CN102798722B - Application of protein NT5E in preparation of reagent for diagnosing gastric cancer and diagnosis kit - Google Patents
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Abstract
The invention provides an application of protein NT5E in preparation of reagent for diagnosing gastric cancer and a diagnosis kit. According to the invention, the existing ELISA (enzyme-linked immunosorbent assay) technology is adopted, and an IgG antibody for resisting the protein NT5E in human serum is qualitatively detected by using an indirect method; purified human protein NT5E antigen is used for coating a microwell plate so that a solid antigen is prepared; serum to be detected is successively added in microwells coated with the antigen, and then an enzyme labeling reagent containing anti-Human IgG labeled by HRP (horse radish peroxidase) is added, so that an NT5E-antibody-enzyme labeled second antibody composite is formed; after the composite is completely washed, an enzyme substrate solution TMB (tetramethylbenzidine) is added in the composite for color development; and TMB is converted into be blue in the presence of the HR enzyme and finally converted into be yellow under the action of acid, and the level of the IgG antibody in a sample is detected by virtue of depth of color. As a means for assisting early diagnosis of gastric cancer, the kit provided by the invention can be used for greatly improving the sensitivity and specificity of the gastric cancer.
Description
Technical field
The present invention relates to bio-science field, more particularly, relate to application and the diagnostic kit of a kind of protein N T5E in the reagent of preparing diagnosis of gastric cancer.
Background technology
Cancer of the stomach is the common cancer of harm humans health, and according to the World Health Organization's statistics of 2008 (World Health Organization's year statistics, 2008), in worldwide, the mortality ratio of Patients with Gastric Cancer is in second.In the statistics of WHO in 2006, show and approach 950,000 new cases, 700,000 people that simultaneously have an appointment die from this kind of disease.In China, the annual new patient's number of cancer of the stomach exceedes 300,000, dies from the number approximately 160,000 of cancer of the stomach, and case fatality rate occupies first of malignant tumour, serious threat people's life health (Chen Zhu, whole nation cause of the death Retrospect spot-check report for the third time, 2008).For reducing mortality ratio, improve the curative effect of cancer of the stomach, key is cancer of the stomach " three early " work, i.e. early detection, early diagnosis and early treatment.But, because most patients with gastric cancer lack specific clinical symptoms, therefore early carcinoma of stomach diagnosis is very low, the not enough 10%(Wu Yun of operability woods: surgery theory and practice, 2005,10:401); The life cycle of postoperative patient is also shorter.At present, overall 5 years survival rates of China's cancer of the stomach are 43.4%, postoperative pathological is (pathological TNM by stages, pTNM) I, II, III, IV phase patient's postoperative 5 years survival rates be respectively 75.65%, 58.73%, 28.Ol% and the bright Asia of Liu 8.42%(: Chinese Journal of Gastrointestinal Surgery, 2010,13:163).Therefore, the early diagnostic rate of raising cancer of the stomach is extremely important.And general health check-up and imaging examination effect are limited, in the time can finding and clarify a diagnosis, often in, late period, lack predictive value.Therefore, from a long-term perspective, should be devoted to find all good tumor markerses of susceptibility and specificity.In addition, heterogeneity in gastric carcinomas is large, and prognosis and larger to the reactive difference for the treatment of, for clinical assessment prognosis and the prediction to therapeutic response and evaluation, all needs good tumor markers.
Desirable tumor markers should meet following condition: (1) susceptibility is high; (2) specificity is high; (3) tumor-marker substrate concentration and tumor size, transfer, grade malignancy are relevant, can assist neoplasm staging and judging prognosis; (4) half life period short, effectively after treatment, concentration declines very soon, can comparatively fast reflect the actual conditions of in-vivo tumour; (5) be present in body fluid, particularly, in blood, be easy to detect (Liu Ping Ya: Chinese Journal of Gastrointestinal Surgery, 2010,13:163).
The research of tumour serum mark is the research emphasis of this area always.Now existing multiple cancer of the stomach tumour marker: as carcinomebryonic antigen (CEA), be present in cancer of the stomach and other gland cancer patient's serum, but less to the diagnostic significance of early carcinoma of stomach, be mainly used in dynamic observation (the Lipkin M:Cancer Research before and after curing gastric cancer, 1988,48:235; Lipkin:JBCSupplymental, 1992,16:1); The part carbohydrate antigen such as CA125, CA19-9, CA50, CA724 and CA242 can raise in part patients with gastric cancer, but only 20～40%(Kodera Y:The American journal of gastroenterology of its susceptibility, 1996,91:49; Chou M:Disease Markers, 2000,16:105;
lai IRetal:
hepato-gastroenterology, 2002,49:115; Edip U et al:Advances in Theray, 2008,25:1075); Also studies have found that tumor-associated glycoprotein antigen-72(TAG-72) be 69% to the susceptibility of cancer of the stomach, specificity is 84%(Liu Jun etc.: Chinese journals of practical medicine, 1999,15:4); Glycoprotein antigen MG7-Ag has the positive rate of 40%-60% in serum, has the positive rate (Ren J:Cancer, 2000,88:280) of 80%-94% in stomach organization; Nucleosome Histones antigen (IPO-38) is 57.4% as the susceptibility of diagnosing gastric cancer, and specificity exceedes 90%(Hao Y etal:J Proteome Res, 2008,7:3668) above these indexs are all conducive to the diagnosis of early carcinoma of stomach.Some oncogene, as DDC, c-myc, c-erb-2, p53 and nm23 etc., generation, the transfer to cancer of the stomach also has the certain significance, but is widely used in clinical still restricted (Liu Qian etc., People's Health Publisher, 2004).Due to the defect of existing cancer of the stomach biological marker in sensitivity and specificity, although there is certain value in curative effect monitoring, prompting recurrence, judging prognosis and people at highest risk's generaI investigation, still can not be used for making a definite diagnosis of cancer of the stomach at present.In order to realize the early stage high sensitivity to cancer of the stomach, the diagnosis of high specific, is badly in need of searching out more responsive, more special cancer of the stomach biomarker on molecular level.
We utilize the advantage of human protein core assembly sheet high flux, express-analysis in early-stage Study, relevant 101 parts of the serum (37 parts of Patients with Gastric Cancer, 14 parts of high risk patients, 50 parts of Healthy Human Serums) of Patients with Gastric Cancer are analyzed, compare the difference in patient and Healthy People sample within a short period of time, provide candidate's serum biomarker, to early diagnosis and effective treatment cancer of the stomach.
Albumen NT5E (CD73) be born of the same parents outer-5'-nucleotidase (Ecto-5'-nucleotidase, eNT) is anchored to a kind of glycoprotein of plasma membrane by glycosyl-phosphatidyl inositol (GPI).NT5E wide expression is in the histocyte such as human endothelial cells, lymphocyte surface.NT5E has Extracellular nucleotidase activity, and hydrolysis AMP produces adenosine.Adenosine and receptors bind can promote angiogenesis, prevention tissue ischemia reperfusion injury, inflammation-inhibiting and immune response.Recent findings adenosine can suppress the proinflammatory reaction (Grunewald JK et al:J Inflamm, 2010,7:10) of mankind's vascular endothelial cell.NT5E can come off from plasma membrane surfaces because of endogenous phospholipase C hydrolysis GPI, and then adenosine generates reduction.NT5E also has non-hydrolytic enzyme activities, participates in sticking and signal transduction of cell.Research finds that NT5E can pass through CD3/TCL compound, participates in the activation (Linden J:Annu Rev Pharmacol Toxicol, 2001,41:775) of T cell.Research shows that NT5E plays a significant role in growth, apoptosis, invasion and attack and the transfer of tumour.Stagg?J?et?al：PNAS,2010,107:1547；Zhi?X?et?al：Clin?Exp?Metastasis,2007,24:439；Wang?L?et?al：J?Cancer?Res?Clin?Oncol,2008,134:365；Mikhailov?A?et?al：J?Immunol,2008,181:464）。
But so far, there is not yet the report using NT5E protein as cancer of the stomach biomarker.
Summary of the invention
For the technical matters existing in above-mentioned prior art, the invention provides application and the diagnostic kit of a kind of protein N T5E in the reagent of preparing diagnosis of gastric cancer, carry out the level of the IgG antibody of the anti-protein NT5E in qualitative detection human serum, as a kind of means of auxiliary cancer of the stomach early diagnosis, greatly improve susceptibility and the specificity of cancer of the stomach early diagnosis.
For achieving the above object, the technical solution adopted in the present invention is as follows:
The application of protein N T5E in the reagent of preparing diagnosis of gastric cancer.
Prepare a diagnostic kit for diagnosis of gastric cancer disease, this kit adopts clinical widely used elisa technique now, the IgG antibody of anti-protein NT5E in application indirect method qualitative detection human serum; Specifically: after diluting by coated damping fluid with the human protein NT5E antigen of purifying, be coated in the micropore in ELISA Plate and make solid phase antigen, add confining liquid; By standard items and test serum sample with adding in antigen measuring hole separately after sample diluting liquid dilution, every hole adds the enzyme marking reagent of the anti-Human IgG antibody that contains horseradish peroxidase (HRP) mark, form NT5E-antibody-ELIAS secondary antibody compound, thoroughly after washing, add enzyme substrate solution colour developing through cleansing solution, the enzyme substrate solution reaction time to after add acid stop buffer; Described enzyme substrate solution changes into blueness under the catalysis of HRP enzyme, and changes into final yellow under sour effect, utilizes the depth of color to detect the level of the IgG antibody of anti-protein NT5E in sample.
The human protein NT5E(band GST label of described purifying) be overexpression from saccharomyces cerevisiae, affinity purification and obtaining, concentration is 25 μ g/mL.
Described standard items comprise 0U/mL standard serum 1 and 100U/mL standard serum 2, are respectively normal human serum and the positive serum of NT5E antibody is diluted in sample diluting liquid.
Described enzyme marking reagent is containing the anti-0.1-1 μ of HRP-bis-g/mL.
Described enzyme substrate solution comprises: in developer A:500mL solution, containing sodium acetate 13.6g, citric acid 1.6g, contains TMB350mg, DMSO20mL, citric acid H in 30% hydrogen peroxide 0.3mL, developer B:500mL solution
2o 5.1g.
Described coated damping fluid is the carbonate buffer solution of 0.05M pH9.6, in 1 liter of solution, contains 1.59g Na
2cO
3, 2.93g NaHCO
3.
Described confining liquid is 0.01mol/L pH7.4 phosphate-NaCl damping fluid (PBS) solution of 0.5%BSA, in 1 liter of solution, contains 5g bovine serum albumin(BSA) (BSA), 8g NaCl, 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
20,0.2g KCl.
Described sample diluting liquid is 0.01mol/L pH7.4 phosphate-NaCl damping fluid (PBS).
Described cleansing solution is 0.01mol/L pH7.4 phosphate-NaCl damping fluid (PBST), and PBST includes 0.05%Tween-20, in 1 liter of solution, contains 8g NaCl, 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
20,0.2g KCl, 0.5mL Tween-20.
Described stop buffer is 2mol/L H
2sO
4solution.
Reagent in described kit all can add antiseptic, so that preserve.
Beneficial effect of the present invention is: 1. the specificity of the serum biomarker providing is 90%, and susceptibility is 92%, has the feature of high specific and hypersensitivity.2. a kind of sensitive, safe, reliable, easy-operating commercial kit is provided, and the level of the IgG antibody of anti-protein NT5E in qualitative determination human serum, contributes to auxiliary early diagnosis cancer of the stomach.
Accompanying drawing explanation
Fig. 1 is that silver dyes expression concentration, the purified condition schematic diagram of identifying NT5E;
In figure:
1 represents that standard BSA solution concentration is 5 μ g/mL
2 represent that standard BSA solution concentration is 10 μ g/mL
3 represent that standard BSA solution concentration is 25 μ g/mL
4 represent that standard BSA solution concentration is 50 μ g/mL
5 represent that standard BSA solution concentration is 100 μ g/mL
6 represent the NT5E protein example (containing GST label) after agarose compatible medium (glutathione) (National Engineering Research Center for Biotechnology) separation and purification.
7 represent protein molecular weight marker, and molecular weight respectively from big to small: 170KD, 130KD, 95KD, 72KD, 55KD.
Fig. 2 is expression, the purified condition schematic diagram that Western-Blotting identifies NT5E;
In figure:
1 represents the NT5E protein example (containing GST label) after agarose compatible medium (glutathione) (National Engineering Research Center for Biotechnology) separation and purification.
2 represent protein molecular weight marker, and molecular weight respectively from big to small: 130KD, 95KD, 72KD, 55KD.
Fig. 3 is the concentration change of the IgG antibody of anti-protein NT5E in normal person, high-risk, Serum Obtained From Advance Gastric Cancer.
Embodiment
The present invention is described further with accompanying drawing in conjunction with the embodiments.
1. expression, purifying and evaluation NT5E:
Protein N T5E is the saccharomyces cerevisiae of the genetic engineering modified mistake of process from early-stage Study, utilize galactose induction overexpression, agarose compatible medium (glutathione) separation and purification, and identify through Western-Blotting, respectively as depicted in figs. 1 and 2.For clearer expression, in figure, adopt the background with gray scale.
2. the preparation of blood serum sample:
Whole blood sample in room temperature place within 2 hours or 4 ℃, spend the night after in 1000g centrifugal about 20 minutes, get supernatant and can detect immediately; Or carry out packing, and sample is put in to-20 ℃ or-80 ℃ of preservations, but should avoid multigelation.Sample after thawing should be again centrifugal, then detects.Detect in sample and can not contain NaN
3, because NaN
3suppress (HRP) activity of horseradish peroxidase.
The compound method of the various damping fluids of 3.ELISA method and reagent:
(1) coated damping fluid: the Na of 0.05 M pH 9.6
2cO
3-NaHCO
3
(2) sample diluting liquid: pH 7.4PBS solution
(3) the PBST solution of cleansing solution: pH 7.4
(4) the pH 7.4PBS solution of confining liquid: 0.5%BSA
(5) enzyme substrate solution: developer A and developer B
(now with the current)
(now with the current)
(6) stop buffer: 2mol/L H
2sO
4solution
(timing slowly splashes into the concentrated sulphuric acid in distilled water, and limit edged mixes)
4.ELISA method is measured the concentration of the IgG antibody of anti-protein NT5E in serum, to assist early diagnosis cancer of the stomach:
Concrete operation step is as follows:
(1) coated: the people NT5E protein solution of purifying to be diluted to 1 μ g/mL with coated damping fluid, to join in 96 hole ELISA Plate, every hole 100 μ L, 37 ℃ of coated spending the night for 2 hours or 4 ℃; Cleansing solution is washed plate 3 times, dries;
(2) sealing: add confining liquid 200 μ L, room temperature insulation 2 hours; Cleansing solution is washed plate 3 times, dries;
(3) dilution of standard items and sample and application of sample: standard items and test serum sample 1:100 are diluted to 100 μ L with sample buffer, join in antigen measuring orifice plate separately.Note not having bubble, sample is added on bottom, plum target hole by application of sample, do not touch hole wall as far as possible, rocks and mix gently, adds a cover or overlay film in ELISA Plate.If test serum sample is more, suggestion is used multitube micropipet application of sample.The preparation in 15 minutes before use of standard items and detected sample, is finished and abandons, and detects next time and uses freshly prepared standard items.
(4) incubation: ELISA Plate is placed in 37 ℃ of reactions 120 minutes, gets rid of liquid in clear opening, need not wash.
(5) enzyme-added: every hole adds the anti-Human IgG antibody 100 μ L of horseradish peroxidase-labeled, 37 ℃, 60 minutes.Get rid of liquid in clear opening, the same plate of washing pats dry for 5 times.
(6) colour developing: pat dry rear each hole and first drip developer A50 μ L, then add developer B50 μ L, light shaking mixes, 37 ℃ of lucifuges develop the color 15 minutes.
(7) stop: sequentially every hole adds stop buffer 50 μ L, cessation reaction.The addition sequence of stop buffer should be as far as possible identical with the addition sequence of substrate solution.The substrate reactions time to after should add as early as possible stop buffer.
(8) result is judged:
I. sequentially measure the optical density (OD value) in each hole at 450nm wavelength with enzyme connection instrument.
* A450 is the abbreviation of 450nm place absorbance.
* at present NT5E antibody there is no the normative reference of the current international practice, has therefore adopted relative unit when this test result calibration.
Ii. the judgement of anti-NT5E value in serum
Iii. quality control
Each testing result must meet following standard:
The A450 of standard serum 1 :≤0.100
The A450 of standard serum 2: >=0.700
As do not meet above-mentioned standard, result is considered as invalidly, must again detect.
Iv. the explanation of assay
The ROC of 50 routine Healthy Human Serums, 37 routine Serum Obtained From Advance Gastric Cancers, 14 routine high-risk patient serum is analyzed and established above reference value.
5. specificity and sensitivity Detection: serum (300 parts of the patients with gastric cancer that adopt 900 parts of cancer of the stomach associated patient, 300 parts of people at highest risk, 300 parts of Healthy Peoples) the present invention has been carried out to specificity and sensitivity Detection, as Fig. 3, the numerical value in figure is the relative level of the IgG antibody concentration of the anti-protein NT5E in Healthy People, high-risk, each 300 parts of serum samples of patients with gastric cancer.
The specificity of auxiliary early diagnosis cancer of the stomach provided by the present invention is 90%, and susceptibility is 92%, is all much higher than the index of diagnosing gastric cancer in prior art.
Disclosed is above only several specific embodiments of the application, but the application is not limited thereto, and the changes that any person skilled in the art can think of, all should drop in the application's protection domain.
Claims (11)
1. the application in the reagent of protein N T5E IgG antibody horizontal of anti-protein NT5E in the detection human serum for the preparation of diagnosis of gastric cancer.
2. a method of preparing the diagnostic kit of diagnosis of gastric cancer disease, is characterized in that, this kit adopts clinical widely used elisa technique now, the IgG antibody of anti-protein NT5E in application indirect method qualitative detection human serum; Specifically: after diluting by coated damping fluid with the human protein NT5E antigen of purifying, be coated in the micropore in ELISA Plate and make solid phase antigen, add confining liquid; By standard items and test serum sample with adding in antigen measuring hole separately after sample diluting liquid dilution, every hole adds the anti-human IgG antibody's of containing horseradish peroxidase (HRP) mark enzyme marking reagent, form NT5E-antibody-ELIAS secondary antibody compound, thoroughly after washing, add enzyme substrate solution colour developing through cleansing solution, the enzyme substrate solution reaction time to after add acid stop buffer; Described enzyme substrate solution changes into blueness under the catalysis of HRP enzyme, and changes into final yellow under sour effect, utilizes the depth of color to detect the level of the IgG antibody of anti-protein NT5E in sample.
3. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, is characterized in that, the human protein NT5E of described purifying is overexpression from saccharomyces cerevisiae, affinity purification and obtaining, and concentration is 25 μ g/mL.
4. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, it is characterized in that, described standard items comprise 0U/mL standard serum 1 and 100U/mL standard serum 2, are respectively normal human serum and the positive serum of NT5E antibody is diluted in sample diluting liquid.
5. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, it is characterized in that, described enzyme substrate solution comprises: in developer A:500mL solution, contain sodium acetate 13.6g, citric acid 1.6g, in 30% hydrogen peroxide 0.3mL, developer B:500mL solution, contain TMB350mg, DMSO20mL, citric acid H
2o5.1g.
6. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, is characterized in that, described coated damping fluid is the carbonate buffer solution of 0.05M pH9.6, in 1 liter of solution, contains 1.59g Na
2cO
3, 2.93g NaHCO
3.
7. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, it is characterized in that the 0.01mol/L pH7.4PBS solution that described confining liquid is 0.5%BSA contain 5g bovine serum albumin(BSA) (BSA) in 1 liter of solution, 8g NaCl, 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
20,0.2g KCl.
8. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, is characterized in that, described sample diluting liquid is 0.01mol/L pH7.4PBS solution.
9. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, it is characterized in that, described cleansing solution is 0.01mol/L pH7.4PBST solution, and described PBST solution includes 0.05%Tween-20, in 1 liter of solution, contain 8g NaCl, 0.2g KH
2pO
4, 2.9g Na
2hPO
412H
2o, 0.2g KCl, 0.5mL Tween-20.
10. the method for the diagnostic kit of preparing diagnosis of gastric cancer disease according to claim 2, is characterized in that, described stop buffer is 2mol/L H
2sO
4solution.
11. according to the method for arbitrary described diagnostic kit of preparing diagnosis of gastric cancer disease in claim 2 to 10, it is characterized in that, the reagent in described kit all adds antiseptic, so that preserve.
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Non-Patent Citations (5)
| Title |
|---|
| Changes of Gene Expression in Gastric Preneoplasia following Helicobacter pylori Eradication Therapy;Chiaojung Jillian Tsai,et al;《Cancer Epidemiol Biomarkers Prev.》;20061231;第15卷(第2期);272-280 * |
| Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2′-deoxycytidine treatment and oligonucleotide microarray;Satoshi Yamashita,et al;《Cancer Sci.》;20060131;第97卷(第1期);64-71 * |
| Chiaojung Jillian Tsai,et al.Changes of Gene Expression in Gastric Preneoplasia following Helicobacter pylori Eradication Therapy.《Cancer Epidemiol Biomarkers Prev.》.2006,第15卷(第2期), |
| Satoshi Yamashita,et al.Chemical genomic screening for methylation-silenced genes in gastric cancer cell lines using 5-aza-2′-deoxycytidine treatment and oligonucleotide microarray.《Cancer Sci.》.2006,第97卷(第1期), |
| Tiannan Guo，et al.Multidimensional identification of Tissue biomarkers of gastric cancer.《J. Proteome Res.》.2012,第11卷(第6期), * |
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