CN107449903B - Application and its kit of the IgG antibody of anti-XRCC3 as gastric cancer serum marker - Google Patents
Application and its kit of the IgG antibody of anti-XRCC3 as gastric cancer serum marker Download PDFInfo
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- CN107449903B CN107449903B CN201710629783.3A CN201710629783A CN107449903B CN 107449903 B CN107449903 B CN 107449903B CN 201710629783 A CN201710629783 A CN 201710629783A CN 107449903 B CN107449903 B CN 107449903B
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention discloses application of the reagent of anti-XRCC3-IgG antibody in detection serum in the kit of preparation diagnosis and indication gastric cancer, the present invention is using anti-XRCC3-IgG antibody as gastric cancer serum marker, the level of anti-protein XRCC3-IgG antibody in qualitative determination human serum, the specificity for detecting gastric cancer is 87%, sensibility is 90%, it can be used as a kind of means of auxiliary early gastric caacer diagnosis, to improve the sensibility and specificity of early gastric caacer diagnosis.It is sensitive, safe and reliable, easy to operate using the cancer diagnosis reagent box of the reagent production of anti-XRCC3-IgG antibody in detection serum.
Description
Technical field
The present invention relates to a kind of marker of cancer, the IgG antibody of specially anti-XRCC3 is as gastric cancer serum marker
Using and its kit.
Background technique
Gastric cancer is to endanger the common cancer of human health, according to World Health Organization's statistics in 2008, in full generation
The death rate of boundary's range, Patients with Gastric Cancer is in second.It is shown in the statistics of the World Health Organization in 2006 close to 950,000
A new cases, while having about 700,000 people dies of a kind of this disease.In China, it is more than 300,000 that annual gastric cancer, which newly sends out patient's number,
The number about 160,000 of gastric cancer is died of, case fatality rate occupies first of malignant tumour, seriously threatens people's life health.To reduce the death rate,
Improve the curative effect of gastric cancer, it is important to " three is early " work, i.e. early detection, early diagnosis and early treatment of gastric cancer.However, due to
Most patients with gastric cancer lack the clinical symptoms of specificity, therefore early carcinoma of stomach diagnosis is very low, and operability is less than 10%；It is postoperative
The life cycle of patient is also shorter.Currently, gastric cancer overall 5 years survival rates in China's are 43.4%, postoperative pathological is by stages
Postoperative 5 years survival rates of (pathological TNM, pTNM) the phase patient of I, II, III, IV are respectively 75.65%, 58.73%,
28.Ol% and 8.42%.Therefore, the early diagnostic rate for improving gastric cancer is extremely important.And general physical examination and imageological examination effect
It is limited, lack predictive value.Therefore from a long-term perspective, should be dedicated to finding the preferable tumor-marker of sensibility and specificity
Object.Furthermore heterogeneity in gastric carcinomas is big, prognosis and larger to the reaction gender gap for the treatment of, anti-for clinical assessment prognosis and to treating
The prediction and evaluation of answering property, are both needed to good tumor markers.
Ideal tumor markers should meet the following conditions: (1) sensibility is high；(2) specificity is high；(3) tumor markers
Concentration is related with tumor size, transfer, grade malignancy, can assist neoplasm staging and judging prognosis；(4) half-life short is effectively controlled
Concentration declines quickly after treatment, can comparatively fast reflect the actual conditions of in-vivo tumour；(5) it is present in body fluid, especially blood, is easy to
Detection.
The research of tumour serum mark is always the research emphasis of this field.There are many gastric cancer tumor markers:
Such as carcinomebryonic antigen (CEA), it is present in the serum of gastric cancer and other adenocarcinoma patients, but smaller to the diagnostic significance of early carcinoma of stomach,
The dynamic observation being mainly used for before and after curing gastric cancer；The parts such as CA125, CA19-9, CA50, CA724 and CA242 sugar antigens exist
It can be increased in the patients with gastric cancer of part, but its sensibility only 20~40%；Also studies have found that tumor-associated glycoprotein antigen -72
It (TAG-72) is 69% to the sensibility of gastric cancer, specificity is 84%；Glycoprotein antigen MG7-Ag has 40%-60% in serum
Positive rate, have the positive rate of 80%-94% in stomach organization；Nucleosome Histones antigen (IPO -38)
Sensibility as diagnosing gastric cancer is 57.4%, and specificity is conducive to examining for early carcinoma of stomach more than 90% the above index
It is disconnected.Certain oncogenes, such as DDC, c-myc, c-erb-2, p53 and nm23 also have certain meaning to generation, the transfer of gastric cancer
Justice, but be widely used in clinical being still restricted.Due to defect of the existing gastric cancer biological marker in sensitivity and specificity,
Although there is certain value in curative effect monitoring, prompt recurrence, judging prognosis and the generaI investigation of people at highest risk, still cannot at present
For making a definite diagnosis for gastric cancer.In order to realize the early stage high sensitivity to gastric cancer, the diagnosis of high specific, urgent need is sought on a molecular scale
Find more sensitive, more special gastric cancer biomarker.
We utilize the advantage that human protein group chip is high-throughput, quickly analyzes in early-stage study, analyze gastric cancer
87 parts of people's associated serum (37 parts of Patients with Gastric Cancer, 50 parts of Healthy Human Serum) compares patient and healthy proper manners within a short period of time
Difference in product gives candidate serum biomarker, to early diagnose and effectively treat gastric cancer.
XRCC3 albumen (X-ray repair cross-complementing protein 3, staggeredly mutually study for a second time courses one has flunked by X-ray
Recoverin 3) major function be to stablize chromosome and DNA plerosis currently, there are no using XRCC3 protein as gastric cancer biology mark
The report of will object.
Summary of the invention
The technical problem to be solved by the present invention is to overcome existing gastric cancer biological markers in sensitivity and specificity
Defect, although there is certain value in curative effect monitoring, prompt recurrence, judging prognosis and the generaI investigation of people at highest risk, at present
It still cannot be used for making a definite diagnosis for gastric cancer, in order to realize the early stage high sensitivity to gastric cancer, the diagnosis of high specific is provided a kind of anti-
Application and its kit of the IgG antibody of XRCC3 as gastric cancer serum marker.
In order to solve the above-mentioned technical problems, the present invention provides the following technical solutions:
The reagent of the IgG antibody of anti-XRCC3 in detection serum answering in the kit of preparation diagnosis and indication gastric cancer
With.The diagnosis and pre- diagnosis, curative effect evaluation or the transfer and relapse of being shown as monitor.
A kind of cancer diagnosis reagent box, including ELISA Plate, human protein XRCC3 0U/ml standard serum, 100U/mL standard
Serum, enzyme marking reagent, enzyme substrate solution, confining liquid, sample diluting liquid, cleaning solution, terminate liquid, wherein human protein XRCC3 packet
By on ELISA Plate.Preferably, human protein XRCC3 is coated in coating buffer used on ELISA Plate is 0.05M pH
9.6 carbonate buffer solution, i.e. Na containing 1.59g in 1 liter of solution2CO3, 2.93g NaHCO3。
The 0U/ml standard serum is that normal human serum is diluted in sample diluting liquid；The 100U/ml standard blood
It is clearly that the serum that XRCC3 antibody is the positive is diluted in sample diluting liquid.
Further, the wine brewing ferment that the human protein XRCC3 (with GST label) was transformed by passing through genetic engineering
Mother is induced overexpression using galactolipin, is isolated and purified and obtained using agarose compatible medium (glutathione).After it is preferred that,
Concentration is 50 μ g/mL.
After it is preferred that, enzyme marking reagent secondary antibody containing the HRP- 0.1-1 μ g/mL, the enzyme substrate solution is TMB solution, including
Color developing agent A and color developing agent B；Contain sodium acetate 13.6g, citric acid 1.6g and 30% hydrogen peroxide in the color developing agent A of every 500mL
350mg containing TMB, DMSO 20mL and Citric Acid Mono 5.1g in the color developing agent B of 0.3mL, every 500mL.
Further, the confining liquid is 7.4 PBS buffer solution of 0.01mol/L pH of 0.5%BSA, i.e. in 1 liter of solution
BSA containing 5g (bovine serum albumin(BSA)), 8g NaCl, 0.2g KH2PO4,2.9g Na2HPO412H20,0.2g KCl
Further, the sample diluting liquid is 7.4 PBS buffer solution of 0.01mol/L pH.
Further, the cleaning solution is slow for the 7.4 PBST phosphate of 0.01mol/L pH containing 0.05%Tween-20
Fliud flushing, i.e. NaCl containing 8g, 0.2g KH in 1 liter of solution2PO4, 2.9g Na2HPO4·12H20,0.2g KCl, 0.5mL Tween-
20。
Further, the terminate liquid is 2mol/L H2SO4Solution.
Further, preservative can be added in each reagent, in order to save.
Kit of the invention is using being present clinical widely used elisa technique, using indirect method qualitative detection people
It is anti-that solid phase is made with the human protein XRCC3 antigen coat microwell plate of purifying in the IgG antibody of anti-protein XRCC3 in serum
Original sequentially adds test serum, then two anti-bindings with HRP label into the micropore of envelope antigen, forms XRCC3- antibody-enzyme
Secondary antibody compound is marked, after thoroughly washing plus substrate TMB colour developing, TMB convert au bleu under the catalysis of HRP enzyme, and in acid
Under the action of be converted to final yellow, the level of the IgG antibody of the anti-protein XRCC3 in the depth and sample of color is in just
It is related.It is measured under 450nm wavelength with microplate reader absorbance (OD value), quantifies testing result.
The present invention is using the IgG antibody of anti-XRCC3 as gastric cancer serum marker, anti-protein in qualitative determination human serum
The level of the IgG antibody of XRCC3, the specificity for detecting gastric cancer is 87%, and sensibility 90% can be used as auxiliary early gastric caacer
A kind of means of diagnosis, to improve the sensibility and specificity of early gastric caacer diagnosis.It is anti-using anti-XRCC3-IgG in detection serum
The cancer diagnosis reagent box of the reagent production of body, it is sensitive, safe and reliable, easy to operate.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment
1. the preparation of blood serum sample:
Whole blood sample in be placed at room temperature for 2 hours or 4 DEG C overnight after in 1000g be centrifuged 20 minutes or so, take supernatant that can stand
Detect；Or it is dispensed, and sample is put in -20 DEG C or -80 DEG C preservations, but multigelation should be avoided.Sample after defrosting
It should be centrifuged, then detect again.NaN3 cannot be contained in institute's test sample, because NaN3 inhibits horseradish peroxidase
(HRP) active.
2, the preparation method of the various buffers of ELISA method and reagent
It is coated with buffer: the Na of 0.05M pH 9.62CO3-NaHCO3
(2) sample diluting liquid: 7.4 PBS solution of pH
(3) cleaning solution: the PBST solution of pH 7.4
(4) confining liquid: 7.4 PBS solution of pH of 0.5%BSA
(5) enzyme substrate solution: color developing agent A and color developing agent B (being both needed to ready-to-use)
(6) terminate liquid: 2mol/L H2SO4Solution
(concentrated sulfuric acid is slowly dropped into distilled water by timing, and side edged mixes)
3, ELISA method measures the concentration of the IgG antibody of anti-protein XRCC3 in serum, to assist early diagnosis gastric cancer:
Specific steps are as follows:
(1) it is coated with: the people XRCC3 protein solution of purifying being diluted to 1 μ g/mL with coating buffer, is added to 96 hole enzymes
In target, every 100 μ L of hole, 37 DEG C of coatings 2 hours or 4 DEG C are overnight；Cleaning solution board-washing 3 times, drying；Protein XRCC3 is in the past
The saccharomyces cerevisiae being transformed by genetic engineering in phase research induces overexpression, agarose compatible medium using galactolipin
(glutathione) isolates and purifies, and identifies by Western-Blotting.
(2) close: be added 200 μ L of confining liquid, incubation at room temperature 2 hours；Cleaning solution board-washing 3 times, drying；
(3) dilution of standard items and sample and sample-adding: standard items and test serum sample 1:100 sample buffer are dilute
It releases to 100 μ L, is added in respective antigen measuring orifice plate.It is careful not to bubble, sample is added on plum target bottom hole by sample-adding
Portion does not touch hole wall as far as possible, shakes gently mixing, capping or overlay film on ELISA Plate.If test serum sample is more, it is proposed that use
Multitube micropipet sample-adding.Standard items and sample to be tested are prepared in 15 minutes before use, are finished discarding, next time, detection used
The standard items of Fresh.
(4) incubate: ELISA Plate is placed in 37 DEG C and reacts 120 minutes, gets rid of liquid in clear opening, does not have to washing.
(5) enzyme: every hole adds the anti-Human IgG antibody 100 μ L of horseradish peroxidase-labeled, and 37 DEG C, 60 points
Clock.Liquid in clear opening is got rid of, is ibid patted dry for board-washing 5 times.
(6) it develops the color: patting dry rear each hole and 50 μ L of color developing agent A is first added dropwise, add 50 μ L of color developing agent B, gently concussion mixes,
37 DEG C are protected from light colour developing 15 minutes.
(7) terminate: sequentially every hole adds 50 μ L of terminate liquid, terminates reaction.The addition sequence of terminate liquid should as far as possible with substrate solution
Addition sequence it is identical.The substrate reactions time, terminate liquid should be added after as early as possible.
(8) result judgement:
I. the optical density (OD value) in each hole is sequentially measured in 450nm wavelength with enzyme-linked instrument.
* A450 is the abbreviation of absorbance at 450nm.
* current XRCC3 antibody there is no the reference standard of the current international practice, therefore use when this test result calibration relatively single
Position.
Ii. in serum anti-XRCC3 value judgement
Iii. quality controls
Each testing result has to comply with following standard:
The A450 of standard serum 1 :≤0.100
The A450 of standard serum 2: >=0.700
Above-mentioned standard is not met such as, then result is considered as in vain, it is necessary to detect again.
Iv. the explanation of inspection result
The ROC analysis of 50 Healthy Human Serums, 37 Serum Obtained From Advance Gastric Cancers is established above with reference to value.
4 specificity and sensitivity Detection:
The present invention is carried out using the serum (300 parts of patients with gastric cancer, 300 parts of Healthy People) of 600 parts of gastric cancer associated patients
Specificity and sensitivity Detection.It is 87% that the present invention, which assists the specificity of early diagnosis gastric cancer, sensibility 90%.
Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention,
Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Claims (9)
1. detecting application of the reagent of anti-XRCC3-IgG antibody in serum in the kit of preparation diagnosis and indication gastric cancer.
2. application as described in claim 1, which is characterized in that described to diagnose and be shown as diagnosis, curative effect evaluation or transfer in advance again
Hair monitoring.
3. a kind of cancer diagnosis reagent box, which is characterized in that including ELISA Plate, human protein XRCC3,0U/ml standard serum,
100U/mL standard serum, enzyme marking reagent, enzyme substrate solution, confining liquid, sample diluting liquid, cleaning solution, terminate liquid, wherein people's egg
White matter XRCC3 is coated on ELISA Plate；The 0U/ml standard serum is that normal human serum is diluted in sample diluting liquid；Institute
The 100U/ml standard serum stated is that XRCC3-IgG antibody is that positive serum is diluted in sample diluting liquid.
4. cancer diagnosis reagent box as claimed in claim 3, which is characterized in that the human protein XRCC3 is by passing through base
Because of the saccharomyces cerevisiae of engineered mistake, overexpression is induced using galactolipin, is isolated and purified and is obtained using agarose compatible medium
It arrives.
5. cancer diagnosis reagent box as claimed in claim 3, which is characterized in that enzyme marking reagent secondary antibody containing the HRP- 0.1-1 μ
G/mL, the enzyme substrate solution are TMB solution, including color developing agent A and color developing agent B；Contain acetic acid in the color developing agent A of every 500mL
In the color developing agent B of sodium 13.6g, citric acid 1.6g and 30% hydrogen peroxide 0.3mL, every 500mL 350mg containing TMB, DMSO 20mL and
Citric Acid Mono 5.1g.
6. cancer diagnosis reagent box as claimed in claim 3, which is characterized in that human protein XRCC3 is coated in ELISA Plate
The carbonate buffer solution that upper coating buffer used is 0.05M pH 9.6.
7. cancer diagnosis reagent box as claimed in claim 3, which is characterized in that the confining liquid is containing 0.5%BSA's
0.01mol/L pH 7.4PBS buffer.
8. cancer diagnosis reagent box as claimed in claim 3, which is characterized in that the sample diluting liquid is 0.01mol/L pH
7.4PBS buffer, the cleaning solution are the 0.01mol/L pH 7.4PBST phosphate buffer containing 0.05%Tween-20.
9. cancer diagnosis reagent box as claimed in claim 3, which is characterized in that the terminate liquid is 2mol/L H2SO4Solution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680709A (en) * | 2012-05-14 | 2012-09-19 | 上海交通大学 | Diagnostic kit and application of coiled-coil domain containing protein 49 (CCDC49) in preparing gastric cancer early-stage diagnostic reagent |
| CN102680708A (en) * | 2012-05-14 | 2012-09-19 | 上海交通大学 | Diagnostic kit and application of RNF19 (ring finger protein 19) in preparation of reagent for early diagnosis of gastric cancers |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680709A (en) * | 2012-05-14 | 2012-09-19 | 上海交通大学 | Diagnostic kit and application of coiled-coil domain containing protein 49 (CCDC49) in preparing gastric cancer early-stage diagnostic reagent |
| CN102680708A (en) * | 2012-05-14 | 2012-09-19 | 上海交通大学 | Diagnostic kit and application of RNF19 (ring finger protein 19) in preparation of reagent for early diagnosis of gastric cancers |
Non-Patent Citations (3)
| Title |
|---|
| Genetic Thr241Met polymorphism of XRCC3 gene and risk of cancer: a meta-analysis of 7845 cases and 9822 controls;Jian Song， et al;《Int J Clin Exp Med》;20161030;第9卷(第10期);第19335-19344页 |
| Relationship between XRCC3 T241M polymorphism and gastric cancer risk:a meta-analysis;Fang Fang et al;《Med Oncol》;20100612;第28卷;第999–1003页 |
| XRCC3 Thr241Met polymorphism and gastric cancer susceptibility: A meta-analysis;Xian-Peng Qin et al;《Clinics and Hepatology and Gastroenterology》;20131204;第38卷;第226-234页 |
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