CN101435822A - Method of oesophagus squama cancer diagnosis of blood serum autoantibody - Google Patents
Method of oesophagus squama cancer diagnosis of blood serum autoantibody Download PDFInfo
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- CN101435822A CN101435822A CNA2008102189135A CN200810218913A CN101435822A CN 101435822 A CN101435822 A CN 101435822A CN A2008102189135 A CNA2008102189135 A CN A2008102189135A CN 200810218913 A CN200810218913 A CN 200810218913A CN 101435822 A CN101435822 A CN 101435822A
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Abstract
The invention discloses a method for diagnosing esophageal squamous cell cancer through a serum autoantibody. The method applies serological histone technology to screen a tumor antigen autoantibody in serum of an esophageal squamous cell cancer patient. Firstly, protein in an ESCC tissue is separated through 2-D PAGE electrophoretic separation and is transferred to a PVDF membrane; and secondly, serum of the patient and serum of a healthy person are used as a primary antibody; an oncoprotein reaction point is detected through western blot; a protein point which only reacts with the serum of the tumor patient and does not react with the serum of the normal person is selected; and an amino acid sequence of the protein point is identified through mass spectrum. By the technology, a CDC25B autoantibody is initially screened out from the serum of the esophageal squamous cell cancer patient; and the specificity of the CDC25B autoantibody is verified. The CDC25B autoantibody is verified to be used as a tumor marker of the diagnosis of the esophageal squamous cell cancer.
Description
Technical field
The present invention finds and verified a kind of new esophageal squamous cell carcinoma (ESCC) tumor markers-change of serum C DC25B autoantibody, and set up the method that reverse elisa technique detects change of serum C DC25B autoantibody, is used for the diagnosis of esophageal squamous cell carcinoma.
Background technology
Esophageal squamous cell carcinoma (ESCC) is a modal type in the cancer of the esophagus, is the fourth-largest tumour that causes death of China.Early diagnosis is the key that promotes the cancer cure rate, but because early stage clinical symptoms is not obvious, and lacking good early diagnosis index and examination technology, esophageal squamous cell carcinoma all is late period when generally making a definite diagnosis.At present, early stage according to tumour, the denier antigen that tumour cell produces (also not having good technology to detect at present), but can be discerned by body immune system, produce special tumour antigen antibody, therefore detect this antibody (, being easy to detect) but the existence of indirect reaction tumour antigen by immune amplification.Many researchs show that also tumor patient is the best index of early diagnosis of tumor at the autoantibody that tumour antigen produces, and the sensitivity of early diagnosis and specificity all are better than the tumor markers of the tumour cell generation of using clinically at present.
In the esophageal squamous cell carcinoma patients serum, have been found that p53, cytokeratins, myomegalin, albumen autoantibodies such as TRIM21, wherein darker to the research of p53 antibody in the serum, find that it can be used as cancer of the esophagus early diagnosis and prognostic indicator.Recently, filter out peroxiredoxin VI autoantibody by the serology protein technique in the ESCC patients serum, the recall rate of this antibody in ESCC is 50%, can be used as cancer of the esophagus early diagnosis index.
At present, the humoral immune reaction searching tumor markers that causes based on tumour antigen has several different methods, the serology protein group be a kind of effectively and reliable technique, it is advantageous that definite oncoprotein antigen is the tumor tissues under the state of nature or the tumour antigen of tumour cell.
Summary of the invention
The present invention uses tumour antigen autoantibody among the serology protein groups technology screening oesophagus phosphorus cancer patients serum.At first, with the albumen in the 2-D PAGE electrophoretic separation ESCC tissue, transfer on the pvdf membrane, anti-with patients serum and healthy human serum then as one, western blot detects the oncoprotein reflecting point, only select with the tumour patient seroreaction not the protein site with the normal human serum reaction, mass spectrum is identified its amino acid sequence.Utilize this technology, from oesophagus phosphorus cancer patients serum, filter out the CDC25B autoantibody first, and verify its specificity.And confirm that the CDC25B autoantibody can be used as the tumor markers of oesophagus phosphorus cancerous diagnose.
Set up the method that easy reverse elisa technique detects change of serum C DC25B autoantibody at last.
Embodiment (used tissue and serum derive from tumour hospital of Zhongshan University in the experiment)
1. find and verified a kind of new esophageal squamous cell carcinoma (ESCC) tumor markers one change of serum C DC25B autoantibody
1). the autoantibody that screening produces at cancer of the esophagus squama tissue tumor proteantigen
Adopt protein technique to screen tumour antigen in the esophageal squamous cell carcinoma tissue (ESCC) with patient's autoserum (antibody that contains tumour antigen).At first, 15 parts of tumor tissues albumen of the parallel separation of 2D electrophoresis, a silver dyes (Figure 1A and B); A electrotransfer to pvdf membrane, respectively with autoserum and pvdf membrane on albumino reaction (Fig. 1 C, a patients serum), the while, 15 portions of normal human serums were as negative control (Fig. 1 D, a normal human serum).The point that in 15 parts of esophageal squamous cell carcinoma patients serums, occurs a strong reaction in 7 parts of samples at same position, and the albumino reaction point appears in neither one in 15 parts of control serums in this position.The molecular weight of this protein site is estimated as 64.0kDa, and PI is 6.0 (figure .1C).7 routine seropositivity patients comprise esophageal squamous cell carcinoma I phases 2 example, II phases 3 example, and III phases 2 example illustrates that this antibody can occur in early days at esophageal squamous cell carcinoma.
2). identify that the albumen with the esophageal squamous cell carcinoma seroreaction is CDC25B antigen
The glue that dyes from silver downcuts this point, and after the trypsinization, the MALDI-TOF mass spectrophotometry identifies that it is CDC25B albumen (NCBI accession no.P30305), and molecular weight 64.89kDa, PI are 6.0 (figure .2).Further behind the tumor tissues albumen two dimensional electrophoresis, use the CDC25B polyclonal antibody to carry out western blotting checking, Fig. 3 A shows this protein site and patients serum and many anti-reactions, is confirmed that it is the CDC25B proteantigen.Fig. 3 B is that 7 esophageal squamous cell carcinoma antibody positive patients' serum dilutes from 1:100 to 1:1600, Western blot measures CDC25B antibody titer in the serum, and titre is the highest reaches 1:1600 (1 example), and 4 routine titres are 1:800,1 routine titre is 1:400, and 1 routine titre is 1:200.
3) high expressed of CDC25B in ESCC tissue and tumor cell line
We verify earlier whether CDC25B expresses in esophageal squamous cell carcinoma cell line and tissue.Expression by CDC25B in westernblotting and 3 esophageal squamous cell carcinoma clones of immunohistochemical study and the tumor tissues, the result is presented at Eca-109, TE-1, with the high expressed all of CDC25B in these 3 clones of TE-10, and the expression among the Eca-109 is the highest, CDC25B also has high expressed in tumor tissues, and normal control tissue is not seen the expression (figure .4A) of CDC25B.Figure .4B is seen in expression in the coupling sample of 15 tumor tissues and cancer beside organism, and CDC25B is high expressed in the tumor tissues of 14/15 example, and cancer beside organism does not express, or expresses extremely low.The expression (T) of CDC25B and comparison (T/N) 3.1-11.2 of cancer beside organism (N) in patient's tumor tissues of CDC25B antibody positive in the serum, and the patient CDC25B (T/N) of CDC25B negative antibody is 0.8-5.4 in the serum.SABC confirms that also CDC25B is at the esophageal squamous cell carcinoma tissue high expressed.Fig. 5 A-B is presented at normal layering oesophagus scaly epithelium and does not express CDC25B.Simultaneously, the patient of change of serum C DC25B negative antibody, CDC25B is painted mainly at nucleus, and endochylema dyeing is weak or cannot see (figure .5C-D).And the patient of CDC25B-antibody positive in the serum, CDC25B dyeing strong positive (figure .5E-F) in the kytoplasm of its tumour cell and the karyon. CDC25B crosses the result who expresses and conforms in these results and other documents in ESCC, and the expression of patient CDC25B in tumor tissues of explanation generation CDC25B antibody significantly raises.
4) CDC25B antibody can be as the tumor markers of esophageal squamous cell carcinoma in the serum
Use CDC25B albumen among the anti-phase Eca-109 that catches of CDC25B antibody as envelope antigen, indirect ELISA is measured 102 routine normal control serum, 124 examples through ESCC patients serum that pathology is determined and the CDC25B antibody in 150 other Infusion in Patients with Digestive serum of example.102 routine normal healthy controls groups, mean light absorbency OD is 0.12 ± 0.07; Antibody positive is defined as the average+3SD sum of OD value more than or equal to the normal healthy controls group, and promptly field value set OD is 0.33.Among 124 routine patients ESCC, 45 parts of (36.29%) CDC25B antibody positives; Colon cancer 8.0% (4/50) positive, cancer of the stomach 12.0% (6/50) positive, liver cancer 6.0% (3/50) positive, 102 parts of none examples of normal healthy controls group are positive, illustrate that CDC25B can be used as the tumor markers (table 1) of oesophagus squama cancer diagnosis.
Table 1 cdc25b autoantibody is in the positive expression contrast of the cancer in digestive system of ESCC and other types
2. set up the method that reverse elisa technique detects change of serum C DC25B autoantibody
Wrap by elisa plate with CDC25B monoclonal antibody (purchasing company) in SANTA, go up plain white after adding esophageal squamous cell carcinoma cell line Eca-109 (this cell line high expressed CDC25B) cracking, CDC25B antigen in the CDC25B antibody capture tumour cell, as envelope antigen, the unconjugated foreign protein of flush away, after the BSA sealing, add serum to be checked, CDC25B antibody in the serum can combine with CDC25B antigen, flush away impurity, the anti-human IgG of adding HRP mark, form the anti-human IgG compound of the anti-CDC25B antibody-HRP-of CDC25B-, after the washing, add the TMB colour developing, microplate reader detects.
Concrete grammar is as follows:
(1) cellular incubation
Esophageal cancer cell strain Eca-109 (purchasing the company in ATCC), RPMI 1640 nutrient culture media contain 10% newborn hyclone, 5% CO2,37 ℃ of cultivations.Be cultured to 10
7Cell.
(2) CDC25B antigen preparation
Collect 10
7The Eca-109 cell, with physiological saline centrifuge washing 3 times, remove supernatant, add lysis buffer (2% NP40 of 300 μ l, 4% CHAPS, 1% DTT, 10 μ l protease inhibitors PMSF), act on 30 minutes on ice, blow and beat repeatedly 20-30 times again, 12000rpm, 4 ℃ centrifugal 20 minutes, collect supernatant.Bradford method protein quantification.
(3) oppositely elisa technique detects change of serum C DC25B autoantibody
1) is cushioned liquid with CDC25B antibody dilution to 1 μ g/100 μ l with 0.01M pH9.0 carbonate bag.Add 100 μ l in the reacting hole of each elisa plate, 4 ℃ are spent the night.Discard solution in the hole next day, washes (1 * TBS-T, compound method sees Table 2, down together) 3 times with lavation buffer solution, each 3 minutes.
2). plain white (20 μ g/ holes, 100 μ l/ holes) is gone up, 4 ℃ of night incubation after adding 100 μ l oesophagus phosphorus cell line Eca-109 cracking in every hole.
3). in each reacting hole, add 5%BSA (pulvis is purchased the company in SIGMA) 200 μ l, and, 4 ℃, spend the night with sealing the membrane closure elisa plate.
4). wash 4 times each 3 minutes with lavation buffer solution.
5) (positive control is a CDC25B antibody to the sample serum 100 μ l of the every hole adding of .ELISA plate 1:100 dilution, negative control is the normal human serum of western blot checking CDC25B negative antibody), incubated at room 2 hours, lavation buffer solution are washed 4 times, each 3 minutes.
6) the .ELISA plate adds goat anti-human igg-HRP (purchasing the company in SANTA) (1:5000), 100 μ l/ holes, and incubated at room 45 minutes is washed 5 times with lavation buffer solution, each 3 minutes.
7). add substrate solution colour developing: in each reacting hole, add each 50ul of tmb substrate A, B liquid (purchasing), 37 ℃ of lucifuges, 10～15 minutes in company of middle China fir Golden Bridge.
8). cessation reaction: in each reacting hole, add 2M sulfuric acid 50ul.
9). microplate reader (ELX800 of BIO-TEX company) 450nm surveys the OD value.
The preparation of table 2 lavation buffer solution
10X?TBS(pH7.6)
1X?TBS-T，2000ml
dH
2O 1800ml
1X?PBS 200ml；
Tween-20 2ml (or 20% tween 10ml) (the interim adding), mixing.
Description of drawings
Fig. 1. the tumour antigen antibody of protein technique among the esophageal squamous cell carcinoma tissue protein screening esophageal squamous cell carcinoma patients serum
Wherein A.2D glue tumor tissues protargin dyes
B.2D glue tumor tissues protargin dyes local the amplification
C.2D electrotransfer is to PVDF behind the electrophoresis, and a patients serum's western blot detects
D.2D electrotransfer is to PVDF behind the electrophoresis, and the western blot of a normal human serum detects
The albumen of Fig. 2 .MALDI-TOF mass spectrophotometry esophageal squamous cell carcinoma seroreaction is CDC25B albumen
The local silver of Fig. 3 A.a. dyes.B. cancer of the esophagus serum western blot, c.CDC25B antibody westernblot
CDC25B antibody titer in the B.7 routine esophageal squamous cell carcinoma antibody positive of Fig. 3 patient's the serum
Fig. 4 A.western blotting detects the expression of CDC25B in 3 kinds of esophageal squamous cell carcinoma clones
The expression of CDC25B in the coupling sample of B.15 individual tumor tissues of Fig. 4 and cancer beside organism
Fig. 5. SABC CDC25B the esophageal squamous cell carcinoma tissue high expressed (A, C, E be 200 *; B, D, F be 400 *)
Wherein A and B. normal control tissue are not seen the expression of CDC25B
The patient of C and D. change of serum C DC25B negative antibody, CDC25B is painted mainly at nucleus, and endochylema dyes weak or cannot see
The patient of CDC25B-antibody positive in E and the F. serum, CDC25B dyeing strong positive in the kytoplasm of its tumour cell and the karyon.
Claims (2)
1. change of serum C DC25B autoantibody is as the oesophagus squama cancer diagnosis mark.
2. reverse ELISA detects the method for change of serum C DC25B autoantibody, carry out according to following steps: use CDC25B monoclonal antibody bag by elisa plate, go up plain white after adding esophageal squamous cell carcinoma cell line Eca-109 cracking, CDC25B antigen in the CDC25B antibody capture tumour cell, as envelope antigen, the unconjugated foreign protein of flush away, after the BSA sealing, add serum to be checked, the CDC25B antibody in the serum can combine with CDC25B antigen, flush away impurity, the anti-human IgG that adds the HRP mark forms the anti-human IgG compound of the anti-CDC25B antibody-HRP-of CDC25B-, after the washing, add the TMB colour developing, microplate reader detects.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109342727A (en) * | 2018-10-15 | 2019-02-15 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma autoantibody molecular marker model and its application |
| CN110687282A (en) * | 2019-08-26 | 2020-01-14 | 中国医学科学院肿瘤医院 | PD-1 and/or p53 autoantibodies as markers for tumor efficacy prediction or prognosis evaluation |
| CN110716044A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN112362871A (en) * | 2020-10-21 | 2021-02-12 | 杭州凯保罗生物科技有限公司 | Biomarkers of esophageal cancer and application thereof |
| CN113721021A (en) * | 2021-09-16 | 2021-11-30 | 郑州大学 | Application of PRKCZ autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
-
2008
- 2008-11-05 CN CNA2008102189135A patent/CN101435822A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109342727A (en) * | 2018-10-15 | 2019-02-15 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma autoantibody molecular marker model and its application |
| CN109342727B (en) * | 2018-10-15 | 2021-11-12 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma autoantibody molecular marker model and application thereof |
| CN110687282A (en) * | 2019-08-26 | 2020-01-14 | 中国医学科学院肿瘤医院 | PD-1 and/or p53 autoantibodies as markers for tumor efficacy prediction or prognosis evaluation |
| CN110716044A (en) * | 2019-10-23 | 2020-01-21 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN110716044B (en) * | 2019-10-23 | 2023-04-18 | 郑州大学 | Serum protein marker, kit and detection method for early screening and diagnosis of esophageal squamous carcinoma |
| CN112362871A (en) * | 2020-10-21 | 2021-02-12 | 杭州凯保罗生物科技有限公司 | Biomarkers of esophageal cancer and application thereof |
| WO2022083673A1 (en) * | 2020-10-21 | 2022-04-28 | 杭州凯保罗生物科技有限公司 | Biomarker for esophageal cancer, and use thereof |
| CN113721021A (en) * | 2021-09-16 | 2021-11-30 | 郑州大学 | Application of PRKCZ autoantibody in auxiliary diagnosis of esophageal squamous cell carcinoma |
| CN113721021B (en) * | 2021-09-16 | 2023-07-07 | 郑州大学 | Application of PRKCZ autoantibody in esophageal squamous carcinoma auxiliary diagnosis |
| CN114167059A (en) * | 2021-11-03 | 2022-03-11 | 郑州大学 | Biomarker for diagnosing esophageal squamous carcinoma and detection kit |
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Application publication date: 20090520 |