CN105759056B - A kind of screening lung cancer kit - Google Patents
A kind of screening lung cancer kit Download PDFInfo
- Publication number
- CN105759056B CN105759056B CN201610210301.6A CN201610210301A CN105759056B CN 105759056 B CN105759056 B CN 105759056B CN 201610210301 A CN201610210301 A CN 201610210301A CN 105759056 B CN105759056 B CN 105759056B
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- reagent
- cerberus
- expressions
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
Abstract
The invention discloses a kind of screening lung cancer kit, and it includes the optional reagent for being used to detect Cerberus expressions.The invention also discloses purposes of the reagent of detection Cerberus expressions in screening lung cancer reagent is prepared.Kit of the present invention is by detecting Cerberus expression, it can be determined that crowd to be checked suffers from the risk of lung cancer, available for the auxiliary diagnosis of clinical lung cancer, takes the remedy measures of correlation or decision-making provides effective foundation, potential applicability in clinical practice good for patient.
Description
Technical field
The present invention relates to a kind of screening lung cancer kit.
Background technology
Lung cancer is one of most common malignant tumour in the world, and its morbidity and mortality is in ascendant trend year by year, at present
The incidence of disease occupies first place in the world, and serious threat human health and life.
The cause of disease of lung cancer 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 eating habit is poor, life stress is big；4. chronic lung disease：Such as pulmonary tuberculosis, pneumoconiosis, silicosis,
Chronic bronchitis；5. human body internal factor：Such as familial inheritance, immunity function reduce, endocrine function imbalance.
Meanwhile lung cancer is a kind of disease for being good at concealment, often develops into late period in disease and just show clinical symptoms, 70
~80% patients with lung cancer has been middle and advanced stage when being diagnosed to be and suffering from Lung Cancer Symptoms, and cancer cell has spread, and misses and most preferably controls
More opportunity, five year survival rate are low.For the patients with lung cancer of early stage, by treatment in time be greatly improved 5 years of patient and more than
Survival rate and life quality.Therefore the early diagnosis and the effective examination of progress of lung cancer are most important.
The examination of lung cancer, refer to there is no the related indication crowd of lung cancer to carry out routine physical examination those, before there is symptom
Lung cancer is found in time.If the lung cancer molecular marker inside blood can be found, for prompting clinician's early stage to patient
The remedy measures or decision-making for taking correlation have great importance.
Cerberus, also known as Cerberus 1, CER-1, it is isolated out of African toad body earliest, is a kind of Wnt
Signal path antagonist, Cerberus with Wnt albumen by being directly connected so as to prevent Wnt from being connected with receptor protein compound.
Have no the report related to lung cancer on Cerberus at present.
The content of the invention
In order to solve the above problems, lung cancer is studied in detail inventor, it was found that Cerberus can be used as it
Molecular marker.Wherein, expressions of the Cerberus in serum is proportionate with lung cancer.Therefore, by detecting in serum
Cerberus expression, the risk that crowd to be checked suffers from lung cancer can be predicted.
Accordingly, the invention provides a kind of screening lung cancer kit, and the reagent of detection Cerberus expression
Purposes in screening lung cancer reagent is prepared.
The screening lung cancer kit of the present invention, it includes the optional reagent for being used to detect Cerberus expressions.
Wherein, the reagent is the reagent for detecting Cerberus expressions in serum.
Wherein, the reagent of the detection Cerberus expressions is protein chip detection method reagent.
Wherein, the reagent of the detection Cerberus expressions is ELISA detection method reagent or Western
Blot detection method reagents.
Present invention also offers purposes of the reagent of detection Cerberus expressions in screening lung cancer reagent is prepared.
Wherein, the reagent is the reagent for detecting Cerberus expressions in serum.
Wherein, the reagent of the detection Cerberus expressions is protein chip detection method reagent.
Wherein, the reagent of the detection Cerberus expressions is ELISA detection method reagent or Western
Blot detection method reagents.
Kit of the present invention is by detecting Cerberus expression, it can be determined that crowd to be checked suffers from the risk of lung cancer：
If Cerberus expression is high, suffers from the risk height of lung cancer, if Cerberus expression is low, suffer from the risk of lung cancer
It is low, it is good available for the auxiliary diagnosis of clinical lung cancer, potential applicability in clinical practice.
Obviously, according to the above of the present invention, according to the ordinary technical knowledge and customary means of this area, do not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The embodiment of form by the following examples, the above of the present invention is remake further specifically
It is bright.But the scope that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to following example.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 is early stage of lung cancer patient and normal healthy controls plasma C erberus testing result figures
Embodiment
The relation of embodiment 1Cerberus expressions and lung cancer
Experimental method is as follows：
First, clinical data
Early stage of lung cancer patient 15 is chosen, normal healthy controls 10, essential information is as follows：
| Essential information | Patients with lung cancer | Normal healthy controls |
| Number | 15 | 10 |
| Age | 61.2±12.2 | 49.5±10.6 |
| Masculinity proportion | 10 (66.7%) | 6 (60.0%) |
2nd, the detection (content for quantitatively detecting Cerberus) of Cerberus expressions
Using the AAH-BLG-CUST kit (article No.s bought in RayBiotech companies：AAH-BLG-CUST), according to
Following method detection Cerberus expression：
1st, sample is dialysed
Patients with lung cancer and the blood of normal healthy controls are extracted, (1200rpm, 10min) is centrifuged after EDTA anti-freezings, supernatant is drawn
Carry out centrifuging (3000rpm, 15min) second afterwards, the supernatant drawn second after centrifuging is blood plasma, as detection sample, is divided
- 80 DEG C of preservations after dress.
Plasma sample needs to be dialysed using dialysis tubing before biotin labeling.The separately sampled μ L of product 100 are added to
Analyse pipe in, then in 4000mL 1 × PBS (pH=8) 4 DEG C dialyse while stirring.Dialyzate is changed once in 3 hours in interval,
Dialysis collects sample to 1.5mL centrifuge tubes afterwards three times.
Note：1 × PBS preparation：1.0g KCl, 40gNaCl, 1.0g KH2PO4,5.75g Na2HPO4 are gone with 4,500ml
Ionized water dissolves, and adjusts pH=8.0 with 1M NaOH, is finally settled to 5,000ml with deionized water.
2nd, biotin labeling sample
In the process of whole biotin labeling sample, any reagent is avoided to be polluted by amine substance or Sodium azide.
1) before using labelled reagent, after labelled reagent (Labeling Reagent) tubule quickly centrifugation, added in pipe
100 1 × PBS of μ L dissolve labelled reagent powder, and piping and druming up and down mixes labelled reagent, is prepared into 1 × labelled reagent solution.
2) 22ul 1 × labelled reagent solution and 155ul mark buffer solutions is added into the centrifuge tube equipped with 35ul samples.
It is quick to mix, 30min is incubated at room temperature on shaking table, centrifuge tube, hybrid reaction reagent are flicked per 5min.
3) after being incubated 30min, 3 μ L terminate liquids (Stop Solution) are added in upper step reaction solution；
4) separately sampled product have added each 100 μ L of sample after terminate liquid and added in dialysis tubing, then 4000mL 1 ×
Dialysed while stirring for 4 DEG C in PBS (pH=8).Change dialyzate once within 3 hours in interval.Sample is collected after dialysis three times.
3rd, slide chip is completely dried
By slide chip (Biotin label-based human antibody array 1and 2) from
Take out in box, after equilibrium at room temperature 20-30min, packaging bag is opened, open sealing strip, chip is then placed on vacuum
Drier or drying at room temperature 1-2 hours.
4th, close and be incubated
1) 100 μ L Block buffer (Blocking Buffer) is added in each chip hole, is incubated on room temperature shaker
1h, avoid producing bubble；
2) confining liquid is pumped, sample after 100 μ L dialysis, one sample of an array, 4 DEG C of oscillation incubations are added in each hole
12-16h。
3) clean
Slide is cleaned using Thermo Scientific Wellwash Versa chip board-washings machine, is divided into two steps：First
Cleaned with 1 × washing lotion I (Wash Buffer I) (being obtained with deionized water dilution 20 × washing lotion I), 1 per the μ L of hole 250 ×
Washing lotion I, clean 5 times, shake 10s every time, impact strength selection is high；Then use 1 × washing lotion II passages instead to be cleaned, per hole
250 μ L 1 × washing lotion II (Wash Buffer II) (being obtained with deionized water dilution 20 × washing lotion II), is cleaned 3 times, every time shake
10s is swung, impact strength selection is high.
4) 1 × washing lotion II is pumped, the Cy3equivalent room temperature lucifuges that 100uL is diluted with confining liquid 1500 are added per hole
Oscillation incubation 2 hours.
5) according to step 3) and 4) washing slide.
5th, fluoroscopic examination
Using laser scanner such as Axon GenePix scanning signals, using Cy3 or green channel (stimulating frequency=
532nm), scanner：The GenePix 4000B Microarray Scanner (places of production：Molecular Devices,LLC；
1311Orleans Drive Sunnyvale, CA94089-1136United States), sweep parameter：PMT:High intensity,
Wavelengh：532nm；resolution：10um.
Analysis software extraction data are carried using instrument, data are carried out using AAH-BLG-CUST DAS
Analysis.After data analysis, the Cerberus protein concentrations of each sample can be immediately arrived at.
3rd, interpretation of result
Difference analysis：Experimental group (patients with lung cancer) and control group (people taking physical examination) are united using SPSS17.0
Meter analysis.
Picture making：Completed with the softwares of GraphPad Prism 5.0.
4th, Cerberus expression and the correlation of lung cancer
The expression testing result of patients with lung cancer and Cerberus in normal healthy controls serum is shown in Fig. 1.
As seen from Figure 1, the change of serum C erberus Average expression levels of patients with lung cancer are 2020pg/ml, the blood of normal healthy controls
Clear Cerberus Average expression levels are 750pg/ml, and Cerberus is significantly raised in early stage of lung cancer patient, with normal healthy controls
Group is compared, and Cerberus expression differences have statistical significance (P<0.05).
As can be seen from the above results, compared with normal population, the Cerberus expressions of early stage of lung cancer patient are notable
Raise (P<0.05), illustrate that lung cancer is proportionate with Cerberus expressions, Cerberus high expression can significantly improve trouble
The possibility of lung cancer, therefore, the Susceptible population of lung cancer can be sieved by the expression for the Cerberus for detecting crowd to be checked
Find out.
The composition and its application method of the kit of the present invention detection blood Cerberus expressions of embodiment 2
First, the composition of Cerberus detection kits
Detection kit (30 person-portion)：
2nd, the application method of Cerberus detection kits
Application method is as follows：
1st, sample is dialysed
Patients with lung cancer and the blood of normal healthy controls are extracted, (1200rpm, 10min) is centrifuged after EDTA anti-freezings, supernatant is drawn
Carry out centrifuging (3000rpm, 15min) second afterwards, the supernatant drawn second after centrifuging is blood plasma, as detection sample, is divided
- 80 DEG C of preservations after dress.
Plasma sample needs to be dialysed using dialysis tubing before biotin labeling.The separately sampled μ L of product 100 are added to
Analyse pipe in, then in 4000mL 1 × PBS (pH=8) 4 DEG C dialyse while stirring.Dialyzate is changed once in 3 hours in interval,
Dialysis collects sample to 1.5mL centrifuge tubes afterwards three times.
Note：1 × PBS preparation：1.0g KCl, 40gNaCl, 1.0g KH2PO4,5.75g Na2HPO4 are gone with 4,500ml
Ionized water dissolves, and adjusts pH=8.0 with 1M NaOH, is finally settled to 5,000ml with deionized water.
2nd, biotin labeling sample
In the process of whole biotin labeling sample, any reagent is avoided to be polluted by amine substance or Sodium azide.
1) before using labelled reagent, after labelled reagent (Labeling Reagent) tubule quickly centrifugation, added in pipe
100 1 × PBS of μ L dissolve labelled reagent powder, and piping and druming up and down mixes labelled reagent, is prepared into 1 × labelled reagent solution.
2) 22ul 1 × labelled reagent solution and 155ul mark buffer solutions is added into the centrifuge tube equipped with 35ul samples.
It is quick to mix, 30min is incubated at room temperature on shaking table, centrifuge tube, hybrid reaction reagent are flicked per 5min.
3) after being incubated 30min, 3 μ L terminate liquids (Stop Solution) are added in upper step reaction solution；
4) separately sampled product have added each 100 μ L of sample after terminate liquid and added in dialysis tubing, then 4000mL 1 ×
Dialysed while stirring for 4 DEG C in PBS (pH=8).Change dialyzate once within 3 hours in interval.Sample is collected after dialysis three times.
3rd, slide chip is completely dried
By slide chip (Biotin label-based human antibody array 1and 2) from
Take out in box, after equilibrium at room temperature 20-30min, packaging bag is opened, open sealing strip, chip is then placed on vacuum
Drier or drying at room temperature 1-2 hours.
4th, close and be incubated
1) 100 μ L Block buffer (Blocking Buffer) is added in each chip hole, is incubated on room temperature shaker
1h, avoid producing bubble；
2) confining liquid is pumped, sample after 100 μ L dialysis, one sample of an array, 4 DEG C of oscillation incubations are added in each hole
12-16h。
3) clean
Slide is cleaned using Thermo Scientific Wellwash Versa chip board-washings machine, is divided into two steps：First
Cleaned with 1 × washing lotion I (Wash Buffer I) (being obtained with deionized water dilution 20 × washing lotion I), 1 per the μ L of hole 250 ×
Washing lotion I, clean 5 times, shake 10s every time, impact strength selection is high；Then use 1 × washing lotion II passages instead to be cleaned, per hole
250 μ L 1 × washing lotion II (Wash Buffer II) (being obtained with deionized water dilution 20 × washing lotion II), is cleaned 3 times, every time shake
10s is swung, impact strength selection is high.
4) 1 × washing lotion II is pumped, the Cy3equivalent room temperature lucifuges that 100uL is diluted with confining liquid 1500 are added per hole
Oscillation incubation 2 hours.
5) according to step 3) and 4) washing slide.
5th, fluoroscopic examination
Using laser scanner such as Axon GenePix scanning signals, using Cy3 or green channel (stimulating frequency=
532nm), scanner：The GenePix 4000B Microarray Scanner (places of production：Molecular Devices,LLC；
1311Orleans Drive Sunnyvale, CA94089-1136United States), sweep parameter：PMT:High intensity,
Wavelengh：532nm；resolution：10um.
Analysis software extraction data are carried using instrument, data are carried out using AAH-BLG-CUST DAS
Analysis.After data analysis, the Cerberus protein concentrations of each sample can be immediately arrived at.
To sum up, kit of the present invention can suffer from lung cancer by detecting Cerberus expression with examination crowd to be checked
Risk：If Cerberus expression is high, suffers from the risk height of lung cancer, if Cerberus expression is low, suffer from lung cancer
Risk it is low, available for the auxiliary diagnosis of clinical lung cancer, take the remedy measures of correlation or decision-making to provide effectively for patient
Foundation, potential applicability in clinical practice are good.
Claims (4)
1. detect purposes of the reagent of Cerberus expressions in screening lung cancer reagent is prepared.
2. purposes according to claim 1, it is characterised in that：The reagent is to be used to detect Cerberus in serum to express
Horizontal reagent.
3. purposes according to claim 1 or 2, it is characterised in that：The reagent of the detection Cerberus expressions is
Protein chip detection method reagent.
4. purposes according to claim 1 or 2, it is characterised in that：The reagent of the detection Cerberus expressions is
ELISA detection method is with reagent or Western Blot detection method reagents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610210301.6A CN105759056B (en) | 2016-04-06 | 2016-04-06 | A kind of screening lung cancer kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610210301.6A CN105759056B (en) | 2016-04-06 | 2016-04-06 | A kind of screening lung cancer kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105759056A CN105759056A (en) | 2016-07-13 |
| CN105759056B true CN105759056B (en) | 2017-11-14 |
Family
ID=56334206
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610210301.6A Active CN105759056B (en) | 2016-04-06 | 2016-04-06 | A kind of screening lung cancer kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105759056B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106546739A (en) * | 2016-10-18 | 2017-03-29 | 石永录 | A kind of screening lung cancer test kit |
| CN106916900B (en) * | 2017-05-05 | 2021-04-16 | 成都医学院第一附属医院 | Kit for screening cancer brain metastasis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998049296A1 (en) * | 1997-04-29 | 1998-11-05 | Regeneron Pharmaceuticals, Inc. | Human cerberus protein |
| WO2008048120A2 (en) * | 2006-10-17 | 2008-04-24 | Synergenz Bioscience Limited | Methods and compositions for assessment of pulmonary function and disorders |
| CN101589137A (en) * | 2005-03-31 | 2009-11-25 | 斯丹姆涅恩有限公司 | Amnion-derived cell compositions, methods of making and uses thereof |
-
2016
- 2016-04-06 CN CN201610210301.6A patent/CN105759056B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998049296A1 (en) * | 1997-04-29 | 1998-11-05 | Regeneron Pharmaceuticals, Inc. | Human cerberus protein |
| CN101589137A (en) * | 2005-03-31 | 2009-11-25 | 斯丹姆涅恩有限公司 | Amnion-derived cell compositions, methods of making and uses thereof |
| WO2008048120A2 (en) * | 2006-10-17 | 2008-04-24 | Synergenz Bioscience Limited | Methods and compositions for assessment of pulmonary function and disorders |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105759056A (en) | 2016-07-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102625915B (en) | Method for measurement of glycoprotein, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein | |
| CN105785036B (en) | A kind of screening lung cancer kit | |
| CN105572353B (en) | A kind of antibody chip kit for detecting liver cancer marker | |
| CN104650234B (en) | Anti- AKR1B10 protein monoclonal antibodies and its application | |
| US20130295580A1 (en) | Oral cancer point of care diagnostics | |
| CN110196329A (en) | A kind of cancer of the esophagus early stage combined detection kit | |
| CN104267185B (en) | Detect the kit of tumour and the material of special identification 2-Acetamido-2-deoxy-D-glucose thereof | |
| CN105759056B (en) | A kind of screening lung cancer kit | |
| CN108603882A (en) | Laminin 2 is used for the purposes of diagnosing hepatocellular carcinoma and cancer of pancreas | |
| CN106680515B (en) | It is combined for the polymolecular marker of pulmonary cancer diagnosis | |
| CN110456076A (en) | The method of multiple antibody immune magnetic beads enrichment detection CTCs | |
| CN109655608A (en) | A kind of excretion body protein for osteosarcoma diagnosis and its instant detection method | |
| CN109507426A (en) | Prostate cancer diagnosis, classification or prognostic marker, detection reagent or kit, system and its application | |
| CN105505877B (en) | The method that the excretion body in tumour cell source is detached in malignant pleural effusion | |
| CN106383229B (en) | Hepatitis B correlation hepatocellular carcinoma early diagnosis kit | |
| CN105675572B (en) | A kind of screening lung cancer kit | |
| CN105548546B (en) | A kind of screening lung cancer kit | |
| CN109085359A (en) | Serum protein markers combine the application in colorectal cancer screening and diagnosis and treatment | |
| CN114724707A (en) | Hepatocellular carcinoma autoantibody marker combined diagnosis model | |
| WO2019169857A1 (en) | Application of igg4 detection reagent in preparation of colorectal cancer diagnostic agent | |
| CN113702637B (en) | Lectin test carrier, kit and prediction model for predicting neoadjuvant chemotherapy curative effect of breast cancer | |
| CN114544955B (en) | Application of GASP-2 detection reagent in preparation of early diagnosis and susceptibility detection kit for lung cancer | |
| TWI656344B (en) | Method for screening circulating tumor cells in blood | |
| CN109975547A (en) | Application of the RANTES detection reagent in terms of preparing diagnosis of colorectal carcinoma agent | |
| CN105954517B (en) | A kind of screening lung cancer kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |