CN109085355A - Application of serum protein marker combination in screening, diagnosis and treatment of lung cancer - Google Patents
Application of serum protein marker combination in screening, diagnosis and treatment of lung cancer Download PDFInfo
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Abstract
本发明属于分子生物学、临床检测技术领域，涉及一种蛋白组合物，其包含选自CYFRA21‑1、NSE、IL‑2、IL‑8、IFN‑γ、MIP‑1β和VEGF‑A中任意的两种、三种、四种、五种、六种或七种。本发明还涉及与蛋白组合物中的蛋白特异性结合的配体组合物，以及所述蛋白组合物或配体组合物在制备肺癌筛查、辅助诊断、治疗监测和预后判断试剂盒中的应用。本发明涉及本发明可用于肺癌的筛查、辅助诊断、治疗监测和预后判断，尤其是早期肺癌的筛查和辅助诊断，其敏感性高、特异性高，优于单一蛋白标志物以及临床常用血清标志物CYFRA21‑1和NSE的检测。
The invention belongs to the technical fields of molecular biology and clinical detection, and relates to a protein composition comprising any Two, three, four, five, six or seven of the The present invention also relates to a ligand composition specifically binding to the protein in the protein composition, and the application of the protein composition or the ligand composition in the preparation of lung cancer screening, auxiliary diagnosis, treatment monitoring and prognosis judgment kits . The present invention relates to the screening, auxiliary diagnosis, treatment monitoring and prognosis judgment of lung cancer, especially the screening and auxiliary diagnosis of early lung cancer. It has high sensitivity and high specificity, and is superior to single protein markers and commonly used clinical Detection of serum markers CYFRA21‑1 and NSE.
Description
Technical field
The invention belongs to molecular biology and clinical detection technique field, and in particular, to comprising selected from CYFRA21-1,
NSE, IL-2, IL-8, IFN-γ, the marker combination of seven albumen of MIP-1 β and VEGF-A and marker combination exist
Prepare the application in screening lung cancer, auxiliary diagnosis, Treatment monitoring and Index for diagnosis blood serum designated object kit.
Background technique
Lung cancer is the most common malignant tumour of countries in the world today, and male lung cancer morbidity and mortality account for all pernicious
First of tumour, women disease incidence accounts for second, and the death rate accounts for second.In China, according to the newest of National Cancer Center
Data, China's new hair lung cancer 70.48 ten thousand in 2012, disease incidence 52.06/106；Death is 56.94 ten thousand, and the death rate is
42.05/106, malignant tumour first place is occupy in male and female.
Although there is new development in terms of diagnostic method, surgical technic and chemotherapeutics in recent years, patients with lung cancer
Overall 5 years survival rates are still very low, are only 16.1% in China.Most clinical diagnosis cases of lung cancer have been mostly advanced stage, are lost
Remove the chance of operative treatment, poor prognosis.But the postoperative 10 years survival rates of studies have shown that I phase lung cancer are up to 92%.Therefore, it drops
The key of the low patients with lung cancer death rate is early warning, early detection, so as to early intervention.
Currently used screening lung cancer method includes chest x-ray, the spiral computerized tomoscan (low-dose of low dosage
Spiral CT, LDCT), expectorative cytology inspection, brushing piece or take biopsy, BALF cytology under bronchoscope
Check etc..2011, American National screening lung cancer test (National Lung Screening Trial, NLST) was reported for the first time
The death rate of lung cancer can be significantly reduced in people at highest risk by accusing the spiral computerized tomoscan screening of low dosage.But above-mentioned various inspections
Look into means sensibility, specificity, relevance grade and in terms of exist limitation.What these inspection items required sets
Standby complicated, operation professional technique requires high and instrument price expensive, it is therefore desirable to effective and inexpensive pervasive detection side
Method.
Other than above physical test mode, clinically also have at present using blood serum tumor marker Diagnosis of pulmonary
Cancer.Compared with above-mentioned inspection method, serum mark analyte detection has many advantages, such as that quick, wound is small, is easy to receive, and is suitble to extensive
Mass screening, and it is convenient for long-term dynamics monitoring, facilitate early detection malignant tumour.Common marker includes that cancer is thin
Born of the same parents' keratin 21-1 segment (CYFRA21-1), neuronspecific enolase (NSE) etc..But these tumor markers lists
It is very limited solely to detect its sensibility and specificity.
In consideration of it, there is an urgent need in the art to develop to can be used for screening lung cancer, auxiliary diagnosis, Treatment monitoring and Index for diagnosis
Blood serum designated object or marker combination, to improve the diagnosis and recall rate of lung cancer, improve the whole prognostic level of lung cancer.Separately
On the one hand, this field also there is an urgent need to develop can be used for lung cancer early stage auxiliary diagnosis marker or marker combination, to
The early stage of lung cancer can be effectively detected, improve early diagnosis and early control effect.
Summary of the invention
The present inventor passes through in-depth study and creative labor, has obtained a kind of protein composition.The present inventor is frightened
It finds oddly, which can be used in screening, auxiliary diagnosis, Treatment monitoring and/or the Index for diagnosis of lung cancer, and have
There are good sensibility and/or specificity.Thus provide following inventions:
One aspect of the present invention is related to a kind of protein composition, it includes selected from CYFRA21-1, NSE, IL-2, IL-8,
At least any two kinds in IFN-γ, MIP-1 β and VEGF-A albumen.In some embodiments of the present invention, the protein groups
Closing object includes at least any two kinds, three kinds, four kinds, five kinds, six kinds or seven kinds therein.
In one or more embodiments of the invention, the protein composition, it includes CYFRA21-1 and NSE；
Optionally, the protein composition also includes arbitrary one kind, two in IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A
Kind, three kinds, four kinds or five kinds.
In one or more embodiments of the invention, the protein composition, it includes IL-2 and IL-8,；It can
Selection of land, the protein composition also include in CYFRA21-1, NSE, IFN-γ, MIP-1 β and VEGF-A it is arbitrary a kind of,
Two kinds, three kinds, four kinds or five kinds.
In one or more embodiments of the invention, the protein composition, it includes IL-2, IL-8 and
NSE,；Optionally, the protein composition also includes arbitrary one in CYFRA21-1, IFN-γ, MIP-1 β and VEGF-A
Kind, two kinds, three kinds or four kinds.
In one or more embodiments of the invention, the protein composition, it includes IL-2, IL-8 and IFN-
γ；Optionally, the protein composition also includes arbitrary one kind, two in CYFRA21-1, NSE, MIP-1 β and VEGF-A
Kind, three kinds or four kinds.
In one or more embodiments of the invention, the protein composition, it includes IL-2, IL-8 and MIP-
1β；Optionally, the protein composition also includes arbitrary one kind, two in CYFRA21-1, NSE, IFN-γ and VEGF-A
Kind, three kinds or four kinds.
In some embodiments of the present invention, the protein composition is comprising following albumen or by following protein groups
At:
(1) NSE, IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A albumen；
(2) CYFRA21-1, IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A albumen；
(3) IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A albumen；
(4) NSE, IL-2, IL-8, IFN-γ and VEGF-A albumen；
(5) NSE, IL-8, IFN-γ, MIP-1 β and VEGF-A albumen；
(6) NSE, IL-2, IL-8, MIP-1 β and VEGF-A albumen；
(7) NSE, IL-2, IL-8, IFN-γ, MIP-1 β albumen；
(8) CYFRA21-1, IL-2, IL-8, IFN-γ and VEGF-A albumen；
(9) IL-2, IL-8, IFN-γ and VEGF-A albumen；
(10) IL-2, IL-8, MIP-1 β and VEGF-A albumen；
(11) NSE, IL-8, IFN-γ and VEGF-A albumen；
(12) IL-8, IFN-γ, MIP-1 β and VEGF-A albumen；
(13) NSE, IL-8, MIP-1 β, VEGF-A albumen；Or
(14)CYFRA21-1、NSE、IL-2、IFN-γ。
In the present invention, the protein composition is the composition that can be used as significant albumen, for the detection of lung cancer, tool
Body includes screening, auxiliary diagnosis, Treatment monitoring and/or the Index for diagnosis of lung cancer (especially non-small cell lung cancer).The albumen
Composition is alternatively referred to as protein combination, mark compositions or marker combination.When as marker composition or marker group
When conjunction, each albumen is considered as marker.Each albumen or each marker may be mixed together, and can not also mix, such as independent packet
Dress.The protein composition can be also simply referred to as composition；In certain embodiments of the present invention, the composition is a kind of use
In the composition of the screening of lung cancer, auxiliary diagnosis, Treatment monitoring and/or Index for diagnosis.
Another aspect of the present invention relates to a kind of ligand combination objects, are and egg described in protein composition of the invention
The composition of the ligand of white specific binding, for detecting the protein composition.The ligand combination object can be used in lung
The detection of cancer specifically includes screening, auxiliary diagnosis, Treatment monitoring and/or the Index for diagnosis of lung cancer.The ligand combination object can also
Referred to as ligand combination.
In the present invention, the ligand is the molecule in conjunction with the protein-specific in protein composition of the invention.This
The ligand of invention can be nucleic acid, polypeptide (including antibody) or compound.In some embodiments of the present invention, the ligand
For antibody such as monoclonal antibody；Ligand combination object is antibody compositions at this time.Antibody can be human antibody, chimeric antibody, again
Group antibody, humanized antibody, monoclonal antibody or its antigen-binding fragment or polyclonal antibody.
Another aspect of the present invention relates to a kind of kits, and it includes ligand combination objects of the invention.
It (is especially non-that another aspect of the present invention relates to protein compositions of the invention or ligand combination object in preparation lung cancer
Small Cell Lung Cancer) purposes in screening, auxiliary diagnosis, Treatment monitoring and/or Index for diagnosis kit.
In the present invention, the kit is sentenced for screening, auxiliary diagnosis, Treatment monitoring and/or the prognosis of serum sample
It is disconnected.In embodiments of the invention, the blood serum sample is patients with lung cancer and/or normal human serum sample.
A kind of method of screening lung cancer, the diagnosis such as monitoring of auxiliary diagnosis lung cancer, lung cancer therapy or lung cancer for prognosis judgement, packet
Include the step of detecting protein composition of the invention.If the testing result of at least two albumen is the positive, it is judged as lung cancer sample
This.
In the present invention, the judgment method is, when in ligand combination object of the invention at least two ligands with it is to be measured
When example reaction is presented positive, that is, it is judged as lung cancer sample.In certain embodiments of the present invention, according to albumen (marker)
Concentration is defined as Cut-off value as positive and negative judgment criteria；Albumen (marker) concentration is greater than Cut-off value
Sample is denoted as the positive, and the sample less than Cut-off value is denoted as feminine gender.In certain embodiments of the present invention, above-mentioned seven eggs
The Cut-off value of white (marker) concentration is respectively as follows: 2.71ng/ml (CYFRA21-1), 8.07ng/ml (NSE), 5.63pg/ml
(IL-2)、0.29pg/ml(IL-8)、0.46pg/ml(IFN-γ)、45.13pg/ml(MIP-1β)、VEGF-A(456.00pg/
ml)。
The method of detection protein marker is related to being detected by the interaction with protein-specific antibody.Such as
It can the use of enzyme-linked immunosorbent assay (ELISA) reagent for protein marker (wherein include as described herein the albumen
Antibody).Standard technique well known to those skilled in the art can be used and generate antibody, or the antibody of purchase can be used.
These antibody can be polyclonal antibody, or preferably monoclonal antibody.
The presence of the method quantitative detection protein marker of immunoassays can be used.The immunoassays generally include by
Biological sample is incubated with antibody, and detects binding antibody by known technology ELISA.
In embodiments of the invention, the method, ELISA method and positive judgment criteria for preparing sample are as follows:
(1) sample is prepared:
All patients in group and normal control extract 5ml peripheric venous blood on an empty stomach, are added in non-anticoagulant heparin tube, acquisition
After be stored at room temperature 30 minutes, carry out serum separation.Room temperature is centrifuged later, serum is moved into new centrifuge tube, is numbered, juxtaposition
It is saved in -80 DEG C of refrigerators to be measured.Multigelation is avoided before final detection.
(2) ELISA: the specific detection method is as follows for each albumen:
(a) all reagents and required lath CYFRA21-1: are being placed into 30min at room temperature using preceding.Dissolution mark
Quasi- product and quality-control product prepare cleaning solution, and the lath of number needed for testing is fixed on grillage.The buffering of 50 μ l is added in every hole
The enzyme-linked object of liquid and 10 μ l (this is the antibody of CYFRA21-1, has been coupled the enzyme that can decompose substrate thereon).50 μ l are marked
Quasi- product, quality-control product and blood serum sample are added in corresponding detection hole, are thoroughly mixed, and concussion is incubated for 60 minutes at room temperature.Quickly
The content in detection hole is discarded, every hole is added 350 μ l cleaning solutions and cleans 3 times, and firmly clappers are remained to remove on blotting paper
Liquid.100 μ l substrate solutions are added in every hole, at room temperature avoid light place 15 minutes.Every hole is added 100 μ l terminate liquids and terminates instead
It answers.After adding terminate liquid in 10 minutes, at room temperature, absorptance is read at 450nm using microplate reader.
(b) all reagents and required lath NSE: are being placed into 30min at room temperature using preceding.Dissolve standard items and
Quality-control product prepares cleaning solution, and the lath of number needed for testing is fixed on grillage.By 25 μ l standard items, quality-control product and serum
Sample is added in corresponding detection hole, is thoroughly mixed.Enzyme-linked object that every hole is added after 100 μ l dilution (this is the antibody of NSE,
On be coupled the enzyme that can decompose substrate), at room temperature concussion be incubated for 60 minutes.The content in detection hole is quickly discarded, often
Hole is added 300 μ l cleaning solutions and cleans 5 times, firmly has the final say on blotting paper to remove residual liquid.100 μ l substrates are added in every hole
Liquid, at room temperature avoid light place 15 minutes.Every hole is added 100 μ l terminate liquids and terminates reaction.After adding terminate liquid in 5 minutes, room
Under temperature, absorptance is read at 450nm using microplate reader.
(c) IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A: standard items are redissolved and are collected (to antigen standard tubule
In every pipe 50 μ l specificity buffer 1 × Mniversal Assay Buffer of sample be added redissolve standard items.By antigen standard
Product tubule be placed on ice 10 minutes to ensure to redissolve completely.The entire contents of each tubule are collected into a tubule, and
Add 1 × Mniversal Assay Buffer to 250 μ l).Prepare 4 times of dilution standard product (centrifuge tube mark is 1~8 spare, to
200 μ l are added in No. 1 pipe and redissolve antigen standard, 150 μ 1 × Mniversal of l Assay are added into 2~No. 8 pipes
Buffer.It from taking 50 μ l to be added in No. 2 pipes in No. 1 pipe, mixes, until there is standard items in No. 7 pipes, No. 8 are managed this step in repetition
For blank well).50 μ l magnetic bead liquid are added in every hole.96 orifice plates (are fixed on hand-held magnetic bead cleaning machine, and ensure by cleaning magnetic bead
96 hole boards are good, 2min stood, so that magnetic bead accumulates in every hole board bottom.It is inverted, to discard the liquid in 96 orifice plates and clap net residual
Stay liquid.Every hole is added 150 μ 1 × Wash of l Buffer and stands 2min, so that magnetic bead accumulates in every hole board bottom.It is inverted, to abandon
Fall the liquid in 96 orifice plates and claps net Liquid Residue.96 orifice plates are removed from hand-held magnetic bead cleaning machine).It is loaded and is incubated for (every
25 μ l 1 × Mniversal Assay Buffer are added in hole, and then the ready standard items of 25 μ l or sample is added in every hole.
It sticks sealing plate film and covers black lid for micro plate, 500r/min is incubated at room temperature 120 minutes).Clean 96 orifice plates.Detection antibody is added
And it is incubated for (25 μ l 1 × Detection Antibody Mixture of every hole addition.96 orifice plates are sealed with new sealing plate film, are covered
Black lid for micro plate, the concussion of 500r/min room temperature are incubated for 30 minutes.Clean 96 orifice plates.SAPE is added and is incubated for (50 μ of every hole addition
l SAPE.96 orifice plates are sealed with new sealing plate film, cover black lid for micro plate, the concussion of 500r/min room temperature is incubated for 30 minutes).Clearly
Wash 96 orifice plates.Data are read on Luminex instrument, and (120 μ l Reading Buffer are added in every hole.It is close with new sealing plate film
96 orifice plates are sealed, black lid for micro plate is covered, the concussion of 500r/min room temperature is incubated for 5 minutes.Sealing plate film is removed, in Luminex instrument
Read 96 orifice plate data).
(3) positive judgment criteria: according to protein concentration as positive and negative judgment criteria, it is defined as Cut-off value.
The sample that protein concentration is greater than Cut-off value is denoted as the positive, and the sample less than Cut-off value is denoted as feminine gender.Above-mentioned seven albumen
The Cut-off value of concentration be respectively as follows: 2.71ng/ml (CYFRA21-1), 8.07ng/ml (NSE), 5.63pg/ml (IL-2),
0.29pg/ml(IL-8)、0.46pg/ml(IFN-γ)、45.13pg/ml(MIP-1β)、VEGF-A(456.00pg/ml)。
In the present invention:
CYFRA21-1 is cytokeratin 19 fragment antigen, is current clinically used Sera of Lung Cancer marker, is also used for
Head and neck neoplasm, tumor in digestive tract, cervical carcinoma etc..
NSE is one of the enolase for participating in glycolytic pathway, is presently believed to be the head of detection Small Cell Lung Cancer
Marker is selected, neuroblastoma, thyroid gland medullary substance cancer etc. are also used for.
IL-2 is interleukin 2, and physiological action includes stimulation and the differentiation and proliferation for maintaining T cell；Stimulate NK cell
Proliferation enhances NK killing activity and generates cell factor, and induction LAK cell generates；Promote B cell proliferation and secretory antibody；Swash
Macrophage living.
IL-8 is interleukin 8, is the cell factor of the secretions such as macrophage and epithelial cell.IL-8 combination chemotactic
Factor acceptor Interleukin-8 receptor α (IL8RA) and Interleukin-8 receptor β (IL8RB), has cell to neutrophil leucocyte
Chemotaxis and realize its adjusting to inflammatory reaction.Meanwhile there are also very strong angiogenesispromoting effects, promote the life of tumour
Long and transfer.
IFN-γ is interferon, is a very strong inflammatory factor, in the tumours such as liver cancer, lung cancer, kidney, bladder cancer
Expression increases, and can promote tumour growth.
MIP-1 β be macrophage inflammatory factor, to panimmunity effector cell include T cell, B cell, monocyte,
Neutrophil leucocyte and Dendritic Cells etc. all have significant chemotaxis.It expresses and increases in the malignant tumours such as lung cancer, breast cancer,
Promote the invasion and transfer of tumour.
VEGF-A is anti-vascular endothelial cell growth factor, angiogenesis, tumour growth and transfer process in play
Important function.
In the present invention, when referring to the amino acid sequence of any of the above-described a albumen comprising the overall length of the albumen is also wrapped
Include its fusion protein.However, it will be appreciated by those skilled in the art that in the amino acid sequence of the albumen, it can naturally-produced or people
Work introduces mutation or variation (including but not limited to replacing, be deleted and/or added), without influencing its biological function.In this hair
In a bright embodiment, which is people's albumen.
Above-mentioned albumen has the meaning known to those skilled in the art known, and specific amino acid sequence can use this field
The means that technical staff knows obtain, such as with reference to NCBI (National Center for Biotechnology
Information the ID number in): CYFRA21-1 (3880), NSE (2026), IL-2 (3558), IL-8 (3576), IFN-γ
(3458), (6351) MIP-1 β, VEGF-A (7422).
In the present invention, term " sensibility ", which refers to, makes a definite diagnosis trouble by goldstandard (routine clinical histopathological diagnosis method)
The ratio (%) of detected positive case number, the i.e. true positive rate of this laboratory diagnosis in the case group of disease.Sensibility=kidney-Yang
Venereal disease number of cases/total the case load of case group.
In the present invention, term " specificity " refers to be diagnosed as by goldstandard (routine clinical histopathological diagnosis method)
The ratio (%) of detected feminine gender number, i.e. the true negative rate of this diagnostic test in disease-free control group.Specificity=Kidney-Yin
Property number/control group total number of persons.
In the present invention, the lung cancer is preferably the early stage of lung cancer.
In the present invention, the lung cancer is preferably non-small cell lung cancer；The more preferably non-small cell lung cancer of early stage.
According to the TNM stage of the 8th edition malignant tumour of UICC (International Union Against Cancer) publication.Lung cancer by stages usually
By three, element is formed by stages substantially, i.e. T by stages, N by stages, M by stages.By these three elements, codetermine it is last by stages.This
It is method by stages general at present.T is by stages: T refers to the case where primary tumors, with gross tumor volume increase and adjacent tissue by
The increase of tired range, is successively indicated with T1~T4.N is by stages: N refers to regional lymph nodes involvement.Lymph node by for a long time, is not used
N0 is indicated.With the increase of lymph node involvement degree and range, successively indicated with N1~N2.M is by stages: M refers to DISTANT METASTASES IN (usually
It is Blood route metastasis), no DISTANT METASTASES IN person indicates have DISTANT METASTASES IN person to be indicated with M1 with M0.
In the present invention, term " early stage of lung cancer " includes T1aN0M0, T1bN0M0, T1cN0M0 and T2aN0M0.
Advantageous effect of the invention
The present invention achieves one of following technical effect or a variety of:
(1) the present invention provides one kind can effectively detect Sera of Lung Cancer protein composition, which has the feature that
(a) level in Serum of Patients with Lung Cancer is relatively high；(b) level in normal human serum is relatively low.
(2) present invention utilizes CYFRA21-1, NSE, the IL-2, IL-8, IFN-γ, MIP-1 in antibody combined test sample
β and VEGF-A albumen can effectively detect lung cancer, and sensibility and specificity, which is significantly higher than, uses any one single albumen mark
The testing result of will object and clinical common blood serum designated object CYFRA21-1 and NSE.
(3) method of the invention can significantly improve the recall rate of the early stage of lung cancer, be conducive to patients with lung cancer early discovery, early control
It treats, improves the survival rate of patients with lung cancer.
Detailed description of the invention
Figure 1A-Fig. 1 G: patients with lung cancer and normal human serum marker ELISA detection.It can be seen that from figure Figure 1A-Fig. 1 G
CYFRA21-1 (Figure 1A), NSE (Figure 1B), IL-2 (Fig. 1 C), IL-8 (Fig. 1 D), IFN-γ (Fig. 1 E), MIP-1 β (Fig. 1 F) and
Seven levels of the albumen in Serum of Patients with Lung Cancer sample of VEGF-A (Fig. 1 G) are higher than normal human serum sample.
Fig. 2A-Fig. 2 D: blood serum designated object ELISA detection before and after Operation for Lung Cancer.It can be seen that from Fig. 2A-Fig. 2 D
The level of CYFRA21-1 (Fig. 2A and Fig. 2 B), NSE (Fig. 2 C and Fig. 2 D) albumen after Operation for Lung Cancer in serum sample is obvious
It reduces.Wherein:
Fig. 2A is the difference of patients with lung cancer preoperative and postoperative CYFRA21-1 serum levels mean value；Fig. 2 B is that same patient is preoperative
The variation of postoperative CYFRA21-1 serum levels, the left end of every straight line and right end are respectively the preoperative and postoperative CYFRA21-1 of patient
Serum levels.
Fig. 2 C is the difference of the horizontal mean value of patients with lung cancer preoperative and postoperative NSE；Fig. 2 D is same patient's preoperative and postoperative NSE serum
Horizontal variation, the left end of every straight line and right end are respectively the serum levels of the preoperative and postoperative NSE of patient.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
In the following examples, the ELISA reagent that detection CYFRA21-1 and NSE is used is purchased from DRG (Germany) company, goods
Number be respectively EIA5070 and EIA4610；The multiple-factor joint that detection IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A are used
Detection reagent is customized in eBioscience (U.S.) company, and article No. is that (this is that this research is special customized to EPX090-19450-801
Product, therefore without this product in the existing product of the said firm).
In the following examples, ELISA method and positive judgment criteria are as follows:
(1) ELISA: the specific detection method is as follows for each albumen:
(a) all reagents and required lath CYFRA21-1: are being placed into 30min at room temperature using preceding.Dissolution mark
Quasi- product and quality-control product prepare cleaning solution, and the lath of number needed for testing is fixed on grillage.The buffering of 50 μ l is added in every hole
The enzyme-linked object of liquid and 10 μ l (this is the antibody of specific protein marker, has been coupled the enzyme that can decompose substrate thereon).By 50
μ l standard items, quality-control product and blood serum sample are added in corresponding detection hole, are thoroughly mixed, and concussion is incubated for 60 minutes at room temperature.
The content in detection hole is quickly discarded, every hole is added 350 μ l cleaning solutions and cleans 3 times, firmly has the final say on blotting paper to remove
Residual liquid.100 μ l substrate solutions are added in every hole, at room temperature avoid light place 15 minutes.Every hole is added 100 μ l terminate liquids and terminates
Reaction.After adding terminate liquid in 10 minutes, at room temperature, absorptance is read at 450nm using microplate reader.
(b) all reagents and required lath NSE: are being placed into 30min at room temperature using preceding.Dissolve standard items and
Quality-control product prepares cleaning solution, and the lath of number needed for testing is fixed on grillage.By 25 μ l standard items, quality-control product and serum
Sample is added in corresponding detection hole, is thoroughly mixed.(this is specific protein mark to the enzyme-linked object that every hole is added after 100 μ l dilution
The antibody of object has been coupled the enzyme that can decompose substrate thereon), concussion is incubated for 60 minutes at room temperature.Quickly discard in detection hole
Content, every hole is added 300 μ l cleaning solutions and cleans 5 times, on blotting paper firmly clappers to remove residual liquid.Every hole adds
Enter 100 μ l substrate solutions, at room temperature avoid light place 15 minutes.Every hole is added 100 μ l terminate liquids and terminates reaction.After adding terminate liquid
In 5 minutes, at room temperature, absorptance is read at 450nm using microplate reader.
(c) IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A: standard items are redissolved and are collected (to antigen standard tubule
In every pipe 50 μ l specificity buffer 1 × Mniversal Assay Buffer of sample be added redissolve standard items.By antigen standard
Product tubule be placed on ice 10 minutes to ensure to redissolve completely.The entire contents of each tubule are collected into a tubule, and
Add 1 × Mniversal Assay Buffer to 250 μ l).Prepare 4 times of dilution standard product (centrifuge tube mark is 1~8 spare, to
200 μ l are added in No. 1 pipe and redissolve antigen standard, 150 μ 1 × Mniversal of l Assay are added into 2~No. 8 pipes
Buffer.It from taking 50 μ l to be added in No. 2 pipes in No. 1 pipe, mixes, until there is standard items in No. 7 pipes, No. 8 are managed this step in repetition
For blank well).50 μ l magnetic bead liquid are added in every hole.96 orifice plates (are fixed on hand-held magnetic bead cleaning machine, and ensure by cleaning magnetic bead
96 hole boards are good, 2min stood, so that magnetic bead accumulates in every hole board bottom.It is inverted, to discard the liquid in 96 orifice plates and clap net residual
Stay liquid.Every hole is added 150 μ 1 × Wash of l Buffer and stands 2min, so that magnetic bead accumulates in every hole board bottom.It is inverted, to abandon
Fall the liquid in 96 orifice plates and claps net Liquid Residue.96 orifice plates are removed from hand-held magnetic bead cleaning machine).It is loaded and is incubated for (every
25 μ l 1 × Mniversal Assay Buffer are added in hole, and then the ready standard items of 25 μ l or sample is added in every hole.
It sticks sealing plate film and covers black lid for micro plate, 500r/min is incubated at room temperature 120 minutes).Clean 96 orifice plates.Detection antibody is added
And it is incubated for (25 μ l 1 × Detection Antibody Mixture of every hole addition.96 orifice plates are sealed with new sealing plate film, are covered
Black lid for micro plate, the concussion of 500r/min room temperature are incubated for 30 minutes.Clean 96 orifice plates.SAPE is added and is incubated for (50 μ of every hole addition
l SAPE.96 orifice plates are sealed with new sealing plate film, cover black lid for micro plate, the concussion of 500r/min room temperature is incubated for 30 minutes).Clearly
Wash 96 orifice plates.Data are read on Luminex instrument, and (120 μ l Reading Buffer are added in every hole.It is close with new sealing plate film
96 orifice plates are sealed, black lid for micro plate is covered, the concussion of 500r/min room temperature is incubated for 5 minutes.Sealing plate film is removed, in Luminex instrument
Read 96 orifice plate data).
(2) positive judgment criteria: according to protein concentration as positive and negative judgment criteria, it is defined as Cut-off value.
The sample that protein concentration is greater than Cut-off value is denoted as the positive, and the sample less than Cut-off value is denoted as feminine gender.Above-mentioned seven albumen
The Cut-off value of concentration be respectively as follows: 2.71ng/ml (CYFRA21-1), 8.07ng/ml (NSE), 5.63pg/ml (IL-2),
0.29pg/ml(IL-8)、0.46pg/ml(IFN-γ)、45.13pg/ml(MIP-1β)、VEGF-A(456.00pg/ml)。
Embodiment 1: sensibility and specificity of the protein combination in Serum of Patients with Lung Cancer pattern detection
For 174 patients with lung cancer (predominantly non-small cell lung cancer is not distinguished and whether belongs to early stage) and 133 normal persons
Serum sample detects CYFRA21-1, NSE, IL-2, IL-8, seven IFN-γ, MIP-1 β and VEGF-A eggs using ELISA method
White level, and analyzed.
Experimental result is as shown in table 1, table 2 and Figure 1A-Fig. 1 G.
Table 1: the sensibility and specificity of single albumen in lung cancer
Note: whole samples are effective sample.
Table 2: the sensibility and specificity of protein combination in lung cancer
Note: 7, which select 2 positives to refer to, 2 antibody positives in 7 antibody；6, which select 2 positives to refer to, 2 antibody positives in 6 antibody；
5, which select 2 positives to refer to, 2 antibody positives in 5 antibody；4, which select 2 positives to refer to, 2 antibody positives in 4 antibody；2 select 1 positive to refer to 2
There is 1 antibody positive in a antibody；Whole samples are effective sample.
The result shows that seven CYFRA21-1, NSE, IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A albumen are in lung cancer
Level in serum is higher than normal human serum (referring to Figure 1A-Fig. 1 G)；Select four, five, six or seven protein antibodies connection
Detection lung cancer is closed, when wherein judging surveyed case there are two antibody positive for the positive, sensibility is up to 86% or more, specifically
Property up to 84% or more, be all remarkably higher than any one single albumen and clinical common blood serum designated object CYFRA21-1 and NSE
Select one and both combination testing result.
Embodiment 2: sensibility of the protein combination in the detection of early stage of lung cancer serum sample
For 29 early stages (I phase, II phase) lung cancer (non-small cell lung cancer), using ELISA method detection CYFRA21-1,
NSE, IL-2, IL-8, IFN-γ, the level of MIP-1 seven albumen of β and VEGF-A, and analyzed.
Experimental result is as shown in Table 3 and Table 4.
Table 3: the sensibility of single albumen in the early stage of lung cancer
| Protein name | Positive case number | Sensibility |
| CYFRA21-1 | 16 | 55.17% |
| NSE | 6 | 20.69% |
| IL-2 | 0 | 0.00% |
| IL-8 | 9 | 31.03% |
| IFN-γ | 24 | 82.76% |
| MIP-1β | 18 | 62.07% |
| VEGF-A | 19 | 65.52% |
Whole samples are effective sample
Table 4: the sensibility of protein combination in the early stage of lung cancer
Note: 7, which select 2 positives to refer to, 2 antibody positives in 7 antibody；6, which select 2 positives to refer to, 2 antibody positives in 6 antibody；
5, which select 2 positives to refer to, 2 antibody positives in 5 antibody；4, which select 2 positives to refer to, 2 antibody positives in 4 antibody；2 select 1 positive to refer to 2
There is 1 antibody positive in a antibody；Whole samples are effective sample.
The result shows that seven CYFRA21-1, NSE, IL-2, IL-8, IFN-γ, MIP-1 β and VEGF-A albumen are in early stage
Level in Serum of Patients with Lung Cancer sample is higher than normal human serum sample；Select four, five, six or seven albumen joint inspections
Survey the early stage of lung cancer, when two of them antibody positive is judged as the positive, sensibility up to 89% (specificity with embodiment 1,
Up to 84% or more), higher than any one single albumen and the detection knot of clinical common blood serum designated object CYFRA21-1 and NSE
Fruit.
Embodiment 3: variation of the protein combination before and after the Operation for Lung Cancer in serum sample
For before 20 lung cancer (non-small cell lung cancer) patients surgeries and postoperative serum sample, ELISA method is used
Detect the level of CYFRA21-1, NSE, six IL-2, IL-8, IFN-γ and VEGF-A albumen.
Shown in result figure 2A- Fig. 2 D and table 5.
Table 5: the low situation of the single proteinemina clear water pancake in Operation for Lung Cancer front and back
Note: whole samples are effective sample.
The results show that the serum levels of CYFRA21-1 and NSE albumen decline more obviously after surgery.By CYFRA21-
1, the albumen such as NSE, IL-2 and IFN-γ are combined analysis, 3 select 1 combination and 4 that 1 combine detection is selected to go out colorectal cancer as the result is shown
The ratio that serum levels reduce after ODP in operation is respectively 95% and 100%, and see Table 6 for details.
Table 6: protein combination serum levels reduce situation before and after Operation for Lung Cancer
Note: 2, which select 1 positive to refer to, has 1 protein level to reduce in 2 antibody；3, which select 1 positive to refer to, 1 albumen in 3 antibody
Level reduces；4, which select 1 positive to refer to, has 1 protein level to reduce in 4 antibody；Whole samples are effective sample.
The above result shows that four CYFRA21-1, NSE, IL-2, IFN-γ protein combinations detect the postoperative serum water of lung cancer
The low sensibility of pancake is higher than any one single albumen and the combination of CYFRA21-1 and NSE, shows above-mentioned protein combination energy
It is enough in treatment of colorectal cancer monitoring.
Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root
According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention
Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.
Claims (10)
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