solution A and developing solution B, and developing solution A is 0.02%(w/v) TMB, developing solution B For 0.006%(w/v) urea peroxide element.

6, terminate liquid；Terminate liquid is 10%(v/v) sulfuric acid solution.

7, cleaning solution；Cleaning solution is that pH is 7.4 and the PBST buffer containing 0.05%(v/v) Tween-20, PBST buffer Concentration be 0.01mol/L.

Embodiment 5: kit uses the foundation of preceding standard curve

The establishment process of the standard curve of the kit detection cancer of the esophagus of the present invention is as follows:

50 μ L biomarker-BioPlex pearl conjugates, biomarker-BioPlex pearl conjugate is added to HTS96 orifice plate Concentration be indicated with the concentration of BioPlex pearl, concentration be 100 pearls/μ L, the biomarker-BioPlex pearl conjugate After first reacting with primary antibody, the secondary antibody of PE conjugation is added into reaction system, specific immune response, process occur for secondary antibody and primary antibody Schematic diagram is as shown in figure 4, the secondary antibody that finally formed biomarker-BioPlex pearl conjugate and primary antibody and PE are conjugated is answered It is as shown in Figure 5 to close object.

The dilution of the commercially available primary antibody for biomarker group is prepared, the preparation process of an anti-serum samples is 37 1h is stood at DEG C solidifies whole blood sample, after being centrifuged 10min with 6000rpm/min room temperature, takes supernatant, which is packet Serum containing autoantibody isolates and purifies the antibody in serum, followed by the Sample dilution in kit to pure Antibody after change is diluted the primary antibody dilution for preparing various concentration gradient, and the concentration gradient of primary antibody dilution is successively are as follows: 8000ng/mL、6000ng/mL、4000ng/mL、2000ng/mL、1000ng/mL、500ng/mL、250ng/mL、125ng/mL、 75ng/mL and 30ng/mL；Every kind of primary antibody dilution takes 50 μ L to be added into each every hole；To be added without primary antibody as first Negative control is added without primary antibody and secondary antibody as second negative control；Then foil closed plate is used, experimental group and control group are put It sets on the oscillator, under the conditions of being protected from light, 30min is incubated for the speed oscillation of 6000rpm/min.

After incubation, is washed BioPlex pearl 3 times with the PBS buffer solution of 150 μ L, then add the secondary antibody of 50 μ L PE conjugation Into every hole, the concentration of the secondary antibody of the PE conjugation of addition is 8000ng/mL, and PE conjugation need not be then added in second negative control Secondary antibody；It is again sealed off plate, plate is shaken in the dark and is incubated for 30min；Then excessive antibody is washed off using PBS buffer solution；With school Positive kit and verifying kit correct BioPlex machine；After HTS plate is loaded on machine, measurement comes from BioPlex pearl With the signal of both PE for being conjugated on secondary antibody.

The signal of both the BioPlex pearl detected according to BioPlex machine and the PE being conjugated on secondary antibody and primary antibody Concentration gradient obtains the standard curve between the signal of both PE being conjugated in the concentration and BioPlex pearl and secondary antibody of primary antibody, such as Shown in Fig. 7.

Embodiment 6: the application method of kit

The application method of kit is as shown in Figure 4 and Figure 6, will contain patients serum and mix to corresponding to biomarker group- In BioPlex pearl conjugate, the secondary antibody of PE conjugation is then added, is detected, specific detection process is as follows:

Serum sample is incubated for

1h is stood at 37 DEG C solidifies whole blood sample, after being centrifuged 10min with 6000rpm/min room temperature, supernatant is taken, on this It include the serum of autoantibody in clear liquid；Serum to be detected is diluted with 1%(w/v) BSA solution in the ratio of 1:100, Then the serum after dilution is added in the loading wells of ELISA Plate, sample-adding amount is 100 holes μ L/, then adds 50 μ LPE conjugation Secondary antibody is placed in 37 DEG C of constant incubators and is incubated for 1h, then discards liquid in loading wells, is washed 5 times with cleaning solution.

It develops the color and terminates and react

Developing solution A and developing solution B are uniformly mixed in equal volume according to 1:1, mixed developing solution is then rapidly added enzyme mark In the loading wells of plate, sample-adding amount is 100 holes μ L/, is placed in 37 DEG C of water-baths and is protected from light colour developing 15min, then again into each loading wells 50 μ L terminate liquids are added, then color development stopping reaction reads OD value, and use blank with automatic microplate reader at 450nm and 595nm Hole zeroing.

Result judgement

Add two standard deviations as cutoff value using the average of the OD value of all Normal groups, is then determined as higher than this cutoff value The positive is then determined as feminine gender lower than this cutoff value.

Embodiment 7: kit reliability demonstration in the present invention

This research has collected the serum for the patient with esophageal carcinoma that 500 are not done pathological section verifying and 300 were done pathological section It is after verifying patient with esophageal carcinoma (control group).500 are not done in the patient with esophageal carcinoma of pathological section verifying, and male 300, women 200, average age is 60.3 ± 8.4 years old, and the range of age is 38-82 years old；300 cancer of the esophagus done after pathological section verifying Patient, male 150, women 150, average age is 58.7 ± 9.2 years old, and the range of age is 35-80 years old.All cancer of the esophagus are suffered from Person's serum be all in patient be initially diagnosed as the cancer of the esophagus and not yet by any chemicotherapy when collect, the time being diagnosed is 2010 January in December, 2015 in year.

Through the invention kit to above-mentioned 500 do not do pathological section verifying patient with esophageal carcinoma serum and 300 The esophagus cancer patient blood serum for doing case slice verifying carries out cancer of the esophagus checkout and diagnosis, antibody positive rate, the sensitivity of testing result Property and specificity are as shown in table 3.Table 3 shows, the increase of antigen combination number and the sensibility of early stage esophagus cancer diagnosis are also therewith Increase, when 7 kinds of tumor associated antigens combine, sensibility is up to 89.3%, that is to say, that in early stage patient with esophageal carcinoma using this 7 The ratio that the early stage cancer of the esophagus is correctly diagnosed as when a tumor associated antigen is diagnosed is 89.3%, simultaneously, though specificity It decreases, but still up to 88.4%, it is true when this result prompts 7 kinds of tumor associated antigen joint-detections of non-patient with esophageal carcinoma Be set to be not suffering from cancer of the esophagus person ratio be 88.4%.The cancer of the esophagus is diagnosed using this 7 kinds of tumor associated antigens combination, it can be with Make the raising of the sensitivity and specificity of diagnosis all by a relatively large margin, therefore, this 7 kinds of tumor associated antigen combinations are ideal Cancer of the esophagus early diagnosis and screening method and means.

The different tumor associated antigen combinatorial association testing results of table 3

Embodiment 8: the foundation of Staging in Esophageal Carcinoma characteristic spectrum

According to the standard curve between the signal of both PE being conjugated in the concentration of primary antibody and BioPlex pearl and secondary antibody (such as Fig. 7 institute Show), Staging in Esophageal Carcinoma characteristic spectrum is obtained, it is specific as shown in table 4.

4 Staging in Esophageal Carcinoma characteristic spectrum of table

Claims (8)

1. application of one group of tumor associated antigen in cancer of the esophagus early screening kit, the tumor associated antigen is CP96 base Because representation, CRP78 gene expression object, MMP-10 gene expression object, cav-1 gene expression object, CDC25B gene expression object, PGK1 gene expression object and HSP105 gene expression object.

2. a kind of cancer of the esophagus early screening kit, which is characterized in that including solid phase carrier and the food being coated on solid phase carrier Pipe tumor related antigen, the cancer of the esophagus tumor associated antigen are CP96 gene expression object, CRP78 gene expression object, MMP- 10 gene expression objects, cav-1 gene expression object, CDC25B gene expression object, PGK1 gene expression object and HSP105 gene expression Object；The kit further includes the secondary antibody of phycoerythrin conjugation.

3. cancer of the esophagus early screening kit according to claim 2, which is characterized in that the oesophagus tumor correlation is anti- BioPlex pearl is conjugated in original.

4. cancer of the esophagus early screening kit according to claim 3, which is characterized in that the kit further includes that sample is dilute Release liquid, developing solution, terminate liquid and cleaning solution.

5. cancer of the esophagus early screening kit according to claim 4, which is characterized in that the Sample dilution be containing 1%(w/v) the PBST buffer of BSA.

6. cancer of the esophagus early screening kit according to claim 5, which is characterized in that the developing solution is by developing solution A It is mixed in equal volume with developing solution B；The developing solution A is 0.02%(w/v) TMB, developing solution B is 0.006%(w/v) peroxidating Urea element.

7. cancer of the esophagus early screening kit according to claim 6, which is characterized in that the terminate liquid is 10%(v/v) Sulfuric acid solution.

8. cancer of the esophagus early screening kit according to claim 7, which is characterized in that the cleaning solution is containing 0.05% (v/v) the PBST buffer of Tween-20；The concentration of the PBST buffer is 0.01mol/L；The pH of the cleaning solution is 7.4.