CN102680708A - Diagnostic kit and application of RNF19 (ring finger protein 19) in preparation of reagent for early diagnosis of gastric cancers - Google Patents
Diagnostic kit and application of RNF19 (ring finger protein 19) in preparation of reagent for early diagnosis of gastric cancers Download PDFInfo
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Abstract
The invention relates to the field of bioscience, in particular to a diagnostic kit and application of an RNF19 (ring finger protein 19) in preparation of a reagent for early diagnosis of gastric cancers. The diagnostic kit is used for early diagnosis of gastric cancers and comprises an ELISA (enzyme-linked immunosorbent assay) plate, a human protein RNF19, standard serums, an ELISA reagent, an enzyme substrate solution, confining liquid, sample diluent, washing liquid and stop liquid, wherein the human protein RNF19 is enveloped on the ELISA plate. Compared with the prior art, the diagnostic kit has the following advantages: 1. a sensitive, safe, reliable and easy-to-operate commercial kit is provided, is used for qualitatively determining the level of the IgA antibody against RNF19 in the human serums, and is conductive to early diagnosis of gastric cancers in an aided manner; and 2. the provided serum biomarker RNF19 has specificity of 86% and sensitivity of 86% and has the characteristics of high specificity and high sensitivity.
Description
Technical field
The present invention relates to bio-science field, particularly a kind of diagnostic kit and the RNF19 application in preparation cancer of the stomach early diagnosis reagent.
Background technology
Cancer of the stomach is the healthy common cancer of harm humans, and according to The World Health Organization's statistics in 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 near 950,000 new cases, 700,000 people that have an appointment simultaneously die from this a kind of disease.In China, annual cancer of the stomach New Development patient number surpasses 300,000, and it is about 160,000 to die from the number of cancer of the stomach, and case fatality rate occupies first of the malignant tumour, serious threat people's life healthy (Chen Zhu, sample survey report, 2008 are looked back in the whole nation cause of the death for the third time).For reducing mortality ratio, improve efficacy in treating gastric carcinoma, key is cancer of the stomach " three early " work, i.e. early detection, early diagnosis and early stage treatment.Yet, because the clinical symptoms of most patients with gastric cancer lack of specific, so the early carcinoma of stomach diagnosis is very low, and operability less than 10% (Wu Yunlin: the 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 staging (pathological TNM; PTNM) 5 years survival rates of postoperative of I, II, III, IV phase patient be respectively 75.65%, 58.73%, 28.01% and 8.42% (Liu Ping Ya: Chinese gastrointestinal surgery magazine, 2010,13:163).Therefore, the early diagnostic rate of raising cancer of the stomach is extremely important.And general health check-up is limited with the imaging examination effect, when treating to find and to clarify a diagnosis, often in, late period, lack early warning value.Therefore, from a long-term perspective, should be devoted to find all tumor markerses preferably of susceptibility and specificity.In addition, cancer of the stomach is heterogeneous big, and prognosis and bigger to the reactive difference of treatment for the clinical assessment prognosis and to the prediction and the evaluation of therapeutic response property, 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 tumour size, transfer, grade malignancy are relevant, can assist neoplasm staging and judging prognosis; (4) half life period weak point, effectively treatment back concentration descends very soon, can comparatively fast reflect the actual conditions of in-vivo tumour; (5) be present in body fluid, particularly in the blood, be easy to detect (Liu Ping Ya: Chinese gastrointestinal surgery magazine, 2010,13:163).
The research of tumour serum mark is the research emphasis of this area always.Existing at present multiple cancer of the stomach tumour marker: like carcinomebryonic antigen (CEA); Be present in cancer of the stomach and other gland cancer patient's the serum, but less, be mainly used in (the Lipkin M:Cancer Research that dynamic observes before and after the curing gastric cancer the diagnostic significance of early carcinoma of stomach; 1988,48:235; Lipkin:JBC Supplymental, 1992,16:1); Part CAs such as CA125, CA19-9, CA50, CA724 and CA242 can raise in the part patients with gastric cancer, but its susceptibility 20～40% (Kodera Y:The American journal of gastroenterology, 1996,91:49 only; Chou M:Disease Markers, 2000,16:105;
Lai IREtal:
Hepato-gastroenterology, 2002,49:115; Edip U et al:Advances in Theray, 2008,25:1075); Discover that in addition tumor-associated glycoprotein antigen-72 (TAG-72) is 69% to the susceptibility of cancer of the stomach, specificity be 84% (Liu Jun etc.: practical medical journal, 1999,15:4); Glycoprotein antigen MG7-Ag has the positive rate of 40%-60% in serum, in stomach organization, have 80%-94% positive rate (Ren J:Cancer, 2000,88:280); Nucleosome histone class antigen (IPO-38) is 57.4% as the susceptibility of diagnosing gastric cancer, specificity surpass 90% (Hao Y etal:J Proteome Res, 2008,7:3668) above these indexs all help the diagnosis of early carcinoma of stomach.Some oncogene also has the certain significance, but is widely used in clinical still restricted (Liu Qian etc., People's Health Publisher, 2004) generation, the transfer of cancer of the stomach like DDC, c-myc, c-erb-2, p53 and nm23 etc.Because the defective of existing cancer of the stomach biological marker on sensitivity and specificity though in curative effect monitoring, prompting recurrence, judging prognosis and people at highest risk's generaI investigation, have certain value, 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 on molecular level, searching out more responsive, more special cancer of the stomach biomarker.
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 subjects serum) of Patients with Gastric Cancer have been analyzed; Different in patient and the healthy subjects sample have been compared within a short period of time; Provided candidate's serum biomarker, in the hope of early diagnosis and effective treatment cancer of the stomach.
Albumen RNF19 (Ring Finger Protein 19) ring finger protein 19 belongs to a member of human Ring Finger ring finger protein family, and the assignment of genes gene mapping of this albumen of encoding is in 8q22.Most Ring family protein members are nucleoprotein, its zinc fingers and protein-DNA, protein-protein interactions relevant (Freemont PS et al:Cell, 1991,64:483).RNF-19 albumen is a kind of fourth finger E3 ubiquitin ligase of the FHA/RING of comprising domain; Play an important role during dna damage is replied in early days; And with E2 ligase U bc13; The phosphorylation of the chromatin GAP-associated protein GAP of inducing through dna damage is raised at the little point of the damage of DNA, the formation of fellowship K63-linked poly ubiquitin chain, main mediating protein phosphorylation and ubiquitin signal pathway.Its C end of RNF-19 albumen contains the Ring-H2 zinc fingers, can combine 2 zinc atoms.This Ring-H2 zinc finger protein possibly have through mediating protein-protein, protein-lipid interact or the cell regulating action reach vacuole or film the albumen sorting (Seol JH et al:Genes Dev, 1999,13:1614).
At present, do not see have with the report of RNF19 protein as the cancer of the stomach biomarker.
Summary of the invention
First purpose of the present invention is to provide a kind of diagnostic kit, can not realize the technical matters of the diagnosis of early stage high sensitivity, high specific to cancer of the stomach to solve stomach cancer diagnosis reagent box of the prior art.
Second purpose of the present invention is to provide a kind of RNF19 application in preparation cancer of the stomach early diagnosis reagent, can not realize the technical matters of the diagnosis of early stage high sensitivity, high specific to cancer of the stomach to solve stomach cancer diagnosis reagent of the prior art.
The object of the invention realizes through following technical scheme:
A kind of diagnostic kit; Be used for the early stage diagnosis of cancer of the stomach; Said diagnostic kit comprises ELISA Plate, human protein RNF19, standard serum, enzyme marking reagent, enzyme substrate solution, confining liquid, sample diluting liquid, cleansing solution and stop buffer; Wherein, said human protein RNF19 encapsulates on said ELISA Plate.
Preferably, said human protein RNF19 is an overexpression from saccharomyces cerevisiae, affinity purification and obtaining, and concentration is 50 μ g/mL.
Preferably, said standard serum comprises 0U/mL standard serum 1 and 100U/mL standard serum 2, and said 0U/mL standard serum 1 is diluted in the sample diluting liquid for healthy subjects serum; Said 100U/mL standard serum 2 is diluted in the sample diluting liquid for the positive serum of RNF19 antibody.
Preferably, said enzyme marking reagent contains HRP-two anti-0.1-1 μ g/mL.
Preferably, said enzyme substrate solution is a TMB solution, and said TMB solution comprises developer A and developer B, contains sodium acetate 13.6g, citric acid 1.6g and 30% oxydol 0.3mL in the developer A:500mL solution; Contain TMB 350mg, DMSO 20mL and citric acid H in the developer B:500mL solution
2O5.1g.
Preferably, said confining liquid is 0.01mol/L pH 7.4 phosphate-NaCl damping fluid (PBS) solution of 0.5%BSA, promptly contains 5g BSA (bovine serum albumin(BSA)), 8g NaCl, 0.2g KH in 1 liter of solution
2PO
4, 2.9g Na
2HPO
412H
2O and 0.2g KCl.
Preferably, said sample diluting liquid is 0.01mol/L pH 7.4 phosphate-NaCl damping fluid (PBS); Said cleansing solution is 0.01mol/L pH 7.4 phosphate-NaCl damping fluid (PBST), and PBST includes 0.05%Tween-20, promptly contains 8g NaCl, 0.2g KH in 1 liter of solution
2PO
4, 2.9g Na
2HPO
412H
2O, 0.2g KCl and 0.5mL Tween-20; Said stop buffer is 2mol/L H
2SO
4Solution.
Preferably, the reagent in the said kit all can add antiseptic.
A kind of diagnostic kit is used for the early stage diagnosis of cancer of the stomach, and what said diagnostic kit adopted is present clinical widely used elisa technique; Use the IgA antibody of anti-protein RNF19 in the indirect method qualitative detection human serum; Specifically: with the antigen coated microwell plate of human protein RNF19, process solid phase antigen, in the micropore of envelope antigen, add test serum; Two resistive connections with the HRP mark close again; Form RNF19-antibody-ELIAS secondary antibody compound, through thoroughly adding substrate TMB colour developing after the washing, TMB changes into blueness under the catalysis of HRP enzyme; And under the effect of acid, changing into final yellow, the level of the IgA antibody of the depth of color and the anti-protein RNF19 in the sample is proportionate.
The application of human protein RNF19 in preparation cancer of the stomach early diagnosis reagent.
Compared with prior art, the present invention has following advantage:
1, a kind of sensitivity, safe, reliable, easy-operating commercial kit are provided, the level of the IgA antibody of anti-protein RNF19 in the qualitative determination human serum helps auxiliary early diagnosis cancer of the stomach;
2, the specificity of the serum biomarker protein RNF19 that provides is 86%, and susceptibility is 86%, has the characteristics of high specific and hypersensitivity.
Description of drawings
Fig. 1 dyes the expression concentration of identifying RNF19, the synoptic diagram of purified condition for silver,
Among the figure:
1 expression standard BSA solution concentration is 5 μ g/mL;
2 expression standard BSA solution concentrations are 10 μ g/mL;
3 expression standard BSA solution concentrations are 25 μ g/mL;
4 expression standard BSA solution concentrations are 50 μ g/mL;
5 expression standard BSA solution concentrations are 100 μ g/mL;
The RNF19 protein example (contain GST label) of 6 expressions after agarose compatible medium (glutathione) (National Engineering Research Center for Biotechnology) separation and purification;
7 expression protein molecular weight marker, molecular weight is respectively from big to small: 170KD, 130KD, 95KD, 72KD, 55KD;
Fig. 2 identifies the expression of RNF19, the synoptic diagram of purified condition for Western-Blotting,
Among the figure:
The RNF19 protein example (contain GST label) of 1 expression after agarose compatible medium (glutathione) (National Engineering Research Center for Biotechnology) separation and purification;
2 expression protein molecular weight marker, molecular weight is respectively from big to small: 130KD, 95KD, 72KD, 55KD;
Fig. 3 is the concentration curve figure of the IgA antibody of anti-protein RNF19 in healthy subjects, high-risk, the patients with gastric cancer serum, annotates: the numerical value among the figure is the level relatively of the IgA AC of the anti-protein RNF19 in healthy subjects, high-risk, each 300 parts of serum sample of patients with gastric cancer.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is done further explanation.
1, expression, purifying and evaluation RNF19:
Protein RNF19 is that the saccharomyces cerevisiae by genetic engineering modified mistake utilizes galactose to induce overexpression; Afterwards; Obtain through agarose compatible medium (glutathione) separation and purification, it is carried out the result that silver dyes quantitatively and Western-Blotting identifies distinguish as depicted in figs. 1 and 2.
2, the preparation of blood serum sample:
Whole blood sample is placed 2 hours or the 4 ℃ backs of spending the night in 1000g centrifugal about 20 minutes in room temperature, gets supernatant and can detect immediately; Or carry out packing, and sample is put in-20 ℃ or-80 ℃ of preservations, but should avoid multigelation.Sample after thawing should be centrifugal once more, detects then.Can not contain NaN in institute's test sample
3, because NaN
3(HRP) activity that suppresses horseradish peroxidase.
3, the compound method of various damping fluids and reagent in the ELISA method:
(1) encapsulates damping fluid: the Na of 0.05M 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
(joining existing usefulness) at present
(joining existing usefulness) at present
(6) stop buffer: 2mol/L H
2SO
4Solution
(timing slowly splashes into the concentrated sulphuric acid in the distilled water, limit edged mixing)
4, the ELISA method is measured the concentration of the IgA antibody of anti-protein RNF19 in the serum, to assist early diagnosis cancer of the stomach:
The concrete operations step is following:
(1) encapsulate: the people RNF19 protein solution of purifying is diluted to 1 μ g/mL with encapsulating damping fluid, joins in the 96 hole ELISA Plates, every hole 100 μ L, 37 ℃ encapsulate and spent the night in 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) standard items and dilution of sample and application of sample: standard items and test serum sample 1:100 are diluted to 100 μ L with sample buffer, join in the antigen measuring orifice plate separately.Note not having bubble, application of sample is added on bottom, plum target hole with sample, does not touch hole wall as far as possible, rocks mixing gently, adds a cover on the ELISA Plate or overlay film.If the test serum sample is more, multitube micropipet application of sample is used in suggestion.Standard items and detected sample face in preceding 15 minutes to be prepared, and uses up to abandon, and detects next time and uses freshly prepared standard items.
(4) incubation: ELISA Plate places 37 ℃ of reactions 120 minutes, gets rid of liquid in the clear opening, need not wash.
(5) enzyme-added: every hole adds the anti-Human IgA antibody 100 μ L of horseradish peroxidase-labeled, 37 ℃, 60 minutes.Get rid of liquid in the clear opening, the same plate of washing is clapped for 5 times dried.
(6) colour developing: bat is done each hole, back and is dripped developer A 50 μ L earlier, adds developer B 50 μ L again, the light shaking mixing, and 37 ℃ of lucifuges developed the color 15 minutes.
(7) stop: every in regular turn hole adds stop buffer 50 μ L, cessation reaction.The addition sequence of the stop buffer addition sequence with substrate solution as far as possible is identical.The substrate reactions time to after should add stop buffer as early as possible.
(8) result judges:
I. join appearance is measured each hole in regular turn at the 450nm wavelength optical density (OD value) with enzyme.
Unit value
* A450 is the abbreviation of 450nm place absorbance.
* at present RNF19 antibody does not still have the normative reference of the current international practice, so has adopted relative unit during the calibration of this testing result.
Ii. the judgement of anti-RNF19 value in the 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, then the result is regarded as invalidly, must detect again.
Iv. the explanation of assay
Above reference value has been established in ROC analysis to 50 routine healthy subjects serum, 37 routine patients with gastric cancer serum, 14 routine high-risk patient serum.
5, specificity and sensitivity Detection: adopt the serum (300 parts of patients with gastric cancer, 300 parts of people at highest risk, 300 parts of healthy subjects) of 900 parts of cancer of the stomach associated patient that diagnostic kit of the present invention has been carried out specificity and sensitivity Detection (like Fig. 3).The specificity of the auxiliary early diagnosis cancer of the stomach of diagnostic kit of the present invention is 86%, and susceptibility is 86%, all is much higher than the index of diagnosing gastric cancer in the prior art.
The present invention also provides human protein RNF19 new purposes in preparation cancer of the stomach early diagnosis reagent.
More than the disclosed several specific embodiments that are merely the application, but the application is not limited thereto, any those skilled in the art can think variation, all should drop in the application's the protection domain.
Claims (10)
1. diagnostic kit; Be used for the early stage diagnosis of cancer of the stomach; It is characterized in that; Said diagnostic kit comprises ELISA Plate, human protein RNF19, standard serum, enzyme marking reagent, enzyme substrate solution, confining liquid, sample diluting liquid, cleansing solution and stop buffer, and wherein, said human protein RNF19 encapsulates on said ELISA Plate.
2. a kind of diagnostic kit as claimed in claim 1 is characterized in that, said human protein RNF19 is an overexpression from saccharomyces cerevisiae, affinity purification and obtaining, and concentration is 50 μ g/mL.
3. a kind of diagnostic kit as claimed in claim 1 is characterized in that, said standard serum comprises 0U/mL standard serum 1 and 100U/mL standard serum 2, and said 0U/mL standard serum 1 is diluted in the sample diluting liquid for healthy subjects serum; Said 100U/mL standard serum 2 is diluted in the sample diluting liquid for the positive serum of RNF19 antibody.
4. a kind of diagnostic kit as claimed in claim 1 is characterized in that, said enzyme marking reagent contains HRP-two anti-0.1-1 μ g/mL.
5. a kind of diagnostic kit as claimed in claim 1; It is characterized in that; Said enzyme substrate solution is a TMB solution, and said TMB solution comprises developer A and developer B, contains sodium acetate 13.6g, citric acid 1.6g and 30% oxydol 0.3mL in the developer A:500mL solution; Contain TMB 350mg, DMSO 20mL and citric acid H in the developer B:500mL solution
2O 5.1g.
6. a kind of diagnostic kit as claimed in claim 1; It is characterized in that; Said confining liquid is 0.01mol/L pH 7.4 phosphate-NaCl damping fluid (PBS) solution of 0.5%BSA, promptly contains 5g BSA (bovine serum albumin(BSA)), 8g NaCl, 0.2g KH in 1 liter of solution
2PO
4, 2.9g Na
2HPO
412H
2O and 0.2g KCl.
7. a kind of diagnostic kit as claimed in claim 1 is characterized in that, said sample diluting liquid is 0.01mol/L pH 7.4 phosphate-NaCl damping fluid (PBS); Said cleansing solution is 0.01mol/L pH7.4 phosphate-NaCl damping fluid (PBST), and PBST includes 0.05%Tween-20, promptly contains 8g NaCl, 0.2g KH in 1 liter of solution
2PO
4, 2.9g Na
2HPO
412H
2O, 0.2g KCl and 0.5mL Tween-20; Said stop buffer is 2mol/L H
2SO
4Solution.
8. a kind of diagnostic kit as claimed in claim 1 is characterized in that the reagent in the said kit all can add antiseptic.
9. a diagnostic kit is used for the early stage diagnosis of cancer of the stomach, it is characterized in that; What said diagnostic kit adopted is present clinical widely used elisa technique, uses the IgA antibody of anti-protein RNF19 in the indirect method qualitative detection human serum, specifically: with the antigen coated microwell plate of human protein RNF19; Process solid phase antigen; In the micropore of envelope antigen, add test serum, two resistive connections with the HRP mark close again, form RNF19-antibody-ELIAS secondary antibody compound; Through thoroughly adding substrate TMB colour developing after the washing; TMB changes into blueness under the catalysis of HRP enzyme, and under the effect of acid, changes into final yellow, and the level of the IgA antibody of the depth of color and the anti-protein RNF19 in the sample is proportionate.
10. the application of human protein RNF19 in preparation cancer of the stomach early diagnosis reagent.
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Cited By (3)
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| CN107449903A (en) * | 2017-07-28 | 2017-12-08 | 上海浦东解码生命科学研究院 | Application and its kit of the anti-XRCC3 IgG antibody as gastric cancer serum mark |
| CN107462725A (en) * | 2017-07-28 | 2017-12-12 | 上海浦东解码生命科学研究院 | Application and its kit of the anti-FNDC4 IgG antibody as gastric cancer serum mark |
| CN115932283A (en) * | 2023-01-31 | 2023-04-07 | 哈尔滨医科大学 | Application of NLN in preparation of alcoholic cardiomyopathy early diagnosis reagent and diagnosis kit thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107449903A (en) * | 2017-07-28 | 2017-12-08 | 上海浦东解码生命科学研究院 | Application and its kit of the anti-XRCC3 IgG antibody as gastric cancer serum mark |
| CN107462725A (en) * | 2017-07-28 | 2017-12-12 | 上海浦东解码生命科学研究院 | Application and its kit of the anti-FNDC4 IgG antibody as gastric cancer serum mark |
| CN107449903B (en) * | 2017-07-28 | 2019-07-05 | 上海浦东解码生命科学研究院 | Application and its kit of the IgG antibody of anti-XRCC3 as gastric cancer serum marker |
| CN107462725B (en) * | 2017-07-28 | 2019-07-05 | 上海浦东解码生命科学研究院 | Application and its kit of the IgG antibody of anti-FNDC4 as gastric cancer serum marker |
| CN115932283A (en) * | 2023-01-31 | 2023-04-07 | 哈尔滨医科大学 | Application of NLN in preparation of alcoholic cardiomyopathy early diagnosis reagent and diagnosis kit thereof |
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