CN106153920A - A kind of screening lung cancer test kit - Google Patents
A kind of screening lung cancer test kit Download PDFInfo
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- CN106153920A CN106153920A CN201610596264.7A CN201610596264A CN106153920A CN 106153920 A CN106153920 A CN 106153920A CN 201610596264 A CN201610596264 A CN 201610596264A CN 106153920 A CN106153920 A CN 106153920A
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- ephb1
- reagent
- protein expression
- lung cancer
- 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
- G01N33/57423—Specifically defined cancers of lung
-
- 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
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
Abstract
The invention discloses a kind of screening lung cancer test kit, it includes the optional reagent for detecting p EphB1 protein expression level.The invention also discloses the reagent detecting p EphB1 protein expression level purposes in preparing screening lung cancer reagent.Test kit of the present invention is by detecting the expression of p EphB1 albumen, it can be determined that person to be checked suffers from the risk of pulmonary carcinoma, can be used for the auxiliary diagnosis of clinical pulmonary carcinoma, takes the remedy measures being correlated with or decision-making to provide effective foundation for patient, and potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to a kind of screening lung cancer test kit.
Background technology
Pulmonary carcinoma is one of the most modal malignant tumor, and its M & M is in ascendant trend year by year, at present
Sickness rate occupies first place in the world, and human health and life in serious threat.
The cause of disease of pulmonary carcinoma is complicated, it is considered that influence factor includes: 1. smoking；2. environmental pollution: such as haze, indoor dress
Repair；3. bad life style: as poor in dietary habit, life stress is big；4. chronic lung disease: such as pulmonary tuberculosis, pneumoconiosis, pneumosilicosis,
Chronic bronchitis；5. human body intrinsic factor: such as Inheritance, immune function reduction, endocrine function imbalance etc..
Meanwhile, pulmonary carcinoma is a kind of disease being good at concealment, often develops into just show clinical symptoms late period in disease, 70
～80% patients with lung cancer when being diagnosed to be and suffering from Lung Cancer Symptoms be in, late period, cancerous cell has spread, and misses and most preferably controls
More on opportunity, five year survival rate is low.For patients with lung cancer in early days, through treatment in time be greatly improved 5 years of patient and more than
Survival rate and life quality.Therefore the early diagnosis of pulmonary carcinoma and to carry out effective examination most important.
The examination of pulmonary carcinoma, refers to do not have the related indication crowd of pulmonary carcinoma to carry out routine physical examination, before there is symptom those
Find pulmonary carcinoma in time.If pulmonary carcinoma molecular marker can be found, it is correlated with for pointing out clinician in early days patient to be taked
Remedy measures or decision-making have great importance.
Summary of the invention
In order to solve the problems referred to above, pulmonary carcinoma is studied in detail by inventor, it was found that phosphorylation EphB1 albumen
(phosphorylated EphB1, p-EphB1) can be as its molecular marker.Wherein, p-EphB1 albumen is in lung tissue
Expression be proportionate with pulmonary carcinoma.Therefore, by the expression of p-EphB1 albumen in detection lung tissue, can predict
Person to be checked suffers from the risk of pulmonary carcinoma.
Accordingly, the invention provides a kind of screening lung cancer test kit, and the examination of the expression of detection p-EphB1 albumen
Agent purposes in preparing screening lung cancer reagent.
The screening lung cancer test kit of the present invention, it includes the optional reagent for detecting p-EphB1 protein expression level.
Wherein, described reagent is for detecting the reagent of p-EphB1 protein expression level in lung tissue.
Wherein, the reagent of described detection p-EphB1 protein expression level is method for immunohistochemical detection reagent.
Wherein, the reagent of described detection p-EphB1 protein expression level is Western Blot or ELISA detection method is used
Reagent.
Present invention also offers the reagent detecting p-EphB1 protein expression level use in preparing screening lung cancer reagent
On the way.
Wherein, described reagent is for detecting the reagent of p-EphB1 protein expression level in lung tissue.
Wherein, the reagent of described detection p-EphB1 protein expression level is method for immunohistochemical detection reagent.
Wherein, the reagent of described detection p-EphB1 protein expression level is Western Blot or ELISA detection method is used
Reagent.
EphB1, i.e. Eph receptor B1, is one of the member of Eph receptor family, is a kind of tyrosine kinase receptor, participates in letter
The Phosphorylation events of protein molecule in number transductive process.At present, the research for EphB1 focuses mostly at it neural
Grow and the transduction aspect of pain signal, see the document of Hafner C about the research of EphB1 Yu relationship between lung cancer,
Differential Gene Expression of Eph Receptors and Ephrins in Benign Human
Tissues and Cancers, points out in document, and it is the most right that total EphB1mRNA content in lung squamous cell carcinoma cancers is substantially less than
According to.And, currently without the report about EphB1 albumen Yu the relation of pulmonary carcinoma, more not about phosphorylation EphB1 albumen with
The report of the relation of pulmonary carcinoma (p-EphB1).
The p-EphB1 protein expression at the test kit of the present invention pulmonary abnormalities position with normal portions by detecting lung exception person
Level, it can be determined that the p-EphB1 protein expression level difference of lung exception person, and then judge the risk that person to be checked suffers from pulmonary carcinoma: if
The expression of p-EphB1 albumen is high, then the risk suffering from pulmonary carcinoma is high, if the expression of p-EphB1 albumen is low, then suffers from pulmonary carcinoma
Risk is low, can be used for the auxiliary diagnosis of clinical pulmonary carcinoma, and potential applicability in clinical practice is good.
Obviously, according to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the present invention above-mentioned basic fundamental thought premise, it is also possible to make the amendment of other various ways, replace or change.
The detailed description of the invention of form by the following examples, remakes the most specifically the foregoing of the present invention
Bright.But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below.All based on foregoing of the present invention
The technology realized belongs to the scope of the present invention.
Detailed description of the invention
Embodiment 1p-EphB1 expression and the relation of pulmonary carcinoma
One, experimental technique
1, clinical data
Choosing patients with lung cancer paraffin section 25 example, far-end compares 16 examples and (refers to the far-end normal lung tissue paraffin of patients with lung cancer
Section, distance cancerous tissue 5cm), essential information is shown in Table 1.
Table 1 clinical elementary information
*Note: other include large cell carcinoma, adenosquamous carcinoma, small cell carcinoma.
2, the detection of p-EphB1 expression
To patients with lung cancer paraffin section, remote organization's comparison paraffin section, carry out the SABC with p-EphB1 as index
(IHC)。
P-EphB1 antibody: purchased from Beijing Bo Aosen Bioisystech Co., Ltd, article No. bs-10781R；
PLSCONFM the most following reagent:
EnVisionTMHRP bis-anti-, antibody diluent, DAB developer, antigen retrieval buffers, Wash Buffer, immune group
Change pen: be purchased from Dako company of Denmark.
Detect the expression of p-EphB1 as follows:
(1) closing peroxidase: take paraffin section and dewax, to aquation, (dewaxing process is as follows: dimethylbenzene I in dewaxing
10min → dimethylbenzene II 10min → dehydrated alcohol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min)
After, use 3%H2O2Solution closes endogenous peroxydase, incubated at room 15min in dark place；
(2) distilled water rinsing: section is placed in distilled water, shaking table is washed 5min/ time, totally three times；
(3) antigen retrieval: add the sodium citrate solution of Tris-EDTA or pH 6.0 of pH 8.0, at 95 DEG C of water-baths
Middle water-bath 50min, carries out antigen retrieval；
(4) distilled water rinsing: take out section, after naturally cooling to room temperature, washes three times with distillation, then uses Wash
Buffer rinse is once；
(5) hatch one to resist: enclosed by tissue with SABC pen, in circle, drip p-EphB1 antibody diluent (for p-
EphB1 antibody and antibody diluent mixed preparing, the two ratio is 1:100), 4 DEG C of overnight incubation；
(6) distilled water rinsing: with distillation washing three times, Wash Buffer rinse is once；
(7) dropping two resists: two anti-working solutions of dropping Dako HRP labelling (resist to mix with antibody diluent for HRP bis-and join
System, the two mixes for 1:1), incubated at room 60min；
(8) distilled water rinsing: distillation washing 3 times, each 5min；
(9) DAB colour developing: dropping chromogenic substrate DAB, examines under a microscope colour developing situation, terminates reaction in distilled water；
(10) haematoxylin is redyed: section is put into dyeing 2min in haematoxylin dye liquor, after 1% hydrochloride alcohol differentiation, and flowing water
Rinse 10min；
(11) be dehydrated transparent: transparent through 80%, 95%, 100% gradient alcohol dehydration, dimethylbenzene, be dried in ventilating kitchen；
(12) mounting: with neutral Instant cement mounting, observe under an optical microscope, DP Controller image acquisition system
System adopts figure.After adopting figure, tumor cell staining power and positive area according to patients with lung cancer and remote organization give SABC
Scoring.
3, SABC standards of grading
Tumor cell staining power * positive tumor cell area=0-9 divides,
Result is added up: negative: 0 point；Low positive: 1-3 divides；High positive > 3 points.
Wherein, tumor cell staining power, positive area scoring as follows:
@
Note: tumor cell staining power result is marked by two veteran pathologist are independent
4, interpretation of result
SPSS17.0 cancerous lung tissue group and remote organization's matched group is used to carry out statistical analysis.
Two, experimental result
During cancerous lung tissue compares with remote organization, the expression testing result of p-EphB1 is shown in Table 2.
The expression of table 2p-EphB1
From table 2, in far-end normal structure, the positive expression rate of p-EphB1 is 56.3%, and High positive expression rate is
0；P-EphB1 positive expression rate in cancerous lung tissue is 92%, and High positive expression rate is 72%；P-EphB1 is at cancerous lung tissue
In significantly raise, compared with far-end normal structure, p-EphB1 expression difference has statistical significance (P < 0.001).
As can be seen from the above results, compared with Ai Pang normal lung tissue, the p-EphB1 expression of cancerous lung tissue is notable
Raising (P < 0.001), illustrate that pulmonary carcinoma is proportionate with p-EphB1 expression, the high expressed of p-EphB1 can significantly improve suffers from lung
The probability of cancer.Owing to the p-EphB1 level of Ai Pang normal lung tissue can reflect the p-EphB1 level of Normal Lung tissue, because of
This, can be by detecting the expression of the p-EphB1 of person to be checked, by Susceptible population's examination of pulmonary carcinoma out.
Embodiment 2 present invention detects test kit composition and the using method thereof of p-EphB1 expression
One, the composition of immunologic combined detection reagent kit
Detection kit (50 person-portion):
| Component | Volume |
| P-EphB1 antibody | 30μl |
| EnVisionTMHRP bis-resists | 100μl |
| Antibody diluent | 10ml |
| DAB developer | 10ml |
| Antigen retrieval buffers | 500ml |
| Wash Buffer* | 500ml |
| SABC pen | 1 |
Note: it is based on 100 μ l that this test kit drips all kinds of experiment reagents by every section, concrete consumption can be according to tissue size
Suitably increase and decrease.
Two, the using method of test kit
By sample lung tissue to be checked, prepare paraffin section, as detection specimen, detect p-EphB1's as follows
Expression:
Detect the expression of p-EphB1 as follows:
(1) closing peroxidase: take paraffin section and dewax, to aquation, (dewaxing process is as follows: dimethylbenzene I in dewaxing
10min → dimethylbenzene II 10min → dehydrated alcohol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min)
After, use 3%H2O2Solution closes endogenous peroxydase, incubated at room 15min in dark place；
(2) distilled water rinsing: section is placed in distilled water, shaking table is washed 5min/ time, totally three times；
(3) antigen retrieval: add the sodium citrate solution of Tris-EDTA or pH 6.0 of pH 8.0, at 95 DEG C of water-baths
Middle water-bath 50min, carries out antigen retrieval；
(4) distilled water rinsing: take out section, after naturally cooling to room temperature, washes three times with distillation, then uses Wash
Buffer rinse is once；
(5) hatch one to resist: enclosed by tissue with SABC pen, in circle, drip p-EphB1 antibody diluent (for p-
EphB1 antibody and antibody diluent mixed preparing, the two ratio is 1:100), 4 DEG C of overnight incubation；
(6) distilled water rinsing: with distillation washing three times, Wash Buffer rinse is once；
(7) dropping two resists: two anti-working solutions of dropping Dako HRP labelling (resist to mix with antibody diluent for HRP bis-and join
System, the two mixes for 1:1), incubated at room 60min；
(8) distilled water rinsing: distillation washing 3 times, each 5min；
(9) DAB colour developing: dropping chromogenic substrate DAB, examines under a microscope colour developing situation, terminates reaction in distilled water；
(10) haematoxylin is redyed: section is put into dyeing 2min in haematoxylin dye liquor, after 1% hydrochloride alcohol differentiation, and flowing water
Rinse 10min；
(11) be dehydrated transparent: transparent through 80%, 95%, 100% gradient alcohol dehydration, dimethylbenzene, be dried in ventilating kitchen；
(12) mounting: with neutral Instant cement mounting, observe under an optical microscope, DP Controller image acquisition system
System adopts figure.After adopting figure, tumor cell staining power and positive area according to patients with lung cancer and remote organization give SABC
Scoring.
To pulmonary abnormalities person, abnormal position, normal portions tissue can be taken respectively, compare p-EphB1 expression, Jin Erping
Valency its suffer from the probability of pulmonary carcinoma, as the auxiliary diagnostic means of clinical pulmonary carcinoma.
To sum up, test kit of the present invention is by detecting the expression of p-EphB1, it can be determined that the pulmonary abnormalities of lung exception person
The p-EphB1 expression difference of position and normal portions, and then examination person to be checked suffers from the risk of pulmonary carcinoma: if the table of p-EphB1
Reach level high, then the risk suffering from pulmonary carcinoma is high, if the expression of p-EphB1 is low, then the risk suffering from pulmonary carcinoma is low, can be used for clinical lung
The auxiliary diagnosis of cancer, takes the remedy measures being correlated with or decision-making to provide effective foundation for patient, and potential applicability in clinical practice is good.
Claims (8)
1. a screening lung cancer test kit, it is characterised in that: it includes optional for detecting phosphorylation EphB1 protein expression water
Flat reagent.
Kit for screening the most according to claim 1, it is characterised in that: described reagent is for detecting phosphoric acid in lung tissue
Change the reagent of EphB1 protein expression level.
Kit for screening the most according to claim 1 and 2, it is characterised in that: described detection phosphorylation EphB1 protein expression
The reagent of level is method for immunohistochemical detection reagent.
Kit for screening the most according to claim 1 and 2, it is characterised in that: described detection phosphorylation EphB1 protein expression
The reagent of level is Western Blot or ELISA detection method reagent.
5. the reagent of detection phosphorylation EphB1 protein expression level purposes in preparing screening lung cancer reagent.
Purposes the most according to claim 5, it is characterised in that: described reagent is for detecting phosphorylation in lung tissue
The reagent of EphB1 protein expression level.
7. according to the purposes described in claim 5 or 6, it is characterised in that: described detection phosphorylation EphB1 protein expression level
Reagent is method for immunohistochemical detection reagent.
8. according to the purposes described in claim 5 or 6, it is characterised in that: described detection phosphorylation EphB1 protein expression level
Reagent is Western Blot or ELISA detection method reagent.
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| CN106153920B CN106153920B (en) | 2018-04-27 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006081418A2 (en) * | 2005-01-27 | 2006-08-03 | The Burnham Institute | Ephb receptor-binding peptides |
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| WO2007149523A2 (en) * | 2006-06-20 | 2007-12-27 | Metaproteomics, Llc | Hexahydro-isoalpha acid based protein kinase modulation cancer treatment |
| CN104039798A (en) * | 2011-12-30 | 2014-09-10 | 韩美药品株式会社 | Thieno[3,2-d]pyrimidine derivatives having inhibitory activity for protein kinases |
-
2016
- 2016-07-25 CN CN201610596264.7A patent/CN106153920B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852974A (en) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | Compositions and methods for treating and diagnosing cancer |
| WO2006081418A2 (en) * | 2005-01-27 | 2006-08-03 | The Burnham Institute | Ephb receptor-binding peptides |
| WO2007149523A2 (en) * | 2006-06-20 | 2007-12-27 | Metaproteomics, Llc | Hexahydro-isoalpha acid based protein kinase modulation cancer treatment |
| CN101505743A (en) * | 2006-06-20 | 2009-08-12 | 麦特普罗泰欧米克斯有限公司 | Hexahydro-isoalpha acid based protein kinase modulation cancer treatment |
| CN104039798A (en) * | 2011-12-30 | 2014-09-10 | 韩美药品株式会社 | Thieno[3,2-d]pyrimidine derivatives having inhibitory activity for protein kinases |
Non-Patent Citations (2)
| Title |
|---|
| CATHERINE WILSON ET AL: "Overcoming EMT-associated resistance to anti-cancer drugs via Src-FAK pathway inhibition", 《ONCOTARGET》 * |
| KLARISA RIKOVA ET AL: "Global Survey of Phosphotyrosine Signaling Identifies Oncogenic Kinases in Lung Cancer", 《CELL》 * |
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