CN108303536A - The quickly kit of detection tumor tissues 2-HG accumulations - Google Patents
The quickly kit of detection tumor tissues 2-HG accumulations Download PDFInfo
- Publication number
- CN108303536A CN108303536A CN201610637388.5A CN201610637388A CN108303536A CN 108303536 A CN108303536 A CN 108303536A CN 201610637388 A CN201610637388 A CN 201610637388A CN 108303536 A CN108303536 A CN 108303536A
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- kit
- accumulations
- tumor tissues
- detection
- quick detection
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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
Abstract
The invention belongs to biotechnologies, are related to the kit and application method of a kind of 2 hydroxyl glutaric acid of quick detection tumor tissues carcinogenic metabolites, 2 HG accumulations, contain the reagent and standard items that detect 2 HG and derivatization reagent in the kit；Alternatively, the specific antibody containing 2 HG in the kit.Using the kit, neoplasmic tissue sample will be obtained and be placed in the pure methanol of addition precooling in centrifuge tube, 14000 rpm are centrifuged after refiner grinding is stood, supernatant is taken to be transferred in vial, it is dried in vacuo after centrifuge is fully dried and derivatization reagent MTBSTFA, 70 DEG C of placements, room temperature cooling is added, upper machine, Agilent 7890A 5975C；Chromatographic column is HP 5MS, and carrier gas is high-purity helium, is identified 2 HG by the way of NIST database search and standard items verification double combination.Testing result shows that kit of the invention can be detected in art in brain tissue accumulates with the presence or absence of 2 HG high levels, helps to realize the rapid differential diagnosis of IDH1 mutation gliomas, and provides important guiding information for postoperative individualized treatment.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of kit of quick detection tumor tissues 2-HG accumulations,
2-HG in tumor tissues is quickly detected by the kit to accumulate, the quick diagnosis for contributing to IDH1 to be mutated.
Background technology
Prior art discloses gliomas（glioma）It is the most common tumour of central nervous system, wherein glioblastoma
Cure rate is low, high recurrence rate, poor prognosis.At present in clinical practice, for glioma that some acatalepsias are true especially in art
Lesion needs to carry out stereo-directional puncture biopsy, and typical person is easier to diagnose, but often frozen section in art in practical operation
Pathology is not true to type simultaneously, and Pathologis can only observe gliosis, and gliosis is in Low grade glioma, normal brain activity group
It knits, inflammation, part lymthoma etc. may occur in which.Although saltant type IDH1 immunohistochemical stainings can differentiate glioma and other feelings
Condition, but generally require 5-7 days, time-consuming, and cannot achieve in art and judge, even needs to puncture repeatedly sometimes, and it is negative to increase patient
Load and operation risk.
Research discloses, often along with isocitric acid dehydrogenase gene in most of diffusivity human gliomas
The mutation clinical practices of (isocitrate dehydrogenase gene) 1 (IDH1) show there is this glioma being mutated
Patient clinically has good outcome, and more sensitive to chemotherapy and targeted therapy., therefore IDH1 mutation are glue
The important molecule parting label of matter tumor one；IDH is catalyzed isocitrate oxidation decarboxylation, generates a-ketoglutaric acid（a-KG）, mutation
Type IDH is while losing its normal enzyme activity, using a-KG as substrate, generates carcinogenic metabolites 2- hydroxyl glutaric acids（2-HG）.
In general, 2-HG is horizontal very low in normal cerebral tissue, and the high accumulation in the glioma of IDH1 mutation, correlative study show
IDH1 mutant cells rely on 2-HG and maintain its carcinogenic nature.Therefore, IDH1 mutation quickly detection, it will help antidiastole colloid
Tumor, and conducive to the formulation and optimization of individualized treatment scheme, clinical meaning is great.Based on this, present inventor is quasi- to be provided
A kind of kit of quick detection tumor tissues 2-HG accumulations, is accumulated by detecting 2-HG in tumor tissues, will realize that IDH1 dashes forward
The quick diagnosis of change.
Invention content
It is the mesh of the present invention the present situation for the prior art, a kind of examination of quick detection tumor tissues 2-HG accumulations is provided
Agent box is accumulated by detecting 2-HG in tumor tissues, to realize that the quick diagnosis of IDH1 mutation provides according to and is postoperative
Bodyization treatment provides important guiding information.
Specifically, the present invention provides a kind of kits of quick detection tumor tissues 2-HG accumulations
Reagent and standard items containing detection 2-HG in the kit and derivatization reagent.
Alternatively, the specific antibody containing 2-HG in the kit.
2-HG of the present invention is saltant type IDH while losing its normal enzyme activity, raw using-KG as substrate
At carcinogenic metabolites 2- hydroxyl glutaric acids（2-HG）.
The present invention also provides a kind of applications of mentioned reagent box, that is, accumulated by detecting 2-HG in tumor tissues
Come assess IDH1 mutation possibility, further realize IDH1 mutation quick diagnosis；It can help antidiastole glue accordingly simultaneously
Matter tumor, and conducive to the formulation and optimization of individualized treatment scheme.
The kit detects 2-HG in tumor tissues by following step and accumulates：
1. obtaining neoplasmic tissue sample, it is placed in centrifuge tube；
2. being added into above-mentioned centrifuge tube and pure methanol being pre-chilled；
3. refiner grinds 1 min, a moment is stood；
4. 14000 rpm are centrifuged；
5. taking supernatant, it is transferred in vial, vacuum drying centrifuge is fully dry；
6. derivatization reagent MTBSTFA, 70 DEG C of placements are added；
7. room temperature cools down, upper machine（Agilent 7890A-5975C；Chromatographic column is HP-5MS, and carrier gas is high-purity helium.
Preferably 2-HG is identified by the way of NIST database search and standard items verification double combination in the present invention.
The detection kit is used in the application, and the in vitro glioma brain tissue obtained in art has been carried out quickly
Detection, testing result show that detection kit using the present invention is detected in brain tissue in art with the presence or absence of 2-HG high levels
Accumulation, helps to realize the rapid differential diagnosis of IDH1 mutation gliomas, and provides important guiding for postoperative individualized treatment
Information.
The advantages of kit of the detection tumor tissues 2-HG accumulations of the present invention, has：
1, it can provide the reference data with specificity for diagnosis IDH1 mutation while being detected to 2-HG；
2, the true lesion of some acatalepsias need not be punctured repeatedly, hence it is evident that reduce patient's danger risk；
3, detection time is quick, and preliminary judgement is can be obtained in art, only need to judge whether there is 2-HG accumulation, and then carry out differentiate be
No is the glioma of glioma or IDH1 mutation, and important information is provided for clinical judgment.
In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.It needs to refer in particular to
Go out, specific example is merely to explanation, it is clear that those skilled in the art can be according to illustrating, in the present invention herein
In the range of various modifications and variations are made to the present invention, these modifications and variations are also included in the scope of the present invention.
Specific implementation mode
Embodiment 1
Detection kit using the present invention has carried out quick detection to the in vitro glioma brain tissue obtained in glioma art,
Neoplasmic tissue sample is cut into 25-30 μ g, is placed in 1.5 ml centrifuge tubes；It is pure that 100 μ l precoolings are added into the centrifuge tube
Methanol；Hand-held refiner grinds 1 min, stands a moment；14000 rpm centrifuge 10 min；40 μ l of supernatant are taken, V is transferred to
In the GC special glass bottles of type bottom, vacuum drying centrifuge is fully dry, takes about 25 min；Derivatization is added in ventilating kitchen
40 μ l MTBSTFA of reagent cover tightly bottle cap, 70 DEG C of 25 min of placement；Room temperature natural cooling, upper machine, Agilent 7890A-
5975C；Chromatographic column is HP-5MS, and carrier gas is high-purity helium.The side of double combination is verified using NIST database search and standard items
Formula identifies 2-HG.
Testing result shows that detection kit using the present invention detects in art and whether there is 2-HG high in brain tissue
Horizontal accumulation, helps to realize the rapid differential diagnosis of IDH1 mutation gliomas, and provides for postoperative individualized treatment important
Tutorial message.
Claims (9)
1. a kind of kit of quick detection tumor tissues 2-HG accumulations, which is characterized in that
Reagent and standard items containing detection 2-HG in the kit and derivatization reagent；
Alternatively, the specific antibody containing 2-HG in the kit.
2. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that pass through following steps
Rapid detection tumor tissues 2-HG accumulations,
1）Neoplasmic tissue sample is obtained, is placed in centrifuge tube；
2）It is added into above-mentioned centrifuge tube and pure methanol is pre-chilled；
3）Refiner grinds 1 min, stands a moment；
4）14000 rpm are centrifuged；
5）Supernatant is taken, is transferred in vial, vacuum drying centrifuge is fully dry；
6）Derivatization reagent MTBSTFA, 70 DEG C of placements are added；
7）Room temperature cools down, upper machine, Agilent 7890A-5975C；Chromatographic column is HP-5MS, and carrier gas is high-purity helium.
3. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection step
In rapid, 2-HG is identified by the way of NIST database search and standard items verification double combination.
4. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection
In step, neoplasmic tissue sample is 25-30 μ g, is placed in 1.5 ml centrifuge tubes.
5. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection
In step, 100 μ l is added into centrifuge tube, pure methanol is pre-chilled.
6. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection
In step, holds refiner and grind 1 min, stand a moment.
7. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection
In step, 10 min are centrifuged using 14000 rpm.
8. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection
In step, 40 μ l of supernatant are taken, are transferred in the GC special glass bottles of V-type bottom, vacuum drying centrifuge dries 25 min.
9. the kit of quick detection tumor tissues 2-HG accumulations as described in claim 1, which is characterized in that the detection
In step, 40 μ l MTBSTFA of derivatization reagent are added in ventilating kitchen, cover tightly bottle cap, 70 DEG C of 25 min of placement.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610637388.5A CN108303536A (en) | 2016-08-07 | 2016-08-07 | The quickly kit of detection tumor tissues 2-HG accumulations |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610637388.5A CN108303536A (en) | 2016-08-07 | 2016-08-07 | The quickly kit of detection tumor tissues 2-HG accumulations |
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| CN108303536A true CN108303536A (en) | 2018-07-20 |
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| CN201610637388.5A Pending CN108303536A (en) | 2016-08-07 | 2016-08-07 | The quickly kit of detection tumor tissues 2-HG accumulations |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647056A (en) * | 2020-06-23 | 2020-09-11 | 山东大学 | L-2-hydroxyglutaric acid biosensor based on specific transcription regulatory factor and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| US20140314728A1 (en) * | 2011-11-18 | 2014-10-23 | Memorial Sloan Kettering Cancer Center | 2-hydroxyglutarate as a biomarker for chronic hypoxia |
| CN104515852A (en) * | 2013-09-29 | 2015-04-15 | 上海交通大学医学院附属瑞金医院 | Use of 2-hydroxyglutaric acid or detection reagent thereof |
-
2016
- 2016-08-07 CN CN201610637388.5A patent/CN108303536A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120040383A1 (en) * | 2010-08-12 | 2012-02-16 | Wei Jia | Methods and Kits Relating To Metabolite Biomarkers For Colorectal Cancer |
| US20140314728A1 (en) * | 2011-11-18 | 2014-10-23 | Memorial Sloan Kettering Cancer Center | 2-hydroxyglutarate as a biomarker for chronic hypoxia |
| CN104515852A (en) * | 2013-09-29 | 2015-04-15 | 上海交通大学医学院附属瑞金医院 | Use of 2-hydroxyglutaric acid or detection reagent thereof |
Non-Patent Citations (4)
| Title |
|---|
| DANG ET AL.: "Cancer-associated IDH1 mutations produce 2-hydroxyglutarate", 《NATURE》 * |
| FELIX SAHM ET AL.: "Detection of 2-Hydroxyglutarate in Formalin-Fixed Paraffin-Embedded Glioma Specimens by Gas Chromatography/Mass Spectrometry", 《BRAIN PATHOLOGY》 * |
| QING-YUN CHENG ET AL.: "Sensitive Determination of Onco-metabolites of D- and L-2-hydroxyglutarate Enantiomers by Chiral Derivatization Combined with Liquid Chromatography/Mass Spectrometry Analysis", 《SCIENTIFIC REPORTS》 * |
| WARD PATRICK S.: "The Common Feature of Leukemia-Associated IDH1 and IDH2 Mutations Is a Neomorphic Enzyme Activity Converting a-Ketoglutarate to 2-Hydroxyglutarate", 《CANCER CELL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647056A (en) * | 2020-06-23 | 2020-09-11 | 山东大学 | L-2-hydroxyglutaric acid biosensor based on specific transcription regulatory factor and application thereof |
| CN111647056B (en) * | 2020-06-23 | 2021-11-23 | 山东大学 | L-2-hydroxyglutaric acid biosensor based on specific transcription regulatory factor and application thereof |
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