CN106442995A - Lung cancer screening check test kit - Google Patents
Lung cancer screening check test kit Download PDFInfo
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- CN106442995A CN106442995A CN201610868210.1A CN201610868210A CN106442995A CN 106442995 A CN106442995 A CN 106442995A CN 201610868210 A CN201610868210 A CN 201610868210A CN 106442995 A CN106442995 A CN 106442995A
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- Prior art keywords
- reagent
- musk
- phosphorylating
- lung cancer
- expression level
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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
Abstract
The invention discloses lung cancer screening check test kit, including the use of random selected reagent for measuring non phosphorylation MUSK protein expression level. The invention also discloses the application of reagent for measuring non phosphorylation MUSK protein expression level in preparing lung cancer screening check test kit. The check test kit through measuring non phosphorylation MUSK expression level, examines the risk of examinee suffering from cancer, the kit applies in assisting diagnoses of clinical cancer, adopts related treatment measure for patient or provides effective bases for decision making, possesses excellent clinical application prospect.
Description
Technical field
The present invention relates to a kind of screening lung cancer test kit.
Background technology
Pulmonary carcinoma is one of modal malignant tumor in the world, and its M ＆ M is in ascendant trend year by year, at present
Sickness rate occupies first place in the world, and seriously threatens human health and life.
The cause of disease complexity of pulmonary carcinoma is it is considered that influence factor includes：1. smoking；2. environmental pollution：As haze, indoor dress
Repair；3. bad life style：As poor in dietary habit, life stress is big；4. chronic lung disease：As pulmonary tuberculosis, pneumoconiosis, pneumosilicosis,
Chronic bronchitiss；5. human body intrinsic factor：As Inheritance, immune function reduction, endocrine function imbalance etc..
Meanwhile, pulmonary carcinoma is a kind of disease be good at and hiding, and often develops into late period in disease and just shows clinical symptoms, 70
～80% patients with lung cancer when being diagnosed to be with Lung Cancer Symptoms be in, late period, cancerous cell has spread, and misses and most preferably controls
More opportunity, five year survival rate is low.For the patients with lung cancer of early stage, through timely treatment be greatly improved 5 years of patient and more than
Survival rate and life quality.Therefore early diagnosiss 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 those, before symptom
Find pulmonary carcinoma in time.If pulmonary carcinoma molecular marker can be found, for pointing out clinician's early stage that patient is taken with correlation
Remedy measures or decision-making have great importance.
MUSK (Muscle-specific tyrosine-protein kinase receptor, MuSK), Chinese
For muscle specific receptor tyrosine kinase, it is one of receptor tyrosine kinase family member, mainly in muscle cell surface table
Reach, the transmission to the reaching maturity of muscle cell joint, Electrophysiology signal plays an important role.At present, have no that MUSK exists
The report of Expressions in Lung Cancer situation.
Content of the invention
Pulmonary carcinoma is studied in detail inventor it was found that non-phosphorylating MUSK albumen (non-phosphorylated
MUSK) can be used as its molecular marker.Wherein, expression in lung tissue for the non-phosphorylating MUSK and pulmonary carcinoma are in positive
Close.Therefore, by detecting the expression of non-phosphorylating MUSK in lung tissue, the risk that person to be checked suffers from pulmonary carcinoma can be predicted.
Accordingly, the invention provides a kind of screening lung cancer test kit, and the expression water of detection non-phosphorylating MUSK albumen
Purposes in preparing screening lung cancer reagent for the flat reagent.
The screening lung cancer test kit of the present invention, it include optional for detecting non-phosphorylating MUSK protein expression level
Reagent.
Wherein, described reagent is the reagent for detecting non-phosphorylating MUSK protein expression level in lung tissue.
Wherein, the reagent of described detection non-phosphorylating MUSK protein expression level is method for immunohistochemical detection reagent.
Wherein, the reagent of described detection non-phosphorylating MUSK protein expression level is that Western Blot or ELISA detects
Method reagent.
Present invention also offers screening lung cancer reagent prepared by the reagent of detection non-phosphorylating MUSK protein expression level
In purposes.
Wherein, described reagent is the reagent for detecting non-phosphorylating MUSK protein expression level in lung tissue.
Wherein, the reagent of described detection non-phosphorylating MUSK protein expression level is method for immunohistochemical detection reagent.
Wherein, the reagent of described detection non-phosphorylating MUSK protein expression level is that Western Blot or ELISA detects
Method reagent.
Test kit of the present invention passes through to detect the non-phosphorylating MUSK table with normal portions for the pulmonary abnormalities position of lung exception person
Reach level it can be determined that the non-phosphorylating MUSK expression difference of lung exception person, and then judge the risk that person to be checked suffers from pulmonary carcinoma：
If the expression of non-phosphorylating MUSK is high, the risk suffering from pulmonary carcinoma is high, if the expression of non-phosphorylating MUSK is low, suffers from lung
The risk of cancer is low, can be used for the auxiliary diagnosis of clinical pulmonary carcinoma, and potential applicability in clinical practice is good.
Obviously, the above according to the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the premise of the present invention above-mentioned basic fundamental thought, modification, replacement or the change of other various ways can also be made.
The specific embodiment of form by the following examples, remakes further specifically to the above of the present invention
Bright.But this scope being interpreted as the above-mentioned theme of the present invention should not be only limitted to Examples below.All based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Specific embodiment
Embodiment 1 non-phosphorylating MUSK expression and the relation of pulmonary carcinoma
First, experimental technique
1st, clinical data
Choose patients with lung cancer paraffin section 20, far-end compares 20 and (refers to the far-end normal lung tissue paraffin of patients with lung cancer
Section, apart from 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.
2nd, the detection of non-phosphorylating MUSK expression
Paraffin section is compareed to patients with lung cancer paraffin section, remote organization, carries out the SABC with MUSK as index
(IHC).
MUSK antibody：Purchased from Affinity Biosciences, article No. DF7040；
EnVisionTMHRP bis- resists, antibody diluent, DAB developer, antigen retrieval buffers, Wash Buffer, SABC
Pen：It is purchased from Dako company of Denmark.
Detect the expression of non-phosphorylating MUSK as follows：
(1) close peroxidase：Paraffin section is taken to be dewaxed, dewaxing, (dewaxing process is as follows to aquation：Dimethylbenzene I
10min → dimethylbenzene II 10min → dehydrated alcohol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min)
Afterwards, 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, in 98 DEG C of water-baths
Middle water-bath 35min, carries out antigen retrieval；
(4) distilled water rinsing：Take out antigen repairing box, after naturally cooling to room temperature, washed three times with distillation, then use Wash
Buffer rinse is once；
(5) incubation one resists：With SABC pen, tissue is enclosed, in circle, Deca MUSK antibody diluent (resists for MUSK
Body and antibody diluent mixed preparing, the two ratio is MUSK/ antibody diluent=1/150), 4 DEG C of overnight incubation；
(6) distilled water rinsing：With distillation washing three times, Wash Buffer rinse is once；
(7) Deca two resists：Two anti-working solutions (i.e. two antigen liquid) of Deca Dako HRP labelling, incubated at room 60min；
(8) distilled water rinsing：Distillation washing 3 times, each 5min；
(9) DAB colour developing：Deca chromogenic substrate DAB, examines under a microscope colour developing situation, terminating reaction in distilled water；
(10) haematoxylin is redyed：Dyeing 2min in haematoxylin dye liquor is put in section, after 1% hydrochloride alcohol differentiation, flowing water
Rinse 10min；
(11) dehydration is transparent：Transparent through 80%, 95%, 100% gradient alcohol dehydration, dimethylbenzene, it is 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, the tumor cell staining power according to patients with lung cancer and remote organization and positive area give SABC
Scoring.
3rd, SABC standards of grading
Tumor cell staining power * positive tumor cell area=0-9 divides,
Result counts：Negative：0 point；Low positive：1-3 divides；High positive>3 points.
Wherein, the scoring of tumor cell staining power, positive area@As follows：
@Note：Independently immunohistochemical staining result is scored by two veteran pathologist
4th, interpretation of result
Statistical analysis are carried out using SPSS17.0 cancerous lung tissue group and remote organization's matched group.
2nd, experimental result
During cancerous lung tissue is compareed with remote organization, the expression testing result of non-phosphorylating MUSK is shown in Table 2.
The expression of table 2 non-phosphorylating MUSK
From table 2, in far-end normal structure, the positive expression rate of non-phosphorylating MUSK is 10%, High positive expression rate
For 0%；Non-phosphorylating MUSK positive expression rate in cancerous lung tissue is 95%, and High positive expression rate is 85%；Non-phosphorylating
MUSK significantly raises in cancerous lung tissue, and compared with far-end normal structure, non-phosphorylating MUSK expression difference has statistics
Learn meaning (P<0.001).
As can be seen from the above results, compared with Ai Pang normal lung tissue, the non-phosphorylating MUSK expression water of cancerous lung tissue
HUD writes and raises (P<0.0001), illustrate that pulmonary carcinoma is proportionate with non-phosphorylating MUSK expression, the height of non-phosphorylating MUSK
Expression can significantly improve the probability suffering from pulmonary carcinoma.Non-phosphorylating MUSK level due to Ai Pang normal lung tissue can reflect normal person
The non-phosphorylating MUSK level of lung tissue, therefore, it can the expression of the non-phosphorylating MUSK by detecting person to be checked, by lung
Susceptible population's examination of cancer is out.
Embodiment 2 present invention detects kit forms and its using method of non-phosphorylating MUSK expression
First, the composition of immunologic combined detection reagent kit
Detection kit (50 person-portion)：
| Component | Volume |
| Non-phosphorylating MUSK antibody | 50μl |
| EnVisionTMHRP bis- resists | 5ml |
| Antibody diluent | 5ml |
| DAB developer | 5ml |
| Antigen retrieval buffers | 1000ml |
| Wash Buffer* | 1000ml |
| SABC pen | 1 |
Note：This test kit is based on 100 μ l by every section all kinds of experiment reagent of Deca, and concrete consumption can be according to tissue size
Suitably increase and decrease.
2nd, the using method of test kit
By sample lung tissue to be checked, prepare paraffin section, as detection specimen, detect non-phosphorylating as follows
The expression of MUSK：
(1) close peroxidase：Paraffin section is taken to be dewaxed, dewaxing, (dewaxing process is as follows to aquation：Dimethylbenzene I
10min → dimethylbenzene II 10min → dehydrated alcohol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min)
Afterwards, 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, in 98 DEG C of water-baths
Middle water-bath 35min, carries out antigen retrieval；
(4) distilled water rinsing：Take out antigen repairing box, after naturally cooling to room temperature, washed three times with distillation, then use Wash
Buffer rinse is once；
(5) incubation one resists：With SABC pen, tissue is enclosed, in circle, Deca MUSK antibody diluent (resists for MUSK
Body and antibody diluent mixed preparing, the two ratio is MUSK/ antibody diluent=1/150), 4 DEG C of overnight incubation；
(6) distilled water rinsing：With distillation washing three times, Wash Buffer rinse is once；
(7) Deca two resists：Two anti-working solutions (i.e. two antigen liquid) of Deca Dako HRP labelling, incubated at room 60min；
(8) distilled water rinsing：Distillation washing 3 times, each 5min；
(9) DAB colour developing：Deca chromogenic substrate DAB, examines under a microscope colour developing situation, terminating reaction in distilled water；
(10) haematoxylin is redyed：Dyeing 2min in haematoxylin dye liquor is put in section, after 1% hydrochloride alcohol differentiation, flowing water
Rinse 10min；
(11) dehydration is transparent：Transparent through 80%, 95%, 100% gradient alcohol dehydration, dimethylbenzene, it is 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, the tumor cell staining power according to patients with lung cancer and remote organization and positive area give SABC
Scoring.
To pulmonary abnormalities person, abnormal position, normal portions tissue can be taken respectively, compare non-phosphorylating MUSK expression,
And then evaluate the probability that it suffers from pulmonary carcinoma, as the auxiliary diagnosis means of clinical pulmonary carcinoma.
To sum up, test kit of the present invention passes through to detect the expression of non-phosphorylating MUSK it can be determined that the lung of lung exception person
Portion's abnormal position and the non-phosphorylating MUSK expression difference of normal portions, and then examination person to be checked suffers from the risk of pulmonary carcinoma：If
The expression of non-phosphorylating MUSK is high, then the risk suffering from pulmonary carcinoma is high, if the expression of non-phosphorylating MUSK is low, suffers from pulmonary carcinoma
Risk low, can be used for the auxiliary diagnosis of clinical pulmonary carcinoma, take the remedy measures of correlation or decision-making to provide effectively for patient
Foundation, potential applicability in clinical practice is good.
Claims (8)
1. a kind of screening lung cancer test kit it is characterised in that：It include optional for detecting non-phosphorylating MUSK protein expression
The reagent of level.
2. kit for screening according to claim 1 it is characterised in that：Described reagent is for detecting non-phosphorus in lung tissue
The reagent of acidifying MUSK protein expression level.
3. kit for screening according to claim 1 and 2 it is characterised in that：Described detection non-phosphorylating MUSK albumen table
The reagent reaching level is method for immunohistochemical detection reagent.
4. kit for screening according to claim 1 and 2 it is characterised in that：Described detection non-phosphorylating MUSK albumen table
The reagent reaching level is Western Blot or ELISA detection method reagent.
5. purposes in preparing screening lung cancer reagent for the reagent of detection non-phosphorylating MUSK protein expression level.
6. purposes according to claim 5 it is characterised in that：Described reagent is for detecting non-phosphorylating in lung tissue
The reagent of MUSK protein expression level.
7. the purposes according to claim 5 or 6 it is characterised in that：Described detection non-phosphorylating MUSK protein expression level
Reagent be method for immunohistochemical detection reagent.
8. the purposes according to claim 5 or 6 it is characterised in that：Described detection non-phosphorylating MUSK protein expression level
Reagent be Western Blot or ELISA detection method reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610868210.1A CN106442995A (en) | 2016-09-30 | 2016-09-30 | Lung cancer screening check test kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610868210.1A CN106442995A (en) | 2016-09-30 | 2016-09-30 | Lung cancer screening check test kit |
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| Publication Number | Publication Date |
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| CN106442995A true CN106442995A (en) | 2017-02-22 |
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| CN201610868210.1A Pending CN106442995A (en) | 2016-09-30 | 2016-09-30 | Lung cancer screening check test kit |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007056244A2 (en) * | 2005-11-04 | 2007-05-18 | Merck & Co., Inc. | Methods of using saha and erlotinib for treating cancer |
| WO2007013665A3 (en) * | 2005-07-27 | 2007-07-05 | Oncotherapy Science Inc | Method of diagnosing small cell lung cancer |
| CN101573380A (en) * | 2006-10-12 | 2009-11-04 | 健泰科生物技术公司 | Antibodies to lymphotoxin-alpha |
-
2016
- 2016-09-30 CN CN201610868210.1A patent/CN106442995A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007013665A3 (en) * | 2005-07-27 | 2007-07-05 | Oncotherapy Science Inc | Method of diagnosing small cell lung cancer |
| WO2007056244A2 (en) * | 2005-11-04 | 2007-05-18 | Merck & Co., Inc. | Methods of using saha and erlotinib for treating cancer |
| CN101573380A (en) * | 2006-10-12 | 2009-11-04 | 健泰科生物技术公司 | Antibodies to lymphotoxin-alpha |
Non-Patent Citations (3)
| Title |
|---|
| ZHEN G. LUO ET AL.: "Regulation of AChR Clustering by Dishevelled Interacting with MuSK and PAK1", 《NEURON》 * |
| 刘岚剑 等: "重症肌无力合并胸腺病变患者血清乙酰胆碱受体抗体、肌联蛋白抗体和肌肉特异性酪氨酸激酶抗体的检测意义", 《临床荟萃》 * |
| 刘文雯 等: "肌肉特异性激酶基因的克隆、表达和纯化", 《生物技术》 * |
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Application publication date: 20170222 |