CN105785036B - A kind of screening lung cancer kit - Google Patents
A kind of screening lung cancer kit Download PDFInfo
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- CN105785036B CN105785036B CN201610190618.8A CN201610190618A CN105785036B CN 105785036 B CN105785036 B CN 105785036B CN 201610190618 A CN201610190618 A CN 201610190618A CN 105785036 B CN105785036 B CN 105785036B
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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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
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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
- G01N33/57484—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
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Abstract
The invention discloses a kind of screening lung cancer kits, it includes the optional reagent for being used to detect FABP1 expressions.The invention also discloses detect purposes of the reagent of FABP1 expressions in screening lung cancer reagent is prepared.Kit of the present invention is by detecting the expression of FABP1, 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 relevant remedy measures or decision 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 kits.
Background technology
Lung cancer is one of most common malignant tumour in the world, and morbidity and mortality are in ascendant trend year by year, at present
Incidence 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. it smokes；2. environmental pollution：Such as haze, indoor dress
It repaiies；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, and often developing to late period in disease just shows clinical symptoms, and 70
~80% patients with lung cancer has been middle and advanced stage when being diagnosed to be with Lung Cancer Symptoms, and cancer cell has been spread, and is missed and is most preferably controlled
More opportunity, five year survival rate are low.For the patients with lung cancer of early stage, by treat in time be greatly improved 5 years of patient and more than
Survival rate and life quality.Therefore the early diagnosis and the effective screening of progress of lung cancer are most important.
The screening of lung cancer refers to do not have 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
Relevant remedy measures or decision is taken to have great importance.
Fatty acid binding protein 1 (fatty acid binding protein 1, FABP1), also known as liver fatty acid
Binding protein is a member of fatty acid binding protein family, is risen in the intake of long chain fatty acids, transhipment, metabolism, adjusting important
Effect.It has no at present about plasma F ABP1 and the relevant report of lung cancer.
Invention content
To solve the above-mentioned problems, lung cancer is studied in detail in inventor, it was found that FABP1 can be used as its molecule
Marker.Wherein, expressions of the FABP1 in serum is proportionate with lung cancer.Therefore, by detecting FABP1 in serum
Expression can predict the risk that crowd to be checked suffers from lung cancer.
Accordingly, the reagent the present invention provides a kind of screening lung cancer kit and the expression for detecting FABP1 is being made
Purposes in standby screening lung cancer reagent.
The screening lung cancer kit of the present invention, it includes the optional reagent for being used to detect FABP1 expressions.
Wherein, the reagent is the reagent for detecting FABP1 expressions in serum.
Wherein, the reagent of the detection FABP1 expressions is protein chip detection method reagent.
Wherein, the reagent of the detection FABP1 expressions is examined for ELISA detection method reagent or Western Blot
Survey method reagent.
The present invention also provides detect purposes of the reagent of FABP1 expressions in screening lung cancer reagent is prepared.
Wherein, the reagent is the reagent for detecting FABP1 expressions in serum.
Wherein, the reagent of the detection FABP1 expressions is protein chip detection method reagent.
Wherein, the reagent of the detection FABP1 expressions is examined for ELISA detection method reagent or Western Blot
Survey method reagent.
Kit of the present invention is by detecting the expression of FABP1, it can be determined that crowd to be checked suffers from the risk of lung cancer：If
The expression of FABP1 is high, then the risk for suffering from lung cancer is high, if the expression of FABP1 is low, the risk for suffering from lung cancer is low, can use
In the auxiliary diagnosis of clinical lung cancer, potential applicability in clinical practice is good.
Obviously, the above according to the present invention according to the ordinary technical knowledge and customary means of this field, is 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 specific embodiment of form by the following examples remakes further specifically the above of the present invention
It is bright.But the range 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 all belongs to the scope of the present invention.
Description of the drawings
Fig. 1 is early stage of lung cancer patient and normal healthy controls plasma F ABP1 testing result figures
Specific embodiment
The relationship of 1 FABP1 expressions of embodiment 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 FABP1) of FABP1 expressions
Using the AAH-BLG-CUST kit (article No.s bought in RayBiotech companies：AAH-BLG-CUST), according to
The expression of following method detection FABP1：
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
It carries out centrifuging (3000rpm, 15min) second afterwards, draws supernatant, that is, blood plasma after centrifuging second, as detection sample, divide
- 80 DEG C of preservations after dress.
Plasma sample needs to dialyse using dialysis tubing before biotin labeling.Separately sampled 100 μ L of product are added to
Analyse pipe in, then in 1 × PBS of 4000mL (pH=8) 4 DEG C dialyse while stirring.The 3 hours replacement dialyzates in interval are primary,
Dialysis collects sample to 1.5mL centrifuge tubes afterwards three times.
Note：The preparation of 1 × PBS：1.0g KCl, 40gNaCl, 1.0g KH2PO4,5.75g Na2HPO4 are gone with 4,500ml
Ion water dissolution adjusts pH=8.0 with 1M NaOH, is finally settled to 5,000ml with deionized water.
2nd, biotin labeling sample
In the process of entire biotin labeling sample, any reagent is avoided to be polluted by amine substance or Sodium azide.
1) it before using labelled reagent, after labelled reagent (Labeling Reagent) tubule quickly centrifugation, is added in pipe
100 1 × PBS of μ L dissolve labelled reagent powder, blow and beat up and down by labelled reagent mixing, are prepared into 1 × labelled reagent solution.
2) 1 × labelled reagent solution of 22ul and 155ul label buffer solutions are added in into the centrifuge tube equipped with 35ul samples.
Quick mixing is incubated at room temperature 30min on shaking table, and 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 in each 100 μ L of sample after terminate liquid and have added in dialysis tubing, then 4000mL 1 ×
It dialyses while stirring for 4 DEG C in PBS (pH=8).It is primary that dialyzate is replaced in 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
It takes 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, it closes and is incubated
1) Block buffer (Blocking Buffer) of 100 μ L is added in each chip hole, is incubated on room temperature shaker
1h avoids generating 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) it cleans
Slide is cleaned using Thermo Scientific Wellwash Versa chip board-washings machine, is divided into two steps：First
It is cleaned with 1 × washing lotion I (Wash Buffer I) (being obtained with deionized water dilution 20 × washing lotion I), 1 per 250 μ L of hole ×
Washing lotion I is cleaned 5 times, shakes 10s every time, and impact strength selection is high；Then it uses 1 × washing lotion II channels instead to be cleaned, per hole
1 × washing lotion II (Wash Buffer II) (being obtained with deionized water dilution 20 × washing lotion II) of 250 μ L, cleans 3 times, shakes every time
10s is swung, impact strength selection is high.
4) 1 × washing lotion II is pumped, 100uL is added in per hole and is protected from light with 1500 diluted Cy3equivalent room temperatures of confining liquid
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 the Data Analysis Software of AAH-BLG-CUST
Analysis.After data analysis, the FABP1 protein concentrations of each sample can be immediately arrived at.
3rd, interpretation of result
Difference analysis：It is united using SPSS17.0 to experimental group (patients with lung cancer) and control group (people taking physical examination)
Meter analysis.
Picture making：It is completed with 5.0 softwares of GraphPad Prism.
4th, the expression of FABP1 and the correlation of lung cancer
The expression testing result of patients with lung cancer and FABP1 in normal healthy controls serum is shown in Fig. 1.
As seen from Figure 1, the serum FABP1 Average expression levels of patients with lung cancer are 154,566pg/ml, the blood of normal healthy controls
Clear FABP1 Average expression levels are 68,350pg/ml), FABP1 is significantly increased in early stage of lung cancer patient, with healthy control group
It compares, FABP1 expressions difference has statistical significance (p<0.0001).
As can be seen from the above results, compared with normal population, the FABP1 expressions of early stage of lung cancer patient significantly increase
(P<0.05), illustrate that lung cancer is proportionate with FABP1 expressions, the high expression of FABP1 can significantly improve the possibility for suffering from lung cancer
Property, therefore, Susceptible population's screening of lung cancer can be come out by the expression for the FABP1 for detecting crowd to be checked.
The composition and its application method of the kit of 2 present invention detection blood FABP1 expressions of embodiment
First, the composition of FABP1 detection kits
Detection kit (30 person-portion)：
2nd, the application method of FABP1 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
It carries out centrifuging (3000rpm, 15min) second afterwards, draws supernatant, that is, blood plasma after centrifuging second, as detection sample, divide
- 80 DEG C of preservations after dress.
Plasma sample needs to dialyse using dialysis tubing before biotin labeling.Separately sampled 100 μ L of product are added to
Analyse pipe in, then in 1 × PBS of 4000mL (pH=8) 4 DEG C dialyse while stirring.The 3 hours replacement dialyzates in interval are primary,
Dialysis collects sample to 1.5mL centrifuge tubes afterwards three times.
Note：The preparation of 1 × PBS：1.0g KCl, 40gNaCl, 1.0g KH2PO4,5.75g Na2HPO4 are gone with 4,500ml
Ion water dissolution adjusts pH=8.0 with 1M NaOH, is finally settled to 5,000ml with deionized water.
2nd, biotin labeling sample
In the process of entire biotin labeling sample, any reagent is avoided to be polluted by amine substance or Sodium azide.
1) it before using labelled reagent, after labelled reagent (Labeling Reagent) tubule quickly centrifugation, is added in pipe
100 1 × PBS of μ L dissolve labelled reagent powder, blow and beat up and down by labelled reagent mixing, are prepared into 1 × labelled reagent solution.
2) 1 × labelled reagent solution of 22ul and 155ul label buffer solutions are added in into the centrifuge tube equipped with 35ul samples.
Quick mixing is incubated at room temperature 30min on shaking table, and 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 in each 100 μ L of sample after terminate liquid and have added in dialysis tubing, then 4000mL 1 ×
It dialyses while stirring for 4 DEG C in PBS (pH=8).It is primary that dialyzate is replaced in 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
It takes 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, it closes and is incubated
1) Block buffer (Blocking Buffer) of 100 μ L is added in each chip hole, is incubated on room temperature shaker
1h avoids generating 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) it cleans
Slide is cleaned using Thermo Scientific Wellwash Versa chip board-washings machine, is divided into two steps：First
It is cleaned with 1 × washing lotion I (Wash Buffer I) (being obtained with deionized water dilution 20 × washing lotion I), 1 per 250 μ L of hole ×
Washing lotion I is cleaned 5 times, shakes 10s every time, and impact strength selection is high；Then it uses 1 × washing lotion II channels instead to be cleaned, per hole
1 × washing lotion II (Wash Buffer II) (being obtained with deionized water dilution 20 × washing lotion II) of 250 μ L, cleans 3 times, shakes every time
10s is swung, impact strength selection is high.
4) 1 × washing lotion II is pumped, 100uL is added in per hole and is protected from light with 1500 diluted Cy3equivalent room temperatures of confining liquid
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 the Data Analysis Software of AAH-BLG-CUST
Analysis.After data analysis, the FABP1 protein concentrations of each sample can be immediately arrived at.
To sum up, kit of the present invention can suffer from the wind of lung cancer by detecting the expression of FABP1 with screening crowd to be checked
Danger：If the expression of FABP1 is high, the risk for suffering from lung cancer is high, if the expression of FABP1 is low, the risk for suffering from lung cancer is low,
Available for the auxiliary diagnosis of clinical lung cancer, relevant remedy measures or decision provides effective foundation are taken for patient, it is clinical
Application prospect is good.
Claims (3)
1. detect purposes of the reagent of 1 expression of fatty acid binding protein in screening lung cancer reagent is prepared；Wherein, it is described
Reagent is the reagent for detecting 1 expression of fatty acid binding protein in serum.
2. purposes according to claim 1, it is characterised in that：The examination of detection 1 expression of fatty acid binding protein
Agent is protein chip detection method reagent.
3. purposes according to claim 1, it is characterised in that：The examination of detection 1 expression of fatty acid binding protein
Agent is ELISA detection method reagent or Western Blot detection method reagents.
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| CN201610190618.8A CN105785036B (en) | 2016-03-29 | 2016-03-29 | A kind of screening lung cancer kit |
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| CN201610190618.8A CN105785036B (en) | 2016-03-29 | 2016-03-29 | A kind of screening lung cancer kit |
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| CN105785036B true CN105785036B (en) | 2018-06-26 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110554192B (en) * | 2019-06-26 | 2020-06-12 | 四川大学华西医院 | Application of LIMCH1 autoantibody detection reagent in preparation of lung cancer screening kit |
| CN110456068B (en) * | 2019-07-31 | 2023-01-06 | 四川大学华西医院 | Application of XIRP1 autoantibody detection reagent in preparation of lung cancer screening kit |
| CN110456081A (en) * | 2019-09-20 | 2019-11-15 | 四川大学华西医院 | IST1 autoantibody detection reagent is preparing the purposes in screening lung cancer kit |
| CN111508563B (en) * | 2020-05-22 | 2023-04-18 | 四川大学华西医院 | Cancer-related alternative splicing database system of long non-coding RNA |
| CN116908451B (en) * | 2023-07-10 | 2024-04-19 | 华中科技大学同济医学院附属协和医院 | Application of protein markers in preparation of reagent for identifying lung metastasis of primary lung adenocarcinoma and colorectal cancer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103529225A (en) * | 2013-11-04 | 2014-01-22 | 武汉华美生物工程有限公司 | Liver fatty acid binding protein content detection kit and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103529225A (en) * | 2013-11-04 | 2014-01-22 | 武汉华美生物工程有限公司 | Liver fatty acid binding protein content detection kit and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Expression of Liver-Type Fatty-Acid-Binding Protein, Fatty Acid Synthase and Vascular Endothelial Growth Factor in Human Lung Carcinoma;Tetsuo Kawamura等;《Pathobiology》;20051231;第72卷;摘要，第234页右栏第1-2段 * |
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