CN107449903A - Application and its kit of the anti-XRCC3 IgG antibody as gastric cancer serum mark - Google Patents
Application and its kit of the anti-XRCC3 IgG antibody as gastric cancer serum mark Download PDFInfo
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- CN107449903A CN107449903A CN201710629783.3A CN201710629783A CN107449903A CN 107449903 A CN107449903 A CN 107449903A CN 201710629783 A CN201710629783 A CN 201710629783A CN 107449903 A CN107449903 A CN 107449903A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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Abstract
The invention discloses application of the reagent of detection serum moderate resistance XRCC3 IgG antibodies in the kit for preparing diagnosis and indication stomach cancer, the present invention is used as gastric cancer serum mark by the use of anti-XRCC3 IgG antibodies, the level of anti-protein XRCC3 IgG antibodies in qualitative determination human serum, the specificity for detecting stomach cancer is 87%, sensitiveness is 90%, can be as a kind of means of auxiliary early gastric caacer diagnosis, to improve the Sensitivity and Specificity of early gastric caacer diagnosis.The cancer diagnosis reagent box made using the reagent for detecting serum moderate resistance XRCC3 IgG antibodies, it is sensitive, safe and reliable, easy to operate.
Description
Technical field
The present invention relates to a kind of mark of cancer, specially anti-XRCC3 IgG antibody is as gastric cancer serum mark
Using and its kit.
Background technology
Stomach cancer is to endanger the common cancer of human health, according to World Health Organization's statistics of 2008, in full generation
Boundary's scope, the death rate of Patients with Gastric Cancer are in second.Shown in the statistics of the World Health Organization in 2006 close to 950,000
Individual new cases, while have about 700,000 people dies from a kind of this disease.In China, annual stomach cancer newly sends out patient's number more than 300,000,
The number about 160,000 of stomach cancer is died from, case fatality rate is occupied first of malignant tumour, serious threat people's life health.To reduce the death rate,
The effect of improving stomach cancer, it is important to " three is early " work, i.e. early detection, early diagnosis and early treatment of stomach cancer.However, due to
Most patients with gastric cancer lack specific clinical symptoms, 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.At present, stomach cancer overall 5 years survival rates in China's are 43.4%, and 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 stomach 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 directed to finding the preferable tumor-marker of Sensitivity and Specificity
Thing.In addition heterogeneity in gastric carcinomas is big, prognosis and larger to the reaction gender gap for the treatment of, for clinical assessment prognosis and anti-to treating
The prediction and evaluation of answering property, are both needed to good tumor markers.
Preferable tumor markers should meet following condition：(1) sensitiveness is high；(2) specificity is high；(3) tumor markers
Concentration is relevant with tumor size, transfer, grade malignancy, can assist neoplasm staging and judging prognosis；(4) half-life short, effectively control
Concentration declines quickly after treatment, can comparatively fast reflect the actual conditions of in-vivo tumour；(5) it is present in body fluid, particularly blood, is easy to
Detection.
The research of tumour serum mark is always the research emphasis of this area.It there is now a variety of gastric cancer tumor markers：
Such as carcinomebryonic antigen (CEA), it is present in the serum of stomach cancer and other adenocarcinoma patients, but it is smaller to the diagnostic significance of early carcinoma of stomach,
The dynamic observation being mainly used in before and after curing gastric cancer；The part such as CA125, CA19-9, CA50, CA724 and CA242 sugar antigens exist
It can be raised in the patients with gastric cancer of part, but its sensitiveness only 20~40%；Also studies have found that tumor-associated glycoprotein antigen -72
(TAG-72) sensitiveness to stomach cancer is 69%, and specificity is 84%；Glycoprotein antigen MG7-Ag has 40%-60% in serum
Positive rate, have 80%-94% positive rate in stomach organization；Nucleosome Histones antigen (IPO -38)
Sensitiveness as diagnosing gastric cancer is 57.4%, and specificity is advantageous to examining for early carcinoma of stomach more than 90% the above index
It is disconnected.Generation, the transfer of some oncogenes, such as DDC, c-myc, c-erb-2, p53 and nm23 to stomach cancer also have certain meaning
Justice, but be widely used in clinical being still restricted.Because existing stomach cancer biological marker is in the defects of sensitivity and specificity,
Although there is certain value in curative effect monitoring, prompting recurrence, judging prognosis and the generaI investigation of people at highest risk, still can not at present
For making a definite diagnosis for stomach cancer.In order to realize the early stage high sensitivity to stomach cancer, the diagnosis of high specific, urgent need is sought on a molecular scale
Find more sensitive, more special stomach cancer biomarker.
We, using human protein group chip high flux, the advantage quickly analyzed, analyze gastric cancer in early-stage Study
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, candidate serum biomarker is given, to early diagnose and effectively treat stomach 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 are current, there are no and marked XRCC3 protein as stomach cancer biology
The report of will thing.
The content of the invention
The technical problem to be solved in the present invention overcomes existing stomach cancer biological marker in sensitivity and specificity
Defect, although there is certain value in curative effect monitoring, prompting recurrence, judging prognosis and the generaI investigation of people at highest risk, at present
Making a definite diagnosis for stomach cancer is still cannot be used for, in order to realize the early stage high sensitivity to stomach cancer, the diagnosis of high specific, there is provided Yi Zhongkang
Application and its kit of the XRCC3 IgG antibody as gastric cancer serum mark.
In order to solve the above-mentioned technical problem, the invention provides following technical scheme：
Detect reagent the answering in the kit for preparing diagnosis and indication stomach cancer of the IgG antibody of the anti-XRCC3 in serum
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 serums, 100U/mL standards
Serum, enzyme marking reagent, enzyme substrate solution, confining liquid, sample diluting liquid, cleaning solution, terminate liquid, wherein human protein XRCC3 bags
By on ELISA Plate.Preferably, it is 0.05M pH human protein XRCC3 to be coated in into coating buffer solution used on ELISA Plate
Na containing 1.59g in 9.6 carbonate buffer solution, i.e. 1 liter of solution2CO3, 2.93g NaHCO3。
Described 0U/ml standard serums are that normal human serum is diluted in sample diluting liquid；Described 100U/ml standard blood
Clear be serum-dilution that XRCC3 antibody is the positive in sample diluting liquid.
Further, the wine brewing ferment that described human protein XRCC3 (with GST labels) was transformed by passing through genetic engineering
Mother, overexpression is induced 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
Developer A and developer B；Contain sodium acetate 13.6g, citric acid 1.6g and 30% hydrogen peroxide in developer A per 500mL
350mg containing TMB, DMSO 20mL and Citric Acid Mono 5.1g in 0.3mL, the developer B per 500mL.
Further, the confining liquid is 0.5%BSA 0.01mol/L pH 7.4PBS buffer solutions, 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 0.01mol/L pH 7.4PBS buffer solutions.
Further, the cleaning solution is the 0.01mol/L pH 7.4PBST phosphate-buffereds containing 0.05%Tween-20
NaCl containing 8g, 0.2g KH in liquid, i.e. 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 preserve.
The kit of the present invention is using being present clinical widely used elisa technique, using indirect method qualitative detection people
Anti-protein XRCC3 IgG antibody in serum, with the human protein XRCC3 antigen coat microwell plates of purifying, solid phase is made and resists
Original, test serum, then two anti-bindings with HRP marks are sequentially added into the micropore of envelope antigen, form XRCC3- antibody-enzyme
Secondary antibody compound is marked, after thoroughly washing plus substrate TMB colour developings, TMB convert au bleu under the catalysis of HRP enzymes, and in acid
In the presence of change into 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.Absorbance (OD values) is determined under 450nm wavelength with ELIASA, quantifies testing result.
The present invention is by the use of anti-XRCC3 IgG antibody as gastric cancer serum mark, anti-protein in qualitative determination human serum
The level of XRCC3 IgG antibody, the specificity for detecting stomach cancer are 87%, sensitiveness 90%, can be used as auxiliary early gastric caacer
A kind of means of diagnosis, to improve the Sensitivity and Specificity of early gastric caacer diagnosis.Resisted using serum moderate resistance XRCC3-IgG is detected
The cancer diagnosis reagent box that the reagent of body makes, it is sensitive, safe and reliable, easy to operate.
Embodiment
The preferred embodiments of the present invention are illustrated below, it will be appreciated 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 is placed 2 hours or 4 DEG C in room temperature and centrifuged 20 minutes or so after 1000g overnight, takes supernatant to stand
Detect；Or dispensed, and sample is put in -20 DEG C or -80 DEG C preservations, but multigelation should be avoided.Sample after defrosting
It should again centrifuge, then detect.NaN3 can not be contained in detected sample, because NaN3 suppresses horseradish peroxidase
(HRP) it is active.
2nd, the compound method of the various buffer solutions of ELISA method and reagent
It is coated with buffer solution：0.05M pH 9.6 Na2CO3-NaHCO3
(2) sample diluting liquid：PH 7.4PBS solution
(3) cleaning solution：PH 7.4 PBST solution
(4) confining liquid：0.5%BSA pH 7.4PBS solution
(5) enzyme substrate solution：Developer A and developer B (being both needed to now with the current)
(6) terminate liquid：2mol/L H2SO4Solution
(concentrated sulfuric acid is slowly dropped into distilled water by timing, and side edged mixes)
3rd, ELISA method determines the concentration of anti-protein XRCC3 IgG antibody in serum, to aid in early diagnosing stomach cancer：Tool
Body operating procedure is as follows：
(1) it is coated with：The people XRCC3 protein solutions of purifying are diluted to 1 μ g/mL with coating buffer solution, are added to 96 hole enzymes
In target, per the μ L of hole 100,37 DEG C of coatings 2 hours or 4 DEG C are overnight；Cleaning solution board-washing 3 times, dry；Protein XRCC3 is in the past
The saccharomyces cerevisiae transformed by genetic engineering in phase research, overexpression, agarose compatible medium are induced using galactolipin
(glutathione) isolates and purifies, and is identified by Western-Blotting.
(2) close：Add the μ L of confining liquid 200, incubation at room temperature 2 hours；Cleaning solution board-washing 3 times, dry；
(3) dilution of standard items and sample and sample-adding：By standard items and test serum sample 1:100 is dilute with sample buffer
Release to 100 μ L, be added in respective antigen measuring orifice plate.Bubble has been careful not to, has been loaded and sample is added on plum target bottom hole
Portion, does not touch hole wall as far as possible, gently rocks mixing, capping or overlay film on ELISA Plate.If test serum sample is more, it is proposed that uses
Multitube micropipet is loaded.Standard items and detected sample 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 reacted 120 minutes, liquid in clear opening is got rid of, without washing.
(5) it is enzyme-added：Add the anti-Human IgG antibodies 100 μ L of horseradish peroxidase-labeled per hole, 37 DEG C, 60 points
Clock.Liquid in clear opening is got rid of, ibid board-washing pats dry for 5 times.
(6) develop the color：Patting dry rear each hole and the μ L of developer A 50 are first added dropwise, add the μ L of developer B 50, gently concussion mixes,
37 DEG C of lucifuges develop the color 15 minutes.
(7) terminate：Sequentially add the μ L of terminate liquid 50, terminating reaction per hole.The addition sequence of terminate liquid should try one's best and substrate solution
Addition sequence it is identical.The substrate reactions time should add terminate liquid as early as possible after.
(8) result judgement：
I. the optical density (OD values) 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 normative reference of the current international practice, therefore be employed during this test result calibration relatively single
Position.
Ii. the judgement of serum moderate resistance XRCC3 values
Iii. quality control
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 invalid, it is necessary to detects again.
Iv. the explanation of assay
ROC analyses to 50 Healthy Human Serums, 37 Serum Obtained From Advance Gastric Cancers are 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 stomach cancer associated patients
Specificity and sensitivity Detection.The specificity of present invention auxiliary early diagnosis stomach cancer is 87%, sensitiveness 90%.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., it should be included in the present invention's
Within protection domain.
Claims (10)
1. detect application of the reagent of serum moderate resistance XRCC3-IgG antibody in the kit for preparing diagnosis and indication stomach cancer.
2. application as claimed in claim 1, it is characterised in that described to diagnose and be shown as diagnosis, curative effect evaluation or transfer in advance again
Hair monitoring.
A kind of 3. cancer diagnosis reagent box, it is characterised in that including ELISA Plate, human protein XRCC3.0 U/ml standard serums,
100 U/mL standard serums, 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.
4. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that described human protein XRCC3 is by by base
Because of the saccharomyces cerevisiae of engineered mistake, overexpression is induced using galactolipin, is isolated and purified and obtained using agarose compatible medium
Arrive.
5. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that enzyme marking reagent secondary antibody containing the HRP- 0.1-1
μ g/mL, the enzyme substrate solution are TMB solution, including developer A and developer B；Contain acetic acid in developer A per 500mL
The g of sodium 13.6, the g of citric acid 1.6 and the mL of 30 % hydrogen peroxide 0.3, per 500mL developer B in mg, DMSO 20 containing TMB 350
The mL and g of Citric Acid Mono 5.1.
6. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that human protein XRCC3 is coated in ELISA Plate
The carbonate buffer solution that upper coating buffer solution used is 0.05 M pH 9.6.
7. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that the confining liquid is the 0.01 of 0.5 % BSA
The PBSs of mol/L pH 7.4.
8. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that the sample diluting liquid is 0.01 mol/L
The PBSs of pH 7.4, the cleaning solution are the PBST phosphate of 0.01 mol/L pH 7.4 containing 0.05 % Tween-20
Buffer solution.
9. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that 0 described U/ml standard serums are normal person
Serum-dilution is in sample diluting liquid；100 described U/ml standard serums are that XRCC3 antibody is positive serum-dilution in sample
In product dilution.
10. cancer diagnosis reagent box as claimed in claim 3, it is characterised in that the terminate liquid is 2 mol/L H2SO4It is molten
Liquid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710629783.3A CN107449903B (en) | 2017-07-28 | 2017-07-28 | Application and its kit of the IgG antibody of anti-XRCC3 as gastric cancer serum marker |
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| CN201710629783.3A CN107449903B (en) | 2017-07-28 | 2017-07-28 | Application and its kit of the IgG antibody of anti-XRCC3 as gastric cancer serum marker |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982868A (en) * | 2018-06-25 | 2018-12-11 | 哈尔滨医科大学 | The application of nucleome Protein S P110 and kit containing the albumen in preparation alcoholic myocardiopathy early diagnosis reagent |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
-
2017
- 2017-07-28 CN CN201710629783.3A patent/CN107449903B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| FANG FANG ET AL: "Relationship between XRCC3 T241M polymorphism and gastric cancer risk:a meta-analysis", 《MED ONCOL》 * |
| JIAN SONG， ET AL: "Genetic Thr241Met polymorphism of XRCC3 gene and risk of cancer: a meta-analysis of 7845 cases and 9822 controls", 《INT J CLIN EXP MED》 * |
| XIAN-PENG QIN ET AL: "XRCC3 Thr241Met polymorphism and gastric cancer susceptibility: A meta-analysis", 《CLINICS AND HEPATOLOGY AND GASTROENTEROLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982868A (en) * | 2018-06-25 | 2018-12-11 | 哈尔滨医科大学 | The application of nucleome Protein S P110 and kit containing the albumen in preparation alcoholic myocardiopathy early diagnosis reagent |
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