CN108026584A - Diagnosis of Non-Small Cell Lung is with protein biomarkers group and utilizes its Diagnosis of Non-Small Cell Lung method - Google Patents
Diagnosis of Non-Small Cell Lung is with protein biomarkers group and utilizes its Diagnosis of Non-Small Cell Lung method Download PDFInfo
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Abstract
This disclosure relates to for the method from the biomarker group comprising biomarker and human diagnostic's lung cancer.Multiple methods for diagnosing are provided, the above method includes the step of at least one of the multiple biomarkers of detection provided in table 2 at least one biomarker values of biomarker from sample, based at least one above-mentioned biomarker values, the above-mentioned mankind are classified as the Asian with non-small cell lung cancer, or determine the possibility with lung cancer.
Description
Technical field
The present invention relates to Diagnosis of Non-Small Cell Lung with protein biomarkers group and to utilize its non-small cell lung cancer
Diagnostic method, is related to N number of in the biomarker protein matter containing stem cell factor acceptor (KIT) in more detail
Protein, for the protein biomarkers group from human diagnostic's non-small cell lung cancer.
Background technology
In the following description, there is provided brief with the relevant information of the application, the multiple information or bibliography provided
The prior art of the application is not identified as.
In South Korea or even the whole world, the main reason for lung cancer is still cancer related mortality (Torre LA, Bray F,
Siegel RL, Ferlay J, Lortet-Tieulent J, et al. (2015) Global cancer statistics,
2012, CA Cancer J Clin.).2011, South Korea was newly diagnosed to be the lung cancer more than 21753, is estimated to be more than 15000
Name dies of lung cancer (Jung KW, Won YJ, Kong HJ, Oh CM, Lee DH, et al. (2014) Can cer statistics
in Korea：Incidence, mortality, survival, and prevalenc e).Although the lung cancer morbidity of developed country
Rate is declining with the death rate, but the developing country that smoking rate persistently rises, especially among countries in Asia, lung cancer morbidity rate
(Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, et are being steeply risen with the death rate
Al. (2015) Global cancer statistics, 2012, CA Cancer J Clin.).
Since most of people with the early stage of lung cancer are not in symptom, the patient more than 60% can not it is curable into
Exhibition is diagnosed (Jemal A, Center MM, DeSantis C, Ward EM (2010) Global patterns of in the stage
cancer incidence and mortali ty rates and trends.Cancer Epidemiol Biomarkers
Prev 19：1893-1907；Jett JR(1993)Current Treatment of Unresectable Lung-
Cancer.Mayo Clinic Proceedings 68：603-611.).5 years survival rates of Patients with Advanced Lung Cancer are less than 10%,
But 5 years survival rates of 1 phase patient can be more than 70% (Hoffman PC, Mau er AM, Vokes EE (2000) Lung
cancer.Lancet 355：479-485.).Therefore, the early stage of the so-called lung cancer critically important for the reduction death rate and incidence
The research purpose of diagnosis has been converted into the research of pulmonary cancer diagnosis method.
(National Lung Screening Trial (NLS T)) (Aberle is tested in American National screening lung cancer
DR, Adams AM, Berg CD, Black WC, Clapp JD, et al. (2011) Reduced lung-cancer
mortality with low-dose computed tomographic screening.N Engl J Med 365：395-
409) in, lung is shown using the spiral computerized tomoscan of low dosage (CT, Computed Tomography) (LDCT) diagnosis
Cancer related mortality reduces by 20%, but the 24.2% of the spiral computerized tomoscan diagnosis of low dosage is the positive, these tubercles
96.4% for mistaken diagnosis (Aberle DR, Adams AM, Berg CD, Black WC, Clapp JD, et al. (2011),
Reduced lung-cancer mortality with low-dose computed tomographic screening.N
Engl J Med 365：395-409.；Aberle DR, Abtin F, Brown K (2013) Comput ed Tomography
Screening for Lung Cancer：Has It Finally ArrivedImplications of the National
Lung Screening Trial.Journal of Clinica l Oncology 31：1002-1008.).Except finding to invade
Outside attacking property tumour, in the experiment of American National screening lung cancer, found using the spiral computerized tomoscan of low dosage all
Lung cancer among lung cancer more than 18% is seemingly painless, when narration lung cancer as caused by the spiral computerized tomoscan of low dosage
During the danger of diagnosis, it is also contemplated that excessive diagnosis (Patz EF, Jr., P insky P, Gatsonis C, Sicks JD,
Kramer BS, et al. (2014) Overdiag nosis in low-dose computed tomography
screening for lung cancer.J AMA Intern Med 174：269-274).
Therefore, there is sensitivity and specific lung with what is measured in the biological specimen such as serum of non-invasive manner collection
Cancer biomarker is possible to contribute to that for high-risk subject, amorphism Lung neoplasm is found particularly by computed tomography
Patient make clinical decision.According to the individual serum biomarker for non-small cell lung cancer (NSCLC), mainly to cell
Data disclosed in Keratin 19 fragment 21.1 (Cyfra 21-1), carcinomebryonic antigen and tissue peptide antigen show that these biologies are marked
Will thing show sensitivity especially to the disease that is only limitted to initial stadium and specificity (Pastor A, Menendez R,
Cremades MJ, Pastor V, Llopis R, et al. (1997) Diagnostic value of SCC, CEA and
CYFRA 21.1in lung cancer：a Bayesian analysis.European Respiratory Journal 10：
603-609.；Buccheri G, Torchio P, Ferrigno D (2003) Clinical equivalence of two
cytokeratin markers in non-small cell lung cancer-A study of tissue
polypeptide antigen and cytokeratin 19fragments.Chest 124：622-632.；Lai RS, Hsu
HK, Lu JY, Ger LP, Lai NS (1996) C YFRA 21-1enzyme-linked immunosorbent
assay.Evaluation as a tum or marker in non-small cell lung cancer.Chest 109：
995-1000.)。
Due to the development and the understanding of genomics to molecular diagnosis method, it was found that existing diagnosis mark can be made up by having
Several lung cancer biomarkers (Hasan N, Kumar R, Kavuru MS (2014) Lung cancer of accurate potentiality
screening beyond low-dose compute d tomography：the role of novel
biomarkers.Lung 192：639-648.；B igbee WL, Gopalakrishnan V, Weissfeld JL, Wilson
DO, Dacic S, et al. (2012) A multiplexed serum biomarker immunoassay panel dis
criminates clinical lung cancer patients from high-risk individuals fou nd to
be cancer-free by CT screening.J Thorac Oncol 7：698-708.；Daly S, Rinewalt D,
Fhied C, Basu S, Mahon B, et al. (2013) D evelopment and validation of a plasma
biomarker panel for discernin g clinical significance of indeterminate
pulmonary nodules.J Thorac Oncol 8：31-36.；Gold L, Ayers D, Bertino J, Bock C,
Bock A, e t al. (2010) Aptamer-based multiplexed proteomic technology for bio
marker discovery.PLoS One 5：e15004.；Ostroff RM, Bigbee WL, Franklin W, Gold L,
Mehan M, et al. (2010) Unlocking biomarker discovery：large scale application of
aptamer proteomic technology fo r early detection of lung cancer.PLoS One 5：
e15003.；Pecot CV, Li M, Zhang XJ, Rajanbabu R, Calitri C, et al. (2012) Added val ue
of a serum proteomic signature in the diagnostic evaluation of lu ng
nodules.Cancer Epidemiol Biomarkers Prev 21：786-792.).Such as O stroff majority report,
They are found that protein biomarkers group (Gold L, Ayers D, Bertino J, Bock for lung cancer early diagnosis
C, Bock A, et al. (2010) Aptamer-based multiplexed proteomic technology for
biomarker disco very.PLoS One 5：e15004.；Ostroff RM, Bigbee WL, Franklin W, Gold
L, Mehan M, et al. (2010) Unlocking biomarker discovery：l arge scale application
of aptamer proteomic technology for early dete ction of lung cancer.PLoS One
5：e15003.).
However, the epidemiology of lung cancer is different according to national characteristic or provincialism background with molecular biology,
And not yet realize the exploitation of protein biomarkers or the integration with Computed tomography model.
Aptamer (aptamer) is a kind of new bio molecule (bio-molecule), is screened in large-scale oligonucleotides
(oligo) storehouse.Operated by the aglucon phyletic evolution technology (SELEX) of a series of index concentration, to extend particular ligand
(ligand) subject nucleic acid aptamer and finally select.Aptamer is can chemically be synthesized and in order to which a variety of purposes are easy to
The single-chain nucleic acid of deformation.Also, aptamer can be by PCR (polymerase chain r eaction)
Method is expanded and analyzed, and is applicable in high power capacity DNA (DN A) array technique.
High power capacity quantitative analysis method based on aptamer has been reported and has proved for screening multivariable protein spy
Sign is come with the outstanding platform for the state of diagnosing the illness.The protein more than 1000 can be measured at the same time on single platform, thus
Can have can analyze the chance of human sample to find disease-specific proteins feature.
The content of the invention
The technical problem of solution
It is an object of the present invention to provide for the method from human diagnostic's non-small cell lung cancer.
Also, it is an object of the present invention to provide the protein biomarkers group for finding non-small cell lung cancer.
The purpose of the present invention is not limited to above mentioned content, general technical staff of the technical field of the invention
Described content it can be clearly understood that other unmentioned purposes from below.
Technical solution
In order to solve the above-mentioned technical problem, one embodiment of the invention, which provides, is used for diagnosing, non-small particularly for diagnosing
The protein biomarkers group of cell lung cancer.In an embodiment of the present invention, multiple biomarkers are utilized in multiple implementations
The detection method based on multiple nucleic acid aptamer that describes in detail is identified in example.Utilization of the present invention is described in the present invention
The above-mentioned detection method based on multiple nucleic acid aptamer, come illustrate the detection to non-small cell lung cancer and help diagnosis non-small cell
Lung cancer biomarker catalogue.In order to identify these biomarkers, from be diagnosed as in the past the presence of non-small cell lung cancer with
The candidate biomarker protein of a small group is determined in no Asian sample.As shown in table 2, by being compared to each other use
In analyzing each biomarker and distinguishing patient's group and the performance of control group, to have selected a small group with more preferable performance
Biomarker.Such as in the detailed description of each embodiment, according to naive Bayesian theorem (Bayesian theorem)
Generate and analyze multivariable classification device.
In order to achieve the object of the present invention, the feature being used for from the method for human diagnostic's lung cancer of one embodiment of the invention exists
In,
Including：There is provided comprising N number of biomarker group in the multiple biomarker protein matter listed in table 2, on
It is the step of being at least 2 integer to state N；And in order to assign N number of biomarker egg with above-mentioned biomarker group respectively
The corresponding biomarker values of white matter, detect multiple above-mentioned biomarker protein matter from from the biological specimen of the mankind
The step of, above-mentioned lung cancer is diagnosed based on multiple above-mentioned biomarker values.
The step of the step of detecting multiple above-mentioned biomarker values may include to carry out biological vivo detection.
Above-mentioned biology vivo detection can include a capture agent corresponding to each above-mentioned biomarker protein matter,
It is above-mentioned be used for from the method for human diagnostic's lung cancer may also include in the group being made of aptamer, antibody and nucleic acid probe selection to
A kind of the step of few capture agent.
Above-mentioned at least one capture agent can be aptamer.
Above-mentioned biological specimen may be selected from the group that is made of whole blood, blood plasma and serum.
Above-mentioned biological specimen can be serum.
The above-mentioned mankind can be Asian.
The above-mentioned mankind can be smoker.
The above-mentioned mankind can have malign lung nodules.
Above-mentioned N can be 3,4,5,6,7 or its more than.
Multiple above-mentioned biomarker values can be complement component C9, carbonic anhydrase (carbonic anhydrase) 6
(CA6), C reactive protein (CRP), ErbB1 (EGFR1), matrix metalloproteinase 7 (MMP7), the anti-eggs of α 1-
The measured value of white enzyme (SERPINA3) and stem cell factor acceptor (KIT).
Multiple above-mentioned biomarker values can be by by real-time polymerase chain reaction (PCR), microarray and Lu Mingkesi
Method in the group of microballoon detection method (Luminex microbead assay) composition is measured.
Above-mentioned lung cancer is diagnosed by statistical method.
Above-mentioned statistical method may be selected from by linear discriminant analysis, logistic regression analysis, Naive Bayes Classification, supporting vector
In the group of machine and random forest (random forest) composition.
Also, as it is a kind of be used for realization the purpose of the present invention, for the albumen from human diagnostic's non-small cell lung cancer
Matter biomarker group is characterized in that, the above-mentioned protein biomarkers group bag being used for from human diagnostic's non-small cell lung cancer
Containing N number of biomarker egg in the multiple biomarker protein matter listed in the table 2 containing stem cell factor acceptor
White matter, N are at least 2.
Above-mentioned N can be 3,4,5,6,7 or its more than.
It is above-mentioned to be used for having complement component C9, carbon from the protein biomarkers group of human diagnostic's non-small cell lung cancer
Acid anhydrides enzyme (carbonic anhydrase) 6 (CA6), C reactive protein (CRP), ErbB1 (EGFR1), base
The measured value of matter metalloproteinases 7 (MM P7), α 1- antiproteases (SERPINA3) and stem cell factor acceptor (KIT).
The above-mentioned mankind can be Asian.
The above-mentioned mankind can be smoker.
The above-mentioned mankind can have malign lung nodules.
The effect of invention
Above-mentioned protein biomarkers group can provide for finding the protein biomarkers group of non-small cell lung cancer.
The effect of the present invention is not limited to above mentioned content, general technical staff of the technical field of the invention
Described content it can be clearly understood that other unmentioned effects from below.
Brief description of the drawings
Fig. 1 is the research flow chart for Algorithm for Training and verification.
Fig. 2 illustrates the Multiple detection method based on aptamer.
Fig. 3 diagrams are suitable for the model of normal cumulative distribution function (CDF) and the example of initial data.
Fig. 4 a to Fig. 4 n are the figure being compared to the candidate markers between patient-control group.
Fig. 5 a to Fig. 5 c diagram to 7- markers Naive Bayes Classifier (Bayes cl assifier) by
Examination person's operating characteristic (ROC) curve.
Fig. 6 a to Fig. 6 g illustrate the Receiver operating curve to 6- marker Naive Bayes Classifiers.
Fig. 7 a and Fig. 7 b are the figure to 7- markers Naive Bayes Classifier compared with the performance of Cyfra 21-1.
Embodiment
With reference to representational embodiment, specifically the present invention is described.The present invention is described with reference to listed embodiment, but
It should be understood that the present invention is not limited to above-described embodiment.In contrast, the present invention is included as by inventing claimed scope institute
Define and may be included in all schemes, change and equivalent in the scope of the present invention.
Those skilled in the art are it will be recognized that similar or equivalent with described content in the present invention
All multi-methods may be included in or be included within the scope of the present invention with material.The present invention is by no means limited to described multiple methods
And material.
Unless otherwise defined, the technology used in the present invention and scientific terminology and the technical field of the invention is common
Technical staff is commonly used to have identical implication.Although similar or equivalent any side with content described in the invention
Method, device and material implementation for use in the present invention or test, but method for optimizing, device and material will now be described.
All documents, publication file and patent application cited in the present invention represents technology neck belonging to the present invention
The level in domain.Cited multiple documents, publication file and patent application are incorporated herein by reference in the present invention, its journey
Degree is with each document, publication file and patent application by specifically and explicitly by being incorporated herein by reference presented journey
Spend identical.
Unless the context clearly determines otherwise, in the list being claimed including appended invention used in the present invention of scope
Number form formula " one (" a ", " an " and " the ") " include plural number expression, with " at least one (at least one) " and " one or
(one or more) more than it " is mixed.Therefore, " aptamer (an aptamer) " includes the mixture of aptamer, " visits
Pin (a probe) " includes the mixture of probe.
Term " about (about) " used in the present invention represents trickle numerical value change or change, its degree is and number
The not changed degree of basic function of the associated project of value.
Used in the present invention term " including (" comprises ", " comprising ", " includes ",
" including ", " contains ", " containing ") " and the version of the term cover, comprising any key element or will
The product (product-by-process) that process, method, the method for plain catalogue limit, or the composition of material not only may include
These key elements, may also include the product that not expressly listed or this process, method, method limit, or the composition institute of material is intrinsic
The nonexcludabilities of other multiple key elements include (nonexclusive inclusion).
In one embodiment, it is based on using for biomarker subset or the quantity of the biomarker of biomarker group
Sensitivity and specificity values in the combination of particular organisms marker levels.Herein, so-called " sensitivity " and the term of " specificity "
With according to from detection of biological samples to one or more biomarker values come find exactly individual whether have it is non-small thin
The ability of born of the same parents' lung cancer is associated use." sensitivity " represents to be used for identify that the mankind's with non-small cell lung cancer is (more exactly
It is a) performance of biomarker." specificity " represents to be used for identify that the mankind's without non-small cell lung cancer is (more exactly
It is a) performance of biomarker.
Include to the more conventional property of the present invention biomarker, method, apparatus, reagent, system and non-for finding and diagnosing
The kit of Small Cell Lung Cancer and cancer.
The term of so-called " lung (lung) " used herein is possible to mixed with the term of so-called " lung (pulmonar y) ".
The term of so-called " smoker " used herein refers to the individual of the suction history with smoke from cigarette.
" biological specimen (biological sample) ", " sample (sample) " and " sample (test sample) " refers to
Obtain from individual or otherwise derived material, biofluid, tissue or cell, can use with the present invention.This is included
Blood (includes whole blood (whole b lood), leucocyte (leukocytes), peripheral blood mononuclear cells (peripheral
Blood mo nonuclear cells), buffy coat (buffy coat), blood plasma (plasma) and serum (serum)),
Phlegm (sputum), tears (tears), mucus (mucus), nasal wash (nasal washes), nasal cavity extract (nasal
Aspirate (breath), urine (urine), sperm (semen), saliva (saliva), peritoneal wash fluid), are breathed
(peritone al washing), cystic fluid (cystic fluid), amniotic fluid (amniotic fluid), gland liquid
(glandular fluid), lymph (lymph fluid), cell liquid (cytologic fluid), ascites (ascites),
Liquor pleurae (pleural fluid), nipple aspirate (nipple aspira te), bronchus aspirate (bronchial
Aspirate), bronchial brushing (bronchial br ushing), synovia (synovial fluid), joint aspirate
(joint aspirate), tissue secretion thing (organ secretions), cell (cell), cell extract (cell
Extra ct) and celiolymph (cerebrospinal fluid).This is also included experimentally from above-mentioned listed material
Separated fragment (fractions).For example, blood sample, which is separable into, includes such as serum, blood plasma or red blood cell or leucocyte
Specific modality blood cell fragment.If it is desired, sample can be the sample combination of individual, such as tissue samples and body
The combination of liquid sample.Above-mentioned term " biological specimen (biological sample) " is also included containing the solid matter to homogenize
Material, for example originating from fecal sample, tissue samples or organize biopsy sample sample.Above-mentioned term " biological specimen " is also wrapped
Containing the material from tissue cultures or cell culture.The proper method for obtaining biological specimen can be used；Representational side
Method includes for example, blood sampling, swab method (for example, mouth epithelial cells swab method) and Fine needle aspiration biopsy.Can be into
The representational tissue of row Fine needle aspiration include lymph node, lung, lung-douching fluid, bronchoalveolar lavage (BAL,
Bronchoalveolar lavage), thyroid gland, breast and liver (liver).Multiple samples can also be for example, by micro- to cut
Cut (for example, detection wind lidar (laser capture microdissection；) or laser microprobe dating LCM
(laser micro dissecti on；LMD)), bladder irrigation, smear (for example, Pasteur (PAP) smear) or latex dust cleaning side
Method gathers.Obtained from individual or " biological specimen " from individual includes carrying out in a suitable approach after obtaining from individual
The sample of processing.
And it will be appreciated that biological specimen can be by obtaining multiple biological specimens and by the sample system from most mankind
Cause the part sample for collecting the biological specimen of (pool) or each individual to be manufactured into collect to export.It is manufactured into the sample collected
Can as the sample process from an individual, once from the presence that cancer is determined by the above-mentioned sample for collecting manufacture, so that it may weight
It is new to measure single sample to determine which (a little) individual has non-small cell lung cancer.
For the purpose of this specification, the sentence of so-called " data for being attributed to the biological specimen of individual " means above-mentioned number
According to any type of data for the biological specimen for being derived from individual or using biological specimen come the data that generate.Above-mentioned data life
Turn into afterwards, being to a certain extent continuously reformatted, change or mathematics modification, such as from the unit of an assay method
Change to the unit of another assay method.However, above-mentioned data are interpreted as being derived from biological specimen or are given birth to using biological specimen
Into.
" target (target) ", " target molecule (target molecule) " and " analyte (analyte) " in the present invention may be used
It is mixed, to censure any molecule interested that may be present in biological specimen." molecule (molecule of interested
Interest) " include：For example, minor variations, disulfide formation in the amino acid sequence in the case of protein
(disulfide bond form ation), glycosylation (glycosylation), esterified (lipidation), acetylation
(acety lation), phosphorylation (phosphorylation) or the essential marker components with not changing molecule substantially
Other operations (manipulati on) or change (modification) of combination between (labeling component) etc.
Deng specific molecular minor variations (minor variation)." target molecule ", " target " or " analyte " is a kind of form or kind
The molecule of class or the copy group of multiple molecular structure." multiple target molecules ", " multiple targets " and " multiple analytes " refer to one with
On this molecule group.In the example of target molecule, protein (proteins), polypeptide (polyp eptides), nucleic acid are included
(nucleic acids), carbohydrate (carbohydrates), lipid (lipids), polysaccharide
(polysaccharides), glycoprotein (glycoproteins), hormone (hormones), acceptor (receptors), antigen
(antigens), antibody (anti bodies), affine body (affibodies), autoantibody (autoantibodies), antibody
Analogies (antibody mimics), viral (viruses), pathogen (pathogens), noxious material (toxic
Substances), matrix (substrates), metabolite (metaboli tes), transition state analog (transition
State analogs), confactor (cofactor s), inhibitor (inhibitors), medicine (drugs), dyestuff
(dyes), nutritional ingredient (nutrients), growth factor (growth factors), cell (cells), tissue (tis
Sues) and the above-mentioned material listed fragment or a part.
" polypeptide (polypeptide) ", " peptide (peptide) " and " protein (protein) " used in the present invention
It can use with the present invention, for use in the polymer for the amino acid for censuring random length.Above-mentioned polymer can be straight chain
Type or branched chain type, and the amino acid of change can be included, it can be cut by non-amino acid (non-amino acid).Above-mentioned term
Also include naturally or artificially for example, by disulfide formation, glycosylation, esterified, acetylation, phosphorylation or and marker components
Between with reference to etc. other operation or change and change amino acid polymers.The definition of above-mentioned term is included for example, (example
Such as, comprising alpha-non-natural amino acid etc.) the similar body of one or the amino acid more than it, and including multiple public in the art
The polypeptide for other changes known.Polypeptide can be single-stranded or connection chain.Protein precursor (preprotein) or full maturity egg
White matter (intact mature protein)；Peptide or polypeptide from mature protein；The fragment of protein；Splice variant
(splice variant)；The restructuring form of protein；Protein variant with amino acid change, missing or substitution；Digestion
Thing (digests)；And (post-translational is changed after such as translation of glycosylation, acetylation, phosphorylation
Modificat ion) it is also included within above-mentioned definition.
" marker (marker) " and " biomarker (biomarker) " used in the present invention can be used with, so as to
In the indication for censuring disease in indication or individual normal in individual or be in progress extremely or other states or show these target point
Son.In more detail, " marker " or " biomarker " is normal or abnormal, and if it is abnormal, then for it is chronic or acute
Specific physiological status or progress there are relevant anatomy, physiology, biochemistry or molecules parameter.Biological marker
Thing can be detected and be measured by a variety of methods including test in laboratory and medical imaging.It is albumen in biomarker
In the case of matter so that the gene for the protein coding that controls the expression of biomarker or above-mentioned biomarker
Under biological specimen or methylation state, the expression of the gene can be used as the amount to the protein biomarkers or presence or absence
Act on behalf of index (surrogate measure).
" biomarker values (biomarker value) ", " value (value) ", " biological marker used in the present invention
Thing level (biomarker level) " and " horizontal (level) " are any point utilized from detection of biological samples biomarker
Analysis method is measured, also, in the above-mentioned biological specimen, in order to censure represent biomarker, for biological marker
Thing or the presence corresponding to biomarker, be not present, absolute magnitude or concentration, relative quantity or concentration, titer (titer),
Horizontal (level), expression, measure horizontal ratio etc. measured value and use with.The correct characteristic of " value " or " level "
Particular design and component depending on the particular analysis method for detecting biomarker.
When biomarker is represents the abnormality progress or disease or other states or its mark in individual, the biology
Marker usually represents being not present for normality progress in individual or disease or other states, or the life with the mark as it
The expression or value of thing marker compare, and show as excessive expression (over-expressed) or cross low expression (under
Expressed it is a kind of in)." up-regulation (up-regulation) ", " (up-regulated) through up-regulation ", " excessive expression
(over-expression) ", " (over-expressed) through excessive expression " and the change type of the performance are to censure ratio
Typically from the value similar to biomarker that is health or being detected in normal individual biological specimen or level (or
Value or level scope) higher biological specimen in biomarker values or level and use with.Multiple above-mentioned terms also can refer to
Claim the value of biomarker than that can be detected in the different step of specified disease or level (or value or horizontal model
Enclose) biomarker values or level in the biological specimen of higher.
" lowering (down-regulation) ", " (down-regulated) through downward ", " cross low expression (under-
Expression) ", " (under-expressed) Jing Guo low expression " and the performance change type are to compare typicalness to censure
Ground from health or normality individual similar detection of biological samples to biomarker value or level (value or water
Flat scope) smaller biological specimen in biomarker values or level and use with.Multiple above-mentioned terms can be also censured than energy
The value or level (or scope of value or level) of enough biomarkers detected from the different step of specified disease
Biomarker values or level in the biological specimen of smaller.
Also, normality progress or the disease being referred to alternatively as through excessive expression or the biomarker Jing Guo low expression in individual
The expression being not present of disease or other states, or with showing " normality " expression or value phase of its biomarker
Compare, there is " expressing to otherness (differentially expressed) " or " level (differential of otherness
) " or " value (differential value) of otherness " level.Therefore, " expression of otherness " of biomarker can also
It is enough to be showed with the change of " normality " expression of biomarker.
So-called " gene expression (differential gene expression) of otherness " and " expression of otherness
The term of (differential expression) " be in order to censure with normal subjects or control object in expression compared with,
The gene of expression activity (or corresponding albumen is carried out with higher or lower level in object with specified disease
Matter expression product) and use with.Above-mentioned term is also included in the different step of same disease with high level or low-level
To carry out the gene of expression activity (or corresponding protein expression product).Above-mentioned term can also become otherness earth's surface
The gene reached is activated or is suppressed in nucleic acid level or protein level, or prepares mutually different polypeptide product
Alternative splicing (alternative spli cing).These differences can be by polypeptide messenger ribonucleic acid levels, surface
Expression, secretion distribute a variety of changes of (partitioning) etc. and become clear and definite.The gene expression of otherness may include two
The comparison of expression between a or gene more than it or their gene outcome；Or two or the gene more than it or they
Gene outcome between expression ratio comparison；It is or on the contrary between normal subjects and the subject with disease or identical
The comparison of different, mutually isogenic two products handled in a different manner between a variety of stages of disease.Otherness
Expression is included for example, in normal and diseased cell, or the mutually different illness events of experience or disease stage is multiple thin
In gene or their expression product between born of the same parents, according to the time or in cellularity expression pattern quantitative differences and qualitative difference
It is different.
" being diagnosed (diagnose) ", " (diagnosing) of diagnosis ", " diagnosis (diagn osis) " and these arts
The change type of language refer to based on individual relevant one or sign, symptom, data or other information more than it, to individual
Health status or situation discovery, judgement or cognition.The health status of individual can be diagnosed as health/it is normal (i.e., not
There are disease or illness), or unsound/abnormal (that is, there are the assessment of disease or illness or characteristic) can be diagnosed as.
Above-mentioned term " diagnosis ", " diagnosis ", " diagnosis " etc. are included with specified disease or illness relatively, the early stage hair of disease
It is existing；The characteristic of disease or classification；Progress, healing or the discovery of recurrence of disease；After the disposal or treatment of individual, to the anti-of disease
The discovery answered.The diagnosis of non-small cell lung cancer includes not suffering from the individual of cancer and the individual difference with cancer.Also, it is also wrapped
Include non-small cell lung cancer and smoker and the difference of positive Lung neoplasm.
" prediction prognosis (prognose) ", " (prognosing) of prediction prognosis ", " prediction prognosis (prognosis) " and
The evolution prediction that the change type of these terms refers to carry out disease or illness in the individual with disease or illness is (for example, prediction
Survival), this term includes the assessment for disease reaction after individual disposal or treatment.
" assessment (evaluate) ", " (evaluating) of assessment ", " assessment (evaluation) " and these terms
Change type includes " diagnosis (diagnose) " and " prediction prognosis (prognose) ", and further includes from the individual for not suffering from disease
Carry out the judgement or prediction of the progress to disease or illness, and having to disease and illness in the individual substantially cured of disease can
The judgement or prediction for the possibility that can be recurred.Above-mentioned term " assessment " further includes pair for example, individual smoothly reacts therapeutic agent, also
It is the prediction for not reacting therapeutic agent (alternatively, for example, toxicity will be undergone or undergo other undesirable side effects), selects to a
The therapeutic agent or observation or the individual for treatment for finding the individual reaction for the treatment of to individual progress etc. that body is offerd medicine
Reaction assessment.Therefore, to non-small cell lung cancer carry out " assessment " may include for example, from individual prediction non-small cell lung cancer into
Exhibition process；The recurrence of non-small cell shape lung cancer is predicted in the patient substantially cured from non-small cell lung cancer；Or judge or predict
Individual reaction for Treatment for Non-small Cell Lung or the survey based on biomarker values derived from the biological specimen as individual
The fixed Treatment for Non-small Cell Lung method to select for individual.
Multiple examples are that " diagnosis " or " assessment " of non-small cell lung cancer can be censured to be as follows below：Early detection is non-small thin
The presence or absence of born of the same parents' lung cancer；Judge moment, type or the hypotype or other classification or characteristic of non-small cell lung cancer；Judge to doubt
Whether it is positive or pernicious non-small cell lung cancer like Lung neoplasm or tumour；Or the progress of non-small cell lung cancer is (for example, observation is swollen
The growth of knurl or transfer velocity), discovery/observation improves or recurrence.
" extra biomedical information (additional biomedical i used in the present invention
Nformation) " refer in addition to the use of biomarker described in the present invention, risk of cancer degree or more specifically
Individual evaluation thing for one related with non-small cell lung cancer risk or more than it." extra biomedical information " wraps
Include the shape of individual, shape, the height and/or body of individual of Lung neoplasm found in computed tomography (CT) image
Weight, the gender of individual, the national characteristic of individual, smoking history, professional history, the known carcinogen that is exposed to are (for example, asbestos, radon
The smog and the emission of industry/factory or automobile/ship/aircraft etc. that gas, chemical substance, fire produce, exposed to can wrap
Include the air pollution of the emission from static father or moving source), second-hand smoking, the family of non-small cell lung cancer (or other cancers)
Race's history, the presence of Lung neoplasm, the size of tubercle, the position of tubercle, the form of tubercle are (for example, from computed tomography image
Found in tubercle, ground-glass-like shade (ground glass opacity；GGO), solid type, non-solid type, tubercle
Interface feature is (for example, smooth (smooth), lobulated (lobulated), clear (sharp and clear), needle-shaped
(spiculated), (infiltrating) is infiltrated) etc..Smoking history is mainly multiplied by the average perfume (or spice) inhaled daily by so-called years of smoking
The viewpoint of " bag year (the pack years) " of cigarette case number is come by quantification.For example, the mankind of average 35 years daily 1 bags of smoking can table
It is now the smoking history with 35 bag years.Extra biomedical information can utilize known routine techniques to be obtained from individual.For example,
Can by daily outpatient service or healthy resume outpatient service etc., by individual with or healthcare practitioners obtain extra biomedical information.Make
For alternative, extra biomedical information can by including computed tomography image (for example, low dosage computerized tomography
Scan-image) and X-ray conventional image technical limit spacing.When the assessment and biomarker that combine extra biomedical information
During the test of level, for example, testing or individually assessing one in extra biomedical information with individually implementation biomarker
Kind of specific project is compared (for example, individually computed tomography image), can make that (or other are non-small thin to non-small cell lung cancer
Born of the same parents' lung cancer associated uses) detection sensitivity, specificity and/or area under the curve (AUC) be improved.
" area under the curve (area under the curve) " or " area under the curve (AUC) " refer in the art
Well-known to Receiver operating curve (receiver operating characteristic curve；ROC)
Lower area.Area under the curve (AUC) measure helps to carry out the accuracy of comparator-sorter via overall data scope.With bigger
Area under the curve (AUC) grader have bigger ability with two groups interested (for example, non-small cell lung cancer sample
This and normal or check sample) between Accurate classification unknown material.It is to distinguish Liang Ge colonies (for example, having non-small cell lung cancer
Group be not non-small cell lung cancer control group) in aspect, Receiver operating curve (ROC) is useful in graphical form
To show specific feature (for example, biomarker described in the present invention and/or extra biomedical information is any
Project) performance.Generally, based on single features value, via the features described above number of whole colony (for example, patient's group and control group)
Arranged according to by ascending order.Then, for each value of features described above, True Positive Rate (the true positi ve for data are calculated
) and false positive rate (false positive rate) rate.By calculate be higher than for its characteristic value more than case load it
Afterwards, divided by total case load measures above-mentioned True Positive Rate.By calculate be higher than for its characteristic value more than control group number it
Afterwards, divided by total control group number measures above-mentioned false positive rate.Although this definition refers to the characteristic of patient's group relative to control group height
Situation, but this definition apply also for patient group characteristic (in this case, can be calculated relative to the low situation of control group
Less than the number of the sample of the value of above-mentioned characteristic).Receiver operating curve (ROC) can be directed to other single calculating, may be used also
Generated for single characteristic, in order to provide single and value (single sum value), for example, can mathematically combine two with
On characteristic (for example, plus, subtract, multiply), which can be represented by Receiver operating curve (R OC).It is additional
Ground, can draw the combination for the multiple characteristic that can export single calculated value with Receiver operating curve (ROC).These are special
Property combination may make up test.Above-mentioned Receiver operating curve (ROC) is represents that (1- is special relative to the false positive rate of test
Property) test True Positive Rate (sensitivity) chart.
" detection (detecting) " or " measure for biomarker values used in the present invention
(determining) " it is used to find and record corresponding to the utensil and use needed for the signal of biomarker values including all
In the use for generating (multiple) material needed for its signal.In various embodiments, above-mentioned biomarker values are glimmering using including
Light (fluorescence), chemiluminescence (chemiluminescence), surface plasma resonance technology (surface
Plasmon r esonance), surface acoustic wave (surface acoustic waves), mass spectrometry (mass
Spectrometry), infra-red sepectrometry (infrared spectroscopy), Raman spectroscopy (r aman
Spectroscopy), atomic force microscope (atomic force microscopy), scanning-tunnelling formula microscope (scanning
Tunneling microscopy), electrochemical assay (e lectrochemical detection methods), nuclear-magnetism it is common
Shake (nuclear magnetic reso nance), quantum dot (quantum dots) the methods of in any appropriate method
To be detected.
In the present invention, " solid support (solid support) " refer to can be by covalent bond or non-common with molecule
A kind of any matrix on surface either directly or indirectly to adhere in valence link." solid support " can have may include for example,
Film (membrane)；Chip (chip) (for example, protein-chip)；Glass slide (slide) is (for example, glass slide or lid glass
Piece)；Column；With cavity form, solid, semisolid, for example, the pore (pore) of microballon (bead) etc. or the grain of chamber (cavity)
Son；Gel；Fiber comprising fiber optic materials；Matrix；And a variety of physical aspects of sample container (receptacle).As sample
The example of container, sample well, pipe, capillary, bottle (vial) and any other can fix the pipe, groove or curvature of sample.Sample
Container can be located at such as on the multiple example platform of microtiter plate (microtiter plate), glass slide, microfluidic device.
Supporter can be formed by natural or synthetic material, organic or inorganic material.It is commonly used for the group of the solid support of capture agent
Into depending on adherence method (for example, covalent bond (covalent attachment)).As the example of other said vesses, bag
Including to adjust can be analyzed in inside and the droplet of relevant operation (microdroplet) and miniflow (mi crofluid) or be dissipated
The oil/water emulsion of dress form.Appropriate solid support is included for example, plastics, resin, polysaccharide, silica or dioxy
SiClx sill, functional glass, modified silica-gel, carbon, metal, unorganic glass, film, nylon, (for example, silk, wool and cotton etc.
) natural fiber, polymer etc..Above-mentioned substance is by that can include carboxyl (carboxy), ammonia for example, for adhering to capture agent
The solid support of the reactive group of base (amino) or hydroxyl (hydroxyl) etc. is formed.Polymolecularity solid support can
Comprising for example, polystyrene (polystyrene), polyethylene terephthalate (polyethylene glycol
Tetrapht halate), polyvinyl acetate (polyvinyl acetate), polyvinyl chloride (polyvinyl c
Hloride), polyvinylpyrrolidone (polyvinyl pyrrolidone), polyacrylonitrile (pol yacrylonitrile),
Polymethyl methacrylate (polymethyl methacrylate), polytetrafluoroethylene (PTFE) (polytetrafluoroethylene)
, butyl rubber (butyl rubber), styrene butadiene ribber (styrenebutadiene rubber), natural rubber
(natural rubber), polyethylene (polyethylene), polypropylene (polypropylene), (poly-) tetrafluoroethene
((poly) tetrafluoroethylene), (poly-) vinylidene fluoride ((poly) vinylide nefluoride), poly- carbonic acid
Ester (polycarbonate) and polymethylpentene (polymethylpe ntene).Workable appropriate solid support grain
Attached bag contain for example,- type encoded particles (- type encoded particles), magnetic-particle
Encoded particles (the encoded of (magnetic par ticles) and glass particle (glass particles) etc.
particles)。
The example of use of biomarker
In various exemplary embodiment, there is provided following is used for from the side of the mankind (individual) diagnosing non-small cell lung cancer
Method：By including any number of analysis method of one in multiple analysis methods described in the invention, to detect quite
One or one of biomarker of such as serum or blood plasma more than it in the circulatory system for being present in individual or its
Biomarker values above.That is, compared with the mankind without non-small cell lung cancer, these biomarkers can be from
Expressed to mankind's otherness with non-small cell lung cancer.The differential expression for finding biomarker from individual can be used for
Following situation：For example, between the early diagnosing of non-small cell lung cancer, positive Lung neoplasm and malign lung nodules differentiation (for example, from
The tubercle that computed tomography Imaging Study arrives), the observation of non-small cell lung cancer recurrence or the observation of other clinical symptoms.
Biomarker described in the present invention can be used for various clinical non-small cell lung cancer sign, and including following feelings
Condition：(in excessive risk individual or group) finds non-small cell lung cancer；That is, pass through non-small cell lung cancer and cellule type
The difference (or simplifying tissue case) of the difference of lung cancer and/or gland cancer and squamous cell carcinoma, the characteristic of non-small cell lung cancer
Provide (for example, non-small cell lung cancer type, hypotype, or stage determine)；It is positive nodule or pernicious lung cancer to determine Lung neoplasm；
The prognosis of non-small cell lung cancer determines；Observe progress or the improvement of non-small cell lung cancer；Observe non-small cell lung cancer recurrence；Transfer
Observation；Treatment method is selected；Observe the reaction to therapeutic agent or other treatment method；Pass through the trouble of computed tomography inspection
Person's classification (for example, non-small cell lung cancer risk is higher, thus can be checked with the computed tomography of recognition helix shape the most by
The mankind of benefit, so that the positive prediction degree increase of computed tomography)；With smoking history etc. and the knot of nodule size
Conjunction, biomarker test and the combination of extra biomedical information (such as, there is provided with individually implementing computed tomography
The situation of test or biomarker test is compared, the analysis experiment that diagnosis performance is improved)；So that Lung neoplasm pernicious or
The diagnosis of positive aspect is simplified；Once Lung neoplasm is observed in computed tomography, so that it may make determining for clinician
Simplify (for example, as independent of tubercle size and according to situation of the test of biomarker for feminine gender, when being judged as tying
When the risk of section is low, order is repeated computed tomography inspection, or such as independent of tubercle size and according to biology
The test of marker is the same for positive situation, when the risk for being judged as tubercle is high, considers biopsy)；And make
For (for example, in computed tomography observe non-calcified tubercle after, be repeated computed tomography inspection,
Eminectomy, or open chest surgery) Clinical Follow-up doctor determine simplify.With only implementing the computerized tomography to excessive risk individual
The situation of scanning or chest x-ray is compared, and biomarker test can improve positive prediction degree.Described life in the present invention
Thing marker not only has serviceability in the case where being used together with computed tomography inspection, also with chest x-ray, branch
Tracheoscopy, fluorescence bronchoscope inspection, Magnetic resonance imaging (MRI) or positron e mission computed tomography
(PET) detect together, and can be connected with other imaging modalities for non-small cell lung cancer to use.Also, by image
Mode or other clinically relevant key elements are come before being found the sign of non-small cell lung cancer, or before symptom shows, in order to
Special-purpose in this purposes and usefully use biomarker as described above.Herein, further include with by meter
Calculation machine tomoscan inspection or other imaging modalities come the individual difference of the amorphism Lung neoplasm observed, for non-small thin
The discriminating of the excessive risk smoker of born of the same parents' lung cancer and the individual diagnosis with non-small cell lung cancer.
On in terms of the diagnosis of non-small cell lung cancer, as the method that biomarker described in the invention can be used
An example, from do not know whether one of individual biomarker as described above with non-small cell lung cancer or its more than
Differential expression can represent that its individual has non-small cell lung cancer, thus, at treatment maximally efficient initial stage, perhaps by it
His means find non-small cell lung cancer or can find non-small cell lung cancer before the appearance of the symptom of non-small cell lung cancer.Non-
In Small Cell Lung Cancer progression, the excessive expression of one or the biomarker more than it is possible to represent non-small cell lung cancer
Progress, for example, the growth and/or transfer (therefore, prognosis is bad) of the tumour of non-small cell lung cancer, on the other hand, one or its
More than biomarker otherness expression degree reduction (that is, subsequent bio marker test in, individual patients court
To or close to " normal " expression situation) can represent for non-small cell lung cancer have improve, for example, non-small cell lung cancer
The size of tumour diminishing (therefore, prognosis bona or better).In the same manner, in the therapeutic process of non-small cell lung cancer
In, the increase of the expression degree of the otherness of one or the biomarker more than it (that is, is tested in subsequent bio marker
In, individual patients are away from the situation of " normal " expression) can represent non-small cell lung cancer be able to be in progress and therapeutic effect not
It is good, conversely, in the therapeutic process of non-small cell lung cancer, the expression degree of the otherness of one or the biomarker more than it
Reduction can represent with for non-small cell lung cancer improvement and treatment be successful.And, hence it is evident that cure non-small cell
After lung cancer, the expression degree of the otherness of one or the biomarker more than it, which increases or decreases, from individual can represent non-
The recurrence of Small Cell Lung Cancer.In this case, with non-small cell lung cancer find again compared with late situation, above-mentioned
Body can re-start treatment in the diseased stage earlier (also, in the case where patient is persistently treated, increases the dosage of medicine
And/or frequency etc., treatment method can be changed).And then by occurring different one or the biology mark more than it according to individual
The expression of will thing, it is contemplated that to the above-mentioned individual reaction to particular therapeutic agent.Observation non-small cell lung cancer recurrence or
In the case of progress, the change of the expression of biomarker can be represented that is, in order to determine that non-small cell lung cancer is lived
Property degree crystallization or treatment method change necessity, it is necessary to image check (for example, computed tomography inspection) repeatedly.
The detection of biomarker described in the invention include treatment method success evaluation or treatment after it is non-small thin
The elimination of born of the same parents' lung cancer, recurrence and/or the observation of progress (including transfer), after Treatment for Non-small Cell Lung or non-small cell lung cancer
Therapeutic process is connected, especially more useful.Treatment for Non-small Cell Lung method is included for example, to individual treatment agent prescription, operation (example
Such as, cut with surgery non-small cell lung tumor at least a portion or excision non-small cell lung cancer and perienchyma), put
Beta ray therapy or used other kinds of Treatment for Non-small Cell Lung method, and the group of these cures in this field
Close.In the treatment method of lung cancer, including for example, giving individual patients prescribed treatment agent, operation (for example, cut with surgery
At least a portion of lung neoplasm), other kinds of Treatment for Non-small Cell Lung side used in radiation cure or the field
Method, and the combination of these treatment methods.For example, small interference ribonucleic acid (siRNA) molecule is the synthesis of blocking gene expression
Double stranded RNA (RNA) molecule, can be used as lung cancer target treatment method.For example, any biological marker in above-mentioned biomarker
Thing can be at least detected once after the treatment, or treatment after be detected repeatedly (for example, periodically), treatment before with treatment after
It can be detected.From the change of the expression according to the time of the arbitrary biomarker in the above-mentioned biomarker in individual
Change to be progress, improvement or the sign of recurrence of non-small cell lung cancer after treatment.In being in progress of above-mentioned non-small cell lung cancer, good
Turn or recurrence example in, including：The expression of biomarker compared with pre-treatment, situation about being increased or decreased after treatment；
The expression of biomarker is compared with earlier time points after treatment, situation about being increased or decreased on time point later；
And a time point goes up the situation different from normal level to the expression of biomarker after the treatment.
As concrete example, to the biomarker water of any biomarker in biomarker described in the present invention
It is flat to be carried out after operation consent or operation (for example, 2 weeks to 16 weeks after operation) in serum or plasma sample.With operation consent sample
Condition ratio, the horizontal increase of (multiple) biomarker expression in sample after surgery, then this can be non-small cell lung cancer into
The sign of exhibition (for example, non-successful property perform the operation), compared with operation consent sample, (multiple) biomarker table in sample after surgery
Reduced up to level, then this can be improvement (for example, removing lung neoplasm) sign of non-small cell lung cancer by successful surgery.Such as
Also can be with similar before and after the other treatment method execution that radiation cure, therapeutic agent dispensing or cancer vaccine are offerd medicine front and rear etc.
Method analysis biomarker level.
Except with single diagnostic test come in addition to carrying out the test of biomarker level, can be with representing the risk to disease
Increased single nucleotide polymorphism (SNP) or other gene lesions or the mobility measure of degree carry out test biology mark in association
Thing level (for example, referring to Amos et al., Nature Genetics 40,616-622 (2009)).
Except with single diagnostic test come in addition to carrying out the test of biomarker level, can be with such as computed tomography
The radiographic test of inspection together carries out biomarker level test.For example, as there is risk to non-small cell lung cancer
Detection of asymptomatic colony (for example, smoker) etc., it is possible to provide appropriate for the therapeutical of computed tomography inspection, economy
The property worked as.For example, for as according to biomarker level, identified that risk is high and breaks with computer for non-small cell lung cancer
Layer scanography is the individual preferentially carried out, and the test of " before computed tomography " biomarker level can be used in high wind
Dangerous individual segregation is the individual for needing to carry out computed tomography inspection.Carrying out the situation of computed tomography test
Under, (for example, being analyzed according to the aptamer of serum or plasma sample) can measure the life of one or the biomarker more than it
Thing marker levels, it is positive in order to improve compared with the situation for individually implementing computed tomography or biomarker test
Premeasure, can will diagnose fraction with extra biomedical information (for example, by computed tomography test Lai determined swollen
Knurl media parameters) it is connected and is assessed.For measuring " after computed tomography " aptamer of biomarker level
Group, which can be used in, determines that Lung neoplasm is positive or negative what is observed in computed tomography (or other imaging modalities)
Possibility.
After the detection of any biomarker can be to computed tomography in biomarker described in the present invention
Test useful.For example, compared with individually carrying out the situation of computed tomography, biomarker test can remove or reduce phase
When the positive test of the sham of quantity.Also, patient can simply be treated by biomarker test.For example, work as Lung neoplasm
Size when being less than 5mm, can be from " check and wait (watch and wait) " in the result that early stage is tested with biomarker
The step of status progression of patient to tissue detection.If Lung neoplasm is 5mm to 9mm, tested by biomarker, can need not
Cut according to the tissue detection or chest of false positive detection.Also, when Lung neoplasm is more than 10mm, marked by biology
Will thing is tested, it is possible to without performing an operation for the individual patients group with positive nodule.It is relevant in the presence of being detected with Nodule tissue
Important ill state, has difficulty according to the position of tubercle to obtain Nodule tissue, therefore is tested according to biomarker, from
The necessity that some patients remove progress tissue detection is possible to advantageously.In the same manner, such as effectively as the positive of tubercle
Situation, some patients need not perform the operation, so as to the relatively unnecessary dangerous and expense that avoids and perform the operation.
Except in excessive risk group with radioactive ray detection in association test biology marker levels (with institute in Image detection
It was observed that Lung neoplasm or tumour size, or other characteristics analyze biomarker level in association) outside, biology is marked
The information of will thing can from different identical data, especially represent to the data of the individual risk of non-small cell lung cancer (for example,
Hazards and/or other lifes whether the clinical history of patient, professional exposes history, symptom, cancer family history, such as smoking
State of thing marker etc.) assessed in association.These a variety of data can be with the meter that is performed in computer or other devices
Calculation machine program/software together, is analyzed by automatic mode.
Described biomarker can be additionally used in image test.For example, among other purposes, for non-small cell lung
Observation, the recurrence observation of cancer diagnosis, progress/improvement or transfer, or the reaction observation to treatment method, make developer (imaging
Agent biomarker) is incorporated into, to play the assisted action of Diagnosis of Non-Small Cell Lung.
The detection of biomarker and biomarker values and measure
For the biomarker described in invention biomarker values can using known various analysis come
It is detected.In one embodiment, biomarker values are detected using capture agent." catching used in the present invention
Obtain agent (capture agent) " or " capture agent (capture reagent) " refer to can specifically with biomarker phase
With reference to molecule.In various embodiments, above-mentioned capture agent is exposed to biomarker in the solution, or by being fixed on
Biomarker is exposed in solid support.In another embodiment, above-mentioned capture agent includes and two on supporter
The characteristic of secondary characteristic reactions.In these embodiments, above-mentioned capture agent is exposed to biomarker in the solution, then in order to
Biomarker is fixed in solid support, can be used together the characteristic in the characteristic and solid support of capture agent.
Capture agent is selected according to the type for the analysis method implemented.Capture agent includes aptamer, antibody, antigen, A De
Nai Ting (adnectins), ankyrin class (ankyrins), other antibody are similar to body and protein scaffolds, autoantibody, chimeric
Body, small molecule, 2 fragments of F (ab '), Fv fragments, Single-Chain Fv Fragment of Murine, single chain antibody fragments, nucleic acid, agglutinin, ligand binding by
Body, affine body (affybody), nano antibody, imprinted polymer, Avimers (avimer), peptide glycan analogies,
Hormone receptor, cytokine receptor, synthesis of receptor and their deformable body and fragment, but it is not limited to this.
In certain embodiments, biomarker values can be detected using biomarker/capture agent complex.
In another embodiment, above-mentioned biomarker values are exported by biomarker/capture agent complex, example
Such as, as being detected situation with the reaction result that interacts with biomarker/capture agent, indirectly it is detected
Survey, but biomarker values depend on the formation of biomarker/capture agent complex.
In certain embodiments, above-mentioned biomarker values can be directly from the biological marker analyte detection of biological sample.
In one embodiment, biomarker is utilized allows two or more biological markers in biological sample
The multiplexing form synchronously detected of thing is detected.In one embodiment of above-mentioned multiplexing form, multiple capture examinations
With direct or be indirectly fixed into covalent bond or non-covalent bond in discrete location of the agent in solid support.In another reality
Apply in example, multiplexing form utilizes mutually different solid support, that is to say, that each solid support and quantum dot one
Together, have and above-mentioned solid support intrinsic capture agent in association.In another embodiment, in order to from biological sample
The detection of each detectable multi-biological marker and use single device.These isolated systems are with biological sample
The mode that can be handled at the same time of each biomarker form.For example, can be by the every of above-mentioned tablet using microtiter plate
A hole (well) is used in biological sample the fixed analysis of one kind in a variety of biomarkers to be checked measured.
One or more in multiple above-described embodiments, represent raw in order to carry out the detection of biomarker values
The component of thing marker/capture complex, can be used fluorescence labels.In various embodiments, using known technology, make above-mentioned glimmering
The intrinsic capture agent of any biomarker that optical label is incorporated into biomarker described in the present invention, so
Afterwards, above-mentioned fluorescence labels can be used in order to detect corresponding biomarker values.Rare earth chela is included in appropriate fluorized marking
Compound, fluorescein and its derivative, rhodamine and its derivative, dansyl, allophycocyanin, PBXL-3, Qdot 605, Liz amine
(Lissamine), phycoerythrin, texas Red and other this compounds.
In one embodiment, above-mentioned fluorized marking is luminescent dye molecule (fluorescent dye molecule).
In some embodiments, above-mentioned luminescent dye molecule includes at least one substituted indoliumring system (indolium ring
System), the substituent on the 3- carbon of indole ring includes chemical reaction base (chemically reactive group) or compound
Material (conjugated substance).In certain embodiments, above-mentioned dye molecule is included for example, AlexaFluor
488th, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700 etc.
AlexaFluor molecules.In another embodiment, above-mentioned dye molecule is included for example, two different AlexaFluor molecules etc.
The first type and Second-Type dye molecule.In another embodiment, above-mentioned dye molecule includes the first type and the second type dye point
Son, two dye molecules have mutually different emission spectrum.
A variety of methods suitable for extensive determination form can be used to measure for fluorescence radiation.For example, Fluorescence Spectrometer with
The mode of analysis microtiter plate, microslide, printed array, cuvette etc. is designed (with reference to Principles of
Fluorescence Spectroscopy by J.R.La kowicz, Springer Science+Business Media,
Inc., 2004.Biolumines cence＆Chemiluminescence：Progress&Current Applications；
Phili p E.Stanley and Larry J.Kricka editors, World Scientific Publishing
Company, January 2002).
In above-described embodiment at one or more than it, chemiluminescence label can be optionally for biomarker values
The mode of detection is alternatively used for the component of mark biomarker/capture complex.Appropriate chemiluminescent substance includes
Oxalyl chloride (oxalyl chloride), rhodamine 6G, Ru (bipy) 32, four (dimethylamino) ethene (TMAE, tetrakis
(dimethylami no) ethylene), pyrogallol (1,2,3- trihydroxy benzenes) (Pyrogallol (1,2,3-trihy
Droxibenzene)), lucigenin (Lucigenin), oxalic acid salt (peroxyoxalates), aryl oxalates (Aryl
Oxalates), acridinium ester (Acridinium esters), dioxetane (dioxetanes) and other materials.
In another embodiment, above-mentioned detection method includes the generation signal that can be detected corresponding to biomarker values
Enzyme/matrix compounds.In general, above-mentioned enzyme be for promote be used for can be by including spectrophotometry
(spectrophotometry), fluorescence radiation (fluorescence) and chemiluminescence (chemiluminescence) is a variety of
Technology is come the chemically change of chromogenic substrate that measures.Appropriate enzyme is included for example, luciferase (luciferases), fluorescence
Plain (luciferi n), malic dehydrogenase (malate dehydrogenase), urease (urease), horseradish peroxidase
Enzyme (horseradish peroxidase；HRPO), alkaline phosphatase (alkali ne phosphatase), beta galactose glycosides
Enzyme (betagalactosidase), glucoamylase (g lucoamylase), lysozyme (lysozyme), glucose oxidase
(glucose oxidase), galactose oxidase (galactose oxidase) and glucose-6-phosphate dehydrogenase (G6PD)
(glucose-6-phosphate dehydrogenase), uricase (uricase), xanthine oxidase (xant hine
Oxidase), lactoperoxidase (lactoperoxidase), microperoxisome (mi croperoxidase) etc..
In another embodiment, above-mentioned detection method can be produce the fluorescence radiation of measurable signal, chemiluminescence,
The combination of radionuclide (radionuclide) or enzyme/matrix compounds.Multimode signaling is in biomarker determination form
Can have the advantages that intrinsic.
In more detail, the biomarker values of the biomarker described in the present invention can be utilized described in following article
Substance aptamer detection method (singleplex aptamer assays), multiple nucleic acid aptamer detection method (multiplexed
Aptamer assays), substance or Multiple immunizations detection method (immunoassays), messenger RNA (mRNA) express spectra
(mRNA e xpression profiling), micro ribonucleic acid (miRNA) express spectra (miRNA expres sion
Profiling), mass spectrometry (mass spectrometric analysis), the sense of organization (histological)/cell
The known analysis method of the property learned (cytological) method etc. detects.
Biomarker values are determined using based on the detection method of aptamer
Molecule physiologically important is used to detect and quantitative detection method is science in biological specimen and other samples
Important means in research and healthcare field.Contain one in solid support including being fixed on as a kind of in this detection method
The use of the microarray of aptamer a or more than it.Each above-mentioned aptamer can in a manner of very special and very
(reference is with " the U.S.Patent of the title of Nucleic Acid Ligand " to be combined with target molecule for high affinity
No.5,475,096 and with the " U.S.Patent of Nucleic Acid Ligand Diagnostic Biochip " titles
No.6,242,246, U.S.Patent No.6,458,543 and U.S.Patent No.6,503,715).Once microarray and sample
Condition contacts, and multiple aptamers realize the life corresponding to biomarker respectively with being present in being combined in sample
The measure of thing marker levels.
" aptamer (aptamer) " used in the present invention refers to the specific bond affinity for target molecule
Nucleic acid.The problem of affinity interaction (affinity interaction) spends (degree) for one, but in this situation
It is lower to understand, to " specific bond affinity (the specific binding affinity) " of the aptamer of target with the sample
Compared with the situation that other compositions are combined, above-mentioned aptamer is usually incorporated into its target with the affinity of the degree of higher
In." aptamer " refers to a kind of a kind of duplication combination of form or nucleic acid molecules with specific nucleotide sequence.Nucleic acid is fitted
Body is included with any number of nucleotide chemically changed, can include an appropriate number of nucleotide." aptamer " refers to one
This molecular combinations more than a.Mutually different aptamer can have the nucleotide of identical or mutually different quantity.Core
Sour aptamer can be DNA or ribonucleic acid or the nucleic acid through chemically changing, and can include single-stranded, double-strand or double-strand
Area, can include the structure of very well ordered.Aptamer can also be light aptamer, in the case, photoreactivity or chemistry
Property reaction sexual function base be contained in aptamer, so as to the mutual covalent bond of corresponding target.It is described in the present invention to appoint
The aptamer method of meaning may include the use for the more than two aptamers being specifically combined with identical target molecule.Such as
Described further herein below, aptamer can include label.If aptamer includes label, the duplication of all aptamers
Need not have same label.And then if mutually different aptamer includes label, the plurality of mutually different nucleic acid respectively
Aptamer can have the label being mutually identical or mutually different label.
Aptamer is using the arbitrary known square of aglucon phyletic evolution technology (SELEX) process including index concentration
Method is differentiated.Once being authenticated, aptamer can be according to the arbitrary public affairs for including chemical synthesis process and enzymatic synthesis method
Perception method is prepared or synthesized.
" sustained release improvement aptamer (SOMAmer) " or slow dissociation rate modification of nucleic acids aptamer used in the present invention
(Slow Off-Rate Modified Aptamer) refers to the aptamer with slow dissociation rate characteristic being improved.It is slow
Release improvement aptamer using with " be used to prepare with improve dissociation rate aptamer method (Method for
Generating Aptamers with Improved Off-Rates) " US publication number the 2009/th of title
In No. 0004667 prepared by aglucon phyletic evolution technology (SELEX) method of described improved index concentration.
Above-mentioned term " the aglucon phyletic evolution technology (SELEX) of index concentration " and " the aglucon phyletic evolution of index concentration
Technology process (SELEX process) " is in order to which (1) is with method for optimizing, for example, by high-affinity in a manner of protein is combined,
To screen the combination for the amplification for being referred to as the above-mentioned nucleic acid through screening with the aptamer of target molecule interaction and (2), and at this
Used with invention.The aglucon phyletic evolution technology process of above-mentioned index concentration can have particular target or biomarker to differentiate
There is the aptamer of high-affinity and use.
The aglucon phyletic evolution technology of index concentration generally includes：The step of preparing the candidate substances mixture of nucleic acid, is
The affine complex of formation (affinity complex) and the step of candidate substances mixture is incorporated into required target molecule；
The step of affine complex being isolated from uncombined candidate substances nucleic acid；Make the step from affine complex seperated nuclear acid and dialysis
Suddenly；The step of purifying nucleic acid；And the step of amplification specific nucleic acid aptamer sequence.Fitted to further improve the nucleic acid through screening
The affinity of body, can repeatedly implement above-mentioned operation.Include amplification step on the process point that above-mentioned operation can be at one or more than it
(for example, referring to " Nucleic Acid ligands " are the U.S.Patent No.5 of title, 475,096).Above-mentioned index is rich
The aglucon phyletic evolution technology process of collection not only can be incorporated into the aptamer of target to generate covalency and use, can also be in order to
It is incorporated into the aptamer of target and uses (for example, referring to " Sy stematic Evolution of generation non-covalent
Nucleic Acid Ligands by Exponential Enrichme nt：Chemi-SELEX " is title
U.S.Patent No.5,705,337).
The aglucon phyletic evolution technology process of above-mentioned index concentration can use for following purpose：For example, in order to improve
(in vivo) stability or transportation characterization in vivo, and in order to differentiate the modification for including the characteristic that improvement is assigned to aptamer
The high-affinity aptamer of nucleotide and use.As the example of this modification, it is included in ribose (ribose) and/or phosphate
Chemical substitute at (phosphat e) and/or base positions.Differentiated by the aglucon phyletic evolution technology process of index concentration
The oligonucleotide that the aptamer gone out includes the nucleotide redundant organism through chemically changing in 5'- the and 2'- positions that description has pyridine
No. 5660985 (" High Affinity Nuc leic Acid Ligands Containing of U.S. Patent No.
Modified Nucleotides ") have it is described.With reference to above, recorded in U.S. Patent No. 5580737 comprising being modified into
2'- amino acid (2'-NH2), one of 2'- fluoro (2'-F) and/or 2'-O- methyl (2'-OMe) or the nucleotide more than it
High specific aptamer.Also, referring to description has the aglucon phyletic evolution technology of index concentration and matching somebody with somebody for optical index enrichment
Widened physics and the chemically U.S. Patent Publication No. of the nucleic acid library of characteristic and their purposes in based system evolution technology
No. 2009/0098549 (" SELEX and P HOTOSELEX ").
Also, the aglucon phyletic evolution technology of index concentration can be in order to differentiate the nucleic acid with preferably slow dissociation rate characteristic
Aptamer and use.Refer to the aglucon that narration is useful for generating the improved index concentration for the aptamer that can be combined in target molecule
No. 2009/0004667 (" Method for Generating of U.S. Patent Application Publication No. of phyletic evolution technical method
Aptamers with Improved Off-Rates").Record for fast with slower dissociation from the generation of each target molecule
The aptamer of rate and the method for light aptamer.The above method includes：Make what target molecule was in contact with candidate substances mixture
Step；The step of forming nucleic acid-target complex；And the step of performing dissociation rate amplification process, herein with fast solution
Nucleic acid-target complex from speed is dissociated without regenerating, but the complex with slow dissociation rate can be steadily
Maintain.Additionally, in order to generate the aptamer with improved slow dissociation rate performance, the above method includes generation candidate
The nucleotide of modification is used during matter mixtures of nucleic acids.
By being deformed to this detection method, using include can make aptamer and the mutual covalent bond of target molecule or
The aptamer for the light reaction functional group that " photo-crosslinking (photocrosslink) " is combined is (for example, referring to U.S.Patent
No.6,544,776, " Nucleic Acid Ligand Diagnostic Biochip ").The photoreactivity aptamer also by
Referred to as light aptamer (for example, respectively referring to entitled " Systematic Evolution of Nucleic Acid
Ligands by Exponential Enrichmen t：PHotoselection of Nucleic Acid Ligands and
The U.S.Patent NO.5,763,177, U.S.Patent No.6,001,577 and U.S.Patent of Solution SELEX "
No.6.291,184；The U.S.Patent No.6 of entitled " Photoselection of Nucleic Acid Ligands ",
458,539).After above-mentioned microarray is in contact with sample, light aptamer obtain can with the chance that target molecule is combined, on
Light aptamer is stated by photolytic activity, solid support is cleaned to remove the molecule of non-specific binding.Because of light core
The effect base through photolytic activity on sour aptamer and the covalent bond produced, with target molecule that light aptamer is combined usually not by
Remove, therefore stringent cleaning condition can be used.In the method, biological marker is corresponded in above-mentioned detection method detectable sample
The biomarker values of thing.
In this all determination forms, above-mentioned aptamer is fixed on solid support before being in contact with sample
On.However, if in certain circumstances, aptamer is fixed before being in contact with sample, then optimal detection can not be realized.
For example, perhaps the preceding immobilization (p re-immobilization) of aptamer can cause with the solid in long reaction time
The mixing of the non-efficiency of target molecule and aptamer in supporting body surface, it is therefore necessary to lengthen incubation time so that
Aptamer is effectively combined with target molecule.Also, light aptamer depends on being used in detection method and is used as solid support
During the material of body, above-mentioned solid support is there is a possibility that the light for the covalent bond between light aptamer and target molecule is produced and dissipated
The tendency penetrated or absorbed.And then according to application method, the surface of solid support is impacted exposed to mark reagent, therefore
There is a possibility that being incorporated into the detection accuracy of the target molecule of aptamer reduces.Finally, in general, aptamer is being exposed to sample
Before product, the immobilization of the aptamer in solid support includes the step of preparing aptamer (that is, immobilization), the preparation
Step is possible to influence the activity degree or feature of aptamer.
Also narration has the following aptamer detection method using separating step：So that aptamer acquisition target in the solution
Afterwards, the special component of aptamer-target mixture is removed before testing (with reference to " Multiplexed Analyses of
Test Samples " are the U.S.Patent Application Publication 2009/0042206 of title).Above-mentioned core
Detection and quantification of the sour aptamer detection method by nucleic acid (that is, aptamer) so that realize the non-nucleic acid target (example from sample
Such as, protein target) detection and quantification.Above method generation is used to be detected non-nucleic acid target and the nucleic acid of quantification replaces
For thing (surrogate) (that is, aptamer), and the multiple nucleic acids technology including amplification step is applicable to include protein
The preferable wide scope target of target.
Aptamer can be with from aptamer biomarker complex (or light aptamer biomarker covalent bond
Complex) it is easily separated detection component and can realizes the isolated mode structure of the aptamer for detection and/or quantification
Into.In one embodiment, which can be included in aptamer sequence the key element that can be cut off or discharge.In another embodiment
In, additional function, for example, labeled or detectable component, being spaced component or particular combination label or immobilization key element can lead
Enter in aptamer.For example, above-mentioned aptamer can by the part (moiety) that can cut off, mark, for separation marking
Interval component and releasable part, to include the label for being connected to aptamer.In one embodiment, cut-off structure
Key element is the destructible connector of optics.The above-mentioned destructible connector of optics can be attached to biotin moiety (biot in
Moiety) and compartment, the n-hydroxysuccinimide base (NHS yls) of the redundant organism for ammonia can be included, available for
Aptamer imports biotin group, so that aptamer can be discharged according to detection method later.
The homogeneous assay (homogenous assays) for handling all detection components in the solution does not require signal detection
The separation of sample and reagent is carried out before.This method speed is fast and easy to use.This method is based on being reacted with particular target
Molecule trapping or binding reagents and generate signal.
In one embodiment, signal method is generated to utilize due to fluorescent marker (fluorophore-l abeled) capture
The change of reagent and the anisotropy signal of the interaction of particular organisms marker target.The capturing agent and target response of above-mentioned mark
When, because the value of the rotary motion of fluorogen that makes to be attached to complex increased molecular weight further slow down.Observe it is each to
Opposite sex change, so that association reaction can be used in solution measuring among biomarker quantitatively.Include in other methods
Fluorescence polarization detection method (fluorescence polarization assay), method of molecular beacons (molecular beacon
Methods), (time resolved fluorescene quenching), chemiluminescence is quenched in time-resolved fluorescence
(chemiluminescence), fluorescence resonance energy transfer (fluorescence resonance energy transfer)
Deng.
In biological specimen, the example that can be used for the detection corresponding to the biomarker values of biomarker
The aptamer detection method based on solution of property includes：Step (a), including the first label, make to have biomarker special
The aptamer of affinity is in contact with biological specimen, so that mixture is prepared, wherein, it is present in sample in biomarker
In the case of, form the affine complex of aptamer；Step (b), makes said mixture be exposed to comprising the first arresting structure key element
The first solid support, and the first label is combined with above-mentioned first solid support；Step (c), remove not with it is above-mentioned
The arbitrary component for the mixture that first solid support is combined；Step (d), the biology of the above-mentioned affine complex of aptamer
Adhere to the second label in marker component；Step (e), makes the affine complex of above-mentioned aptamer from above-mentioned first solid support
Separation；Step (f), makes the above-mentioned affine complex of separated aptamer exposed to second containing the second arresting structure key element etc.
Solid support so that the second label is combined with the second arresting structure key element；Step (g), it is affine multiple from above-mentioned aptamer
(non-complexed) aptamer of fit sub-department's non-complexing, so as to remove the nucleic acid of arbitrary non-complexing from mixture
Aptamer；Step (h), aptamer is leached from above-mentioned solid support；And step (i), it is affine compound to detect above-mentioned aptamer
The aptamer component of body, so as to detect biomarker.
, can in order to detect biomarker values by detecting the aptamer component of the affine complex of above-mentioned aptamer
Use the method known in the art.In order to detect the aptamer component of affine complex, multiple mutually different inspections
Survey method, can be used for example, hybridization assays (hybridization assays), quality analysis (mass spectroscopy)
Or the detection method of real time fluorescent quantitative nucleic acid amplification detection (QPCR) etc..In certain embodiments, nucleic acid base sequence is analyzed
Method can be used for the aptamer component of the detection affine complex of aptamer, and detect biomarker values.In simple terms, it is
One or the sequence of the aptamer more than it for confirming and being quantitatively present in sample, sample can become any nucleic acid alkali
The object of basic sequence analysis method.In certain embodiments, above-mentioned sequence includes whole Nucleic acid aptamer molecules or with intrinsic side
The part for the molecule that formula differentiates above-mentioned molecule and can use.In another embodiment, distinctive base sequence is to make an addition to core
Specific sequence in sour aptamer；Such a sequence is often by " label (tags) ", " bar code (barcodes) " or " postcode
(zipcodes) " censured.In certain embodiments, above-mentioned base sequence analysis method includes amplification of nucleic acid aptamer sequence,
Or the nucleic acid of the ribonucleic acid comprising chemical sex modification and DNA is converted to its that be suitable for base sequence analysis
The enzymatic step of the nucleic acid of his species.
In certain embodiments, above-mentioned base sequence analysis method includes one or the copy step more than it.Another
In embodiment, above-mentioned base sequence analysis method includes the direct base sequence analysis method there is no copy step.
In certain embodiments, above-mentioned base sequence analysis method using in sample with one or the core more than it
Sour aptamer is immediately adjacent to method as the specific primer of target.In another embodiment, above-mentioned base sequence analysis method include with
Multiple applications while all aptamers in sample are as target.
In certain embodiments, above-mentioned base sequence analysis method includes being used to expand for base sequence analysis target
Molecule enzymatic step.In another embodiment, the sequence of the above-mentioned single molecule of base sequence analysis method Direct Analysis.It is raw
Can be used for the detection corresponding to the biomarker values of biomarker in thing sample based on exemplary nucleic acid
The method of base sequence analysis includes：Step (a), using enzymatic step, will include the nucleic acid of the nucleotide through chemically changing
The mixture of aptamer is converted to indeclinable nucleic acid；Step (b), is using following by the indeclinable nucleic acid that its result generates
System carries out base sequence analysis multiplely at the same time, for example, 454 base sequence analysis systems (454Sequencing System)
(454Life Sciences/Roche), Illumina base sequence analysis systems (Illumina Sequenc ing
System) (Illumina), ABI SOLiD base sequence analysis systems (Applied biosystems (Applied
Biosystems)), HeliScope single-molecule sequencers (H eliScope Single Molecule Sequencer)
(Helicos Biosciences), or the real-time unimolecule base sequence analysis system of Pacific Ocean Biological Science Co., Ltd
(Pacific Bioscienc es Real Time Single Molecule Sequencing System)(Pacific
BioScie nces) or Polonator G base sequence analysis systems (Polonator G Sequencing Sy stem)
A large amount of parallel base sequence analysis platform (massively parallel sequencing of (Dover Systems) etc.
platform)；And step (c), differentiated and quantified by particular sequence and Sequence Coefficient (sequence count)
Change the aptamer being present in mixture.
Utilize the measure of the biomarker values of immunodetection
Immunologic detection method is based on the reaction to correspondingly target or the antibody of analyte, can be dependent on particular assay form
To test and analyze thing from sample.In order to based on-reactivity is immunized, specificity and the sensitivity of detection method be improved, because of Dan Ke
Specificity epitope possessed by grand antibody identifies and usually uses monoclonal antibody.Polyclonal antibody compared with monoclonal antibody,
There is increased affinity to target, therefore polyclonal antibody is also successfully used in panimmunity detection method.Immune inspection
Survey method is designed in a manner of being used together with extensive biological specimen matrix.The form of immunodetection is qualitative to provide
The mode of property, semi-quantitative and quantitative result designs.
Quantitative result have known concentration, using specific analyte to be detected, pass through the standard curve of generation
To generate.Reaction from unknown sample acquisition or signal are represented with standard curve, established from unknown sample corresponding to target
Amount or value.
Most immunoassay formats are devised.Enzyme linked immunosorbent assay (ELISA) (enzyme linked immunosorbent assay (ELISA)) or enzyme are immunized
Analytic approach (EIA) can test and analyze thing quantitatively.This method depends on the attachment to mark a kind of in analyte or antibody,
Include enzyme to the above-mentioned direct or indirect property of marker components.Enzyme linked immunosorbent assay (ELISA) test can have for analyte it is direct,
Indirectly, the form of competitive or interlayer detection.Other multiple methods are depended on for example, radioisotope
(radioisotope)(I125) or such as fluorescence radiation mark.Additional technology includes for example, cohesion
(agglutination), turbidimetry (nephelometry), muddy method (turbidi metry), Western blotting (Western
Blot), immuno-precipitation (immunoprecipit ation), immunocytochemical method (immunocytochemistry), exempt from
Epidemic disease histochemical method (immunohistochemistry), flow cytometry (flow cytometry), Lu Mingkesi detection methods
(Luminex assay) and other methods are (with reference to ImmunoAssay：A Practi cal Guide, edited by Brain
Law, published by Taylor＆Francis, L td., 2005edition).
Exemplary determination form includes enzyme linked immunosorbent assay (ELISA) (enzyme linked immu nosorbent
assay；ELISA), radioimmunoassay detection method (radioimmunoassay), fluorescence (fluorescent), chemiluminescence
(chemiluminescence) and fluorescence resonance energy transfer (fluorescence resonance energy transfer；
) or time-resolved fluorescence Resonance energy transfer (time resolved-FRET FRET；TR-FRET) immunodetection.And then
Biomarker immuno-precipitation, the example of biological marker object detecting method include such as gel electrophoresis (gel
Electrophoresis), Capillary Electrophoresis (capillary electrophoresis), plane electrochromatography (planar
) etc. electrochromatography size and the quantitative method that peptide level can be distinguished.
Characteristic of the method for material dependent on mark is produced for the detectable mark of detection and/or quantification or signal.
By appropriate enzyme come the reaction product that is catalyzed can indefinite (herein, when detectable mark is, in reference
Text) be fluorescence, photism or radioactivity, or they can absorb visible lights or ultraviolet.Being suitable for detection like this can examine
The example indefinite of the detector of the mark of survey include x-ray film, radiant tester, scintillation counter, spectrophotometer,
Colorimeter, fluorescence photometer, luminometer and density measuring device.
Detection method can be carried out with can suitably analyze the form of preparation, processing and reaction.This can be more for use example such as
Hole detection tablet (for example, 96 holes or 384 holes) or the situation of appropriate array or microarray.Storing solution for plurality of reagents
Can be by either manually or mechanically preparing, follow-up all liquid reliefs, dilution, mixing, distribution, cleaning, culture, sample reading, data are received
Collection and analysis can be completed mechanically using the detection machine of commercial analytic software, machine ergonomics and detectable mark.
Utilize the measure of the biomarker values of gene expression profile
Messenger RNA measure can be used as being used to detect corresponding albumen in above-mentioned biological specimen in biological specimen
The substitute of the detection of matter level.Therefore, arbitrary biomarker or biomarker described in the present invention can be passed through
Group or appropriate ribonucleic acid are detected to be detected.
Messenger RNA expression passes through RT-polymerase chain reaction (reverse transcr iption
quantitative polymerase chain reaction；RT-PCR) (detected according to real time fluorescent quantitative nucleic acid amplification
(qPCR) RT-polymerase chain reaction (RT-PCR)) measure.RT-polymerase chain reaction is used for from courier's ribose core
Acid is prepared in complementary DNA (cDNA) (cDNA).Above-mentioned complementary DNA (cDNA) is with DNA amplification procedure
Progress, can use to prepare fluorescence in the detection of real time fluorescent quantitative nucleic acid amplification.It is glimmering in real time compared with standard curve
The detection of light quantitative nucleic acid amplification can generate the absolute determinations of the quantity of the messenger RNA duplication such as each cell.With capillary
The combined promise plucked instrument hybridization (Northern blots) of electrophoresis tube, microarray, intrusion detection method (invader assay) and reverse
Record polymerase chain reaction is used to measure the expression of messenger RNA in sample (with reference to Gene E xpression
Profiling：Method and Protocols, Richard A.Shimkets, edi tor, Humana Press, 2004).
Micro ribonucleic acid molecule is non-secret signal, but adjusts the small-sized ribonucleic acid of gene expression.Available for suitable for
Multiple methods of the measure of messenger RNA expression corresponding micro ribonucleic acid.Recently many research institutes just grind
Study carefully the use for the micro ribonucleic acid for carrying out the biomarker for being used as disease.Most disease is with wide scope transcriptional control
(transcriptional regulation), micro ribonucleic acid can play the role of biomarker, and be not to make us frightened
The thing being surprised.Correlation between micro ribonucleic acid concentration and disease is often unlike the correlation between protein level and disease
Clearly, but the value of micro ribonucleic acid biomarker can be substantive.Certainly, as differently expressed during one's sickness
RNA situations, it is necessary to the problems that are faced of when exploitation of occlusion body outer (i n vitro) diagnostic products, micro ribonucleic acid from
Survived in diseased cell and be easily extracted as analysis use, or needed so as to the degree of measure micro ribonucleic acid is abundant
Survive permanent blood or to other Medium Cultures discharge condition.Although the biomarker of many potentialities during one's sickness with
Mode (paracrine fashion) is secreted to be secreted with being intended to property in pathology and functional part in periphery, but protein is given birth to
Thing marker still has similar condition.Many potentiality protein biomarkers are designed to synthesized by protein
Played function on the outside of cell.
Utilize the biological marker analyte detection of internal molecular image technology
Above-mentioned described multiple biomarkers can be additionally used in molecular image test.For example, developer is non-in order to aid in
Diagnosis of small cell lung cancer, can be combined with above-mentioned described biomarker, and above-mentioned described biomarker can
In order to observe the reaction for treatment among progress/improvement or transfer, disease palindromia for disease, or other purposes.
Internal Image Creation technology provides the noninvasive method of the state for the organism specified disease for being used to judge individual.Example
Such as, all parts of organism or whole body can be checked using 3-dimensional image, thus obtained in relation to biological volume morphing and structure
Highly useful information.In order to provide the state of the cancer in relation to individual, more particularly to the information of non-small cell lung cancer state, this
Kind technology can be combined with the detection of biomarker described in the present invention.
Because of the development of various technologies, in organism the use of molecular image technology expanding.In the development of this technology
Include the novel type radiographic contrast that such as radioactive label and/or fluorescent label of strong signal can be provided out of organism in the middle
The exploitation of (contrast agent)；And in order to usefully information is provided and with remarkable sensitivity and accuracy, from
The exploitation of the New video technology of this signal of organism external detection and the strength analyzed.Above-mentioned contrast agent can be appropriate
Image system in visualized, and provide the image at (multiple) position of the organism residing for contrast agent.Above-mentioned contrast agent can
With comprising the capture agent such as aptamer or antibody etc., for example, peptide or protein matter or (for example, for detecting gene expression)
Oligonucleotides, or one or the giant molecule (macromolecules) more than it and/or there are different particulate form (p
Articulate forms) composite bulk phase combine.
Also, above-mentioned contrast agent can have the feature of the radioactive element for Image Creation.Appropriate radioactive element contains
123 (iodine-123) of technetium -99m (techneti um-99m) or iodo- for scintigraphy (scintigraphic) research.
The part that other sections are simply detected include for example, iodo- 123, iodine -131 (iodine-131), indium -111 (indium-111),
Fluoro- 19 (fluori ne-19), carbon -13 (carbon-13), nitrogen -15 (nitrogen-15), oxygen -17 (oxygen-17), gadolinium
(gadolinium), the spin labeling (spin for magnetic resonance imaging (MRI) of manganese (manganese) or iron (iron) etc.
label).This mark is in the art it is well known that and can be by the ordinary skill people of the technical field of the invention
Member is readily selected.
Standard video technology includes magnetic resonance imaging, computed tomography (computed tomography
Scanning), positron emission computerized tomography (positron emission tomography；PET), single photon emission calculates
Machine tomoscan (single photon emission computed tomography；SPECT) etc., but it is not limited to this.
For diagnostic Image Creation in vivo, the type of workable testing agency is to be designated as being used as target (protein, Xin Shihe
Ribosomal ribonucleic acid etc.) radionuclide (radionuclide) and particular organisms marker etc., in the contrast agent side that specifies of selection
It is principal element on face.In general, the form specified of the selected above-mentioned radionuclide by mechanism, has detectable decay
(decay) type.Also, when selection for in-vivo diagnostic radionuclide when, half-life period should fully grow extremely by target tissue and
In the degree that absorption maximum (uptake) can detect in the time, but should be short enough, so that making the harmful radiation to host
Property minimize degree.
Example as image technology is included as radionuclide to individual with comprehensive (syntheti cally) or office
The positron e mission computed tomography and single photon emission computed of the image technology put into portion's property (locally) break
Layer Imaging, but it is not limited to this.Then, the absorption of radionuclide is measured according to the time, and for obtaining the group of target
Knit and the information of biomarker.Due to high energy (gamma ray) release of used specific isotopic element and for detecting it
And the susceptibility and precision (sophistication) of the utensil used, the Two dimensional Distribution of radionuclide can be from vitro
Portion's inference and go out.
The commonly used positive electron release radionuclide in positron e mission computed tomography
(positron-emitting nuclides) include for example, carbon -11 (carbon-11), nitrogen -13 (nitrogen-13), oxygen -
15 (oxigen-15) and Value linear (fluorine-18).(electron capture) is captured by electronics and/or gamma rays is released
The isotopic element put and decayed is used for single photon emission computed tomography, including for example, iodo- 123 and technetium -99m.Profit
It is as follows come the exemplary method of labeled amino acid with technetium -99m：In order to form technetium -99m- chemotactic peptide conjugates
(technetium-99m-chemotactic peptide conjugate) and change (bifu nctionally
Modified) must have the function of the metal combination base of two kinds of Chemotactic Peptide, in order to formed the unstable technetium reacted successively with this-
Composite (technetium-99m-precu rsor complex) before 99m-, in the presence of precursor is chelated cross technetium acid from
The reduction of sub (pertechnetate ion).
For this diagnostic imaging method in vivo, commonly using antibody.Preparation and use for the antibody of in-vivo diagnostic
It is well known in the art.Specifically with listed arbitrary biomarker is combined in table 2 progress
The antibody of mark, be able to can examine to according to the particular organisms marker used for the purpose of diagnosing or assessing the morbid state of individual
Individual injection of the survey and suspection with what type of cancer (for example, non-small cell lung cancer).As previously noted interior
Hold, used mark is according to Image Creation technology to be used and to select.Because of the position of mark, the scope of cancer can be measured.
Also, can be by the amount of the mark in organ or tissue, to determine the presence or absence of the cancer in the organ or tissue.
In the same manner, aptamer can be used for this diagnostic imaging method in vivo.It is for example, described in order to specific in table 2
The discriminating of biomarker and used aptamer (therefore, being specifically combined with above-mentioned particular organisms marker) can
Suitably it is labeled, can be according to particular organisms marker, Xiang Kejian in order to diagnose or judge the non-small cell lung cancer state of individual
The doubtful individual injection with non-small cell lung cancer surveyed.Content as previously noted, used mark can be according to treating
The Image Creation technology that uses selects.The scope of cancer is measured by the position of mark.Also, can be by organ or tissue
Mark amount, determine the presence or absence of the cancer in the organ or tissue.Compared with other developers, aptamer-derivative
Developer (a ptamer-directed imaging agents) can have and tissue intrusion (penetration), tissue point
Cloth (distribution), dynamics (kinetics), remove (elimination), efficiency (potency) and susceptibility correlation
Intrinsic, favourable characteristic.
In order to for example, the detection of the gene expression by using the Image Creation of anti-sequence (antisense) oligonucleotides, this
Kind technology can optionally be implemented using labeled oligonucleotides.This method uses fluorescence molecule for example, as mark
Or radionuclide, it is used in situ hybridization (in situ hybridization).Its other party for the detection of gene expression
Method is included for example, the detection of indicator (reporter gene) activity.
In addition other conventional image technologies pass through the optics device outside subject for the fluorescence signal inside subject
The optical image for having and being detected.The signal can be due to actual fluorescence and/or bioluminescence
(bioluminescence).Because of the sensitivity improving of optical detection utensil, the optical imageization for in-vivo diagnostic detection is useful
Property is improved.
Such as, it is intended to during for novel cancer, such as more rapidly measure the clinical test of clinical effectiveness and/or multiple
Disease the problem of being likely to become moral check using the long-term treatment of placebo of sclerosis (multiple sclerosis) etc.
Among disease, for avoiding using clinical trial of long-term treatment of placebo etc., internal Molecular biomarkers shadow
The use of pictureization gradually increases.
For the comment of other technologies, N.Blow is referred to, Nature Methods, 6,465-469,2009.
Utilize the measure of the biomarker values of histology/cytology
In order to assess non-small cell lung cancer, Various Tissues sample can be used for histology or cytology.The main root of sample
Screened according to the position and metastasis site of tumour.For example, terminal bronchi (endo-bronchial) and through bronchus (trans-
Bronchial) biopsy, fine needle aspiration (fine needle aspirates), cutting needle (cutting
Needles) and central part biopsy (core biopsy) can be used in histology.Bronchus cleaning solution and scrub (b
Rushing), pleura suction (pleural aspiration) and phlegm (sputum) can be used in cytology.Cytological analysis is still
So it is used for the diagnosis of non-small cell lung cancer, on the contrary, it is well known that Histological method can provide the sensitive of higher to cancer detection
Degree.Rising adjusting, the arbitrary biological marker confirmed in the present invention are being shown from the individual with non-small cell lung cancer
Thing can be used for dyeing the histological specimens of the mark people (indicator) as disease.
In one embodiment, specific one of corresponding (multiple) biomarker or the capture agent use more than it
Assessed in the cytology of lung tissue cell sample, it may include one among following or its more than：Cell sample collection, fixation
(fixing), (dehydrating), cleaning (clearing) cell sample, the immobilized cell on microslide are dehydrated
Sample (immobilizing), strengthen cell sample permeability (permeabilizing), for recovering analyte
Processing (treating), dyeing (staining), decoloration (destaining), the cleaning of (analyte retrieval)
(washing), separate and reacted among (blocking) and buffer solution with one or the capture agent more than it.In an embodiment
In, above-mentioned cell sample is generated by cell block (block).
In another embodiment, specific one of corresponding biomarker or the capture agent more than it are used for lung
The Histological assessment of tissue samples, it may include one among following or its more than：Tissue specimen collection, fixation, dehydration, cleaning
Cell sample, immobilized cell sample, the permeability of enhancing cell sample on microslide, for recovering analyte
Processing, dyeing, decoloration, cleaning, partition, among rehydration (rehydrating) and buffer solution with one or catching more than it
Obtain reagent reacting.In one embodiment, fixed and dehydration is replaced with freezing (free zing).
In another embodiment, specific one that makes corresponding (multiple) biomarker or the nucleic acid more than it are fitted
Body is reacted with tissue or cell sample, can be provided in nucleic acid amplification method as nucleic acid target.Appropriate nucleic acid amplification method
Including for example, PCR, q-beta replicase (q-beta replicase), rolling circle amplification (rolling
Circle amplificati on), strand displacement (strand displacement), helicase dependent amplification (helicase
D ependent amplification), ring mediated isothermal amplification (loop mediated isothermal
Amplification), ligase chain reaction (ligase chain reaction) and the limitation of rolling circle amplification is contributed to
(restriction) and it is cyclized (circularization).
In one embodiment, in order to be assessed for histology or cytology, specific the one of corresponding biomarker
A or more than it capture agent be blended in comprising below when any of buffer solution：Barrier material (blocking
Materials), competitor (competitors), lotion (detergents), stabilizer (stabilizers), transport nucleic acid
(carrier nucleic acid), polyanionic material (polyanionic materials) etc..
" cytology agreement (cytology protocol) " generally includes sample collection, sample is fixed, sample immobilization and
Dyeing." cell is prepared (cell preparation) " includes one or slow more than it of the dyeing for the cell through preparation
The use of the aptamer of dissociation rate, it may include each process after sample collection.
Sample collection may include to be directly placed into sample into undressed shipping container, to filling certain type of culture
Sample or any processing are put into the shipping container of liquid or sample (immobilization) is directly placed on loose glass slide.
Sample immobilization (immobilization) is with polylysine (polylysine), gelatin (gelatin) or silicon
A part for the sample that the glass slide adhesion that alkane (silane) is handled is collected, so as to be improved.Glass slide can
By being prepared in the very thin and flat cellular layer of the region-wide smearing (smear ing) of glass slide.In general, in order to make mechanical change
Shape (mechani cal distortion) and dry artifact (drying artifact) minimize and great care.Liquid
Sample can be handled by cell block method.Alternatively, alternatively, liquid sample can at normal temperatures by with fixed solution with
1:1 mixing about 10 minutes.
Cell block can be by remaining diffusate (residual effusion), phlegm (sputum), arena (urine
Sediment), gastro-intestinal Fluid (gastrointestinal fluid), lung liquid (pulmonary fluid), cell scraper (cell
Scraping) or fine needle aspiration generates.Cell has centrifugation or membrane filtration and is concentrated or encapsulates.Exploitation is useful for preparing
All multi-methods of cell block.Representational method fixes (fixed sediment), bacteria Agr (b including sediment
Acterial agar) or membrane filtering method.In sediment fixing means, cell pellet and such as BouinShi liquid, picric acid
After the fixer mixing of (picric acid) or the formalin (buffered forma lin) of buffering etc., above-mentioned mixing
Thing is centrifuged to be granulated to the cell of immobilization.Cell is completely dried as much as possible to be granulated and remove supernatant
Liquid.Collect after being granulated, be put into after being wrapped up with lens paper (lens paper) in tissue cassette (tissue cassette).Will be above-mentioned
Tissue cassette is together put into bottle with additional fixer, is together handled with tissue samples.Agar method (agar method) is very
It is similar, but be granulated and be removed, and it is cut into half after being dried on paper handkerchief.The side of excision is put on a glass slide
One drop dissolving agar on after, cover agar so that above-mentioned granulation does not produce foam fully in agar.Make above-mentioned fine jade
After fat solidification, the agar of arbitrary residual point is packed up.Put it into tissue cassette, then tissue treatment terminates.Alternatively, it is above-mentioned
It is granulated at a temperature of 65 DEG C, directly suspends into 2% agar solution, sample is centrifuged.Above-mentioned agar cell is granulated
When solidification 1 is small at a temperature of 4 DEG C.Above-mentioned solid agar can be removed from centrifugal separating tube, be divided into half.Above-mentioned agar is utilized
After filter paper parcel, it is put into tissue cassette.At this time, processing before is such as above described.Centrifuge and membrane filtration one
Together, a kind of arbitrary method can be replaced by.A kind of arbitrary method can be used for preparing " cell block sample (cell block
Sample in) ".
Cell block is golden using Lowicryl resins (Lowicryl resins), LR whites (L R White), LR is included
It is prepared by the resin through characterization of color (LR Gold), Unicryl and MonoStep.The resin has low viscosity, in low temperature
It is lower to be polymerize using ultraviolet.Embedding treatment (embedding process) cools down sample by easy stages during dehydration, will
Sample is moved in resin, finally has block under the low temperature of appropriate ultraviolet wavelength.
Cell block piece section (cell block sections) is for cytomorphology inspection (cytomorphological
Examination) and using hematoxylin eosin staining method dyed, while additional clip is used for the inspection to special sign thing
Look into.
Whether with being that histology process or cytology process are unrelated, above-mentioned sample divides above-mentioned operation in order to prevent sample
Solution, is immobilized before additional treatments.The processing is referred to as " fixed (f ixation) ", illustrates the extensive thing that can be used with
Matter and method.Sample fixed protocol and reagent are empirically selected based on target to be detected with specific cells/tissue to be analyzed
Select.Sample is fixed dependent on ethanol (ethanol), polyethylene glycol (polyethylene glycol), methanol (metha nol),
The reagent of formalin (formalin) or isopropanol (isopropanol) etc..Above-mentioned sample is being collected and is being added to as far as possible
After in glass slide, it is fixed at once.However, selected fixer can cause the different kinds of molecules target band for making subsequent detection become difficult
Carry out constructive variations.Fixed and immobilization processing and their order can change the shape of cell, and cytoscopy technician is expected that
And recognize this change.Fixer can reduce particular cell types so that cytoplasm shows graininess (granular)
Or netted (reticular).Many fixers can be played a role by being crosslinked combination with cell component.This can make defined epitope
Impaired or change, and new epitope is generated, molecule can be caused to combine (molecular associations), and film can be reduced
Permeability.Formalin is fixed as one kind in most conventional cytology/Histological method.Formalin on protein periphery or
Methyl bridge (methyl bridge) is formed in protein.Precipitation (precip itation) or solidification (coagulation) are also used
In fixation, ethanol is frequently used for such fixation.Crosslinking (crosslinking) and the combination of precipitation can also be used to fix.
Powerful fixing process is to save the method for optimizing of morphologic information from damage, conversely, weak fixing process is the preservation of molecular target
The best approach.
Straight alcohol, polyethylene glycol (PEG), 1.85% formaldehyde of 2mM that representational fixer is 50%.The component tune
Ethanol (50% to 95%), methanol (20%-50%) and formalin (first are individually included with the change on (formulation)
Aldehyde).In PEG1500,50% ethanol and 3% methanol that other common fixers are 2%.Glass slide is at normal temperatures to solid
Determine after about 10 minutes to 15 minutes are placed in liquid, remove and dry.Once glass slide is fixed, just using such as phosphate-buffered salt
The buffer solution of solution (PBS) etc. rinses.
Emphasize to extensive dyestuff otherness or the feature or morphological structure of control cell, subcellular fraction and tissue, can use
In " dyeing (stain) ".It is blueness or black that bush (hematoxylin), which is used for nuclear staining,.Orange G -6 (Orange G-6)
And eosin reddish black (eosin azur e) dyes the cytoplasm of cell.Orange G by keratin (keratin) and can contain
The cell dyeing yellowly of glycogen (glycogen).Eosin W or W S is used to dye kernel (nucleoli), cilium (cilia), red blood cell
(red blood cells) and superficial epithelial squamous cell (super ficial epithelial squamous cells).
Romano this base dyeing (romanowsky stai ns) is used for (air dried) glass slide spontaneously dried, can increase multiform
Property, it is useful to distinguishing the outer material of (intracytoplasmic) the material cell plastid out of cytoplasm.
Dyeing course may include the infiltrative processing of the cell to enhancing dyeing.Utilize the thin of lotion (d etergent)
The processing of born of the same parents can be used for strengthening permeability.In order to strengthen the permeability of cell and tissue, fixed sample also can use solvent
(solvents), saponin(e (saponins) or non-ionic lotion (non-ionic detergents) are handled.Enzyme decomposes
(enzymatic digestio n) can also improve the proximity of the particular target in tissue samples.
After dyeing, above-mentioned sample is rinsed by the increased continuity alcohol of determining alcohol to be dehydrated.Finally cleaning utilizes
Dimethylbenzene (xylene) or the citrus terpene (citrus terpene) with the refractive index close to the coverslip for glass slide
Deng dimethylbenzene substitute complete.The final step is referred to as washing (clearing).Once sample is dehydrated and washes, just make
With sealant (moun ting medium).Above-mentioned sealant selection has close to the refractive index of glass, and can be by lid glass
Piece is attached to glass slide.This may also suppress the additional drying of cell, contraction or decline.
Independent of used dyeing or processing, pneumonocyte sample is finally assessed by certain type of microscopical observation
To complete, which can determine that the visual examination (visual i nspection) in Xingtai and the presence or absence of marker.
Exemplary microscopy includes bright-field (b rightfield), phase difference (phase contrast), fluorescence
(fluorescence) and differential interference contrasts (differential interference contrast).
After inspection, the second test is required on sample, then can remove coverslip, and glass slide is decolourized.Decoloration
(destaining) original solvents system (original solvent s ystem) is included the use of, with the phase of original dyeing course
Reverse-order dyes glass slide, and does not add dyestuff originally.Decoloration can also be straight by the way that glass slide is immersed acid alcohol
Become colorless to cell to complete to decolourize.Once colourless glass slide is rinsed carefully in the sink, and carry out at the second dyeing
Reason.
Also, the discriminating of specific molecular can be tried by using the specific molecular of such as antibody or nucleic acid probe or aptamer etc.
The cytomorphology analysis of agent together carries out.This can improve the accuracy of diagnostic cytology.Micro-dissections (micro-
Dissection) it is particularly useful in the separation to separating abnormality chromosome (abnormal chromosomes), gene expression (gene
Expression) or mutation (mutations) the cell further assessed subset.
Preparation for assessing histological tissue samples includes fixed (fixation), is dehydrated (dehydration), oozes
(infiltration), embedding (embedding) and fragmentation (sectioning) thoroughly.Fixed examination used in histology
Agent has to sacrifice the characterization of molecules identical with each protein as generation with closely similar or identical used in cytology
Valency retains the same problem of morphological feature.It is dehydrated if tissue samples are not fixed, but after frozen, what is freezed
When by fragmentation, then can save the time.This is a kind of safer processing method, can retain more individual marker things.
However, causing subcellular fraction information to be lost due to importing ice crystal, freeze to be not suitable for store tissue sample for a long time.The group freezed
The fragmentation process for generating very thin section can also be disturbed by knitting the ice in sample.In addition to formalin is fixed, four oxidations
Osmium (osmium tetroxide) is additionally operable to fix, and phosphatide (film) is colored.
Washed successively by using the alcohol of gradual increase concentration to make tissue dewatering.Washing includes the use of alcohol and can mix bag
Bury the material of material, alcohol：The ratio of washing accelerating agent (clearing agent) (dimethylbenzene or dimethylbenzene substitute) starts from 50：
50 interim process.Permeating (infilt ration) is included first by tissue and 50：50 embedding reagent：Wash accelerating agent
With embedding reagent (hot wax (warm wax), the nitrocellulose solution of 100% embedding reagent liquid form
(nitrocellulose solution)) together cultivate.Embedding fills fusing by the way that tissue is positioned in mould or box
Embedding reagent such as wax, agar or gelatin are realized.Cure above-mentioned embedding reagent.Then, cured tissue samples can be with
In order to dye and subsequent experimental and cut into thin fragment.
Before dyeing, tissue fragment is removed wax and is dehydrated again.Wax of the dimethylbenzene for removing fragment, alternative one
Dimethylbenzene a or more than it, is organized by the continuous wash in the alcohol for reducing concentration, so as to complete to be dehydrated again.Go paraffin removal it
Before, tissue fragment at a temperature of about 80 DEG C heat fixation in about 20 minutes in glass slide.
Detection wind lidar (laser capture micro-dissection) can separate a part of cell
Collection, further to analyze fragment of tissue.
In order to strengthen the visualization of microscopic features, such as in cytology, tissue fragment or section can use various coloring agents
To dye.Various types of common coloring agents can be used for strengthening or differentiating specific characteristics.
In order to further enhance the interaction of cytology/histological sample and molecular agents, many use have been developed
In the technology of " analyte recovery ".First, fixed sample is heated to high temperature by this first technology.This method is also referred to as heat and lures
Lead epitope reparation (heat-induced epitop e retrieval) or HIER.Various heating techniques are used, including are steamed
Vapour heating (steam h eating), microwave heating (microwaving), autoclaving (autoclaving), water bath with thermostatic control
(wa ter bath) and pressure cooking (pressure cooking), or the combination of these heating means.Analyte recovers solution
Including such as water, citrate (citrate) and brine buffer solution (saline buf fers).Analyte recover core be
High-temperature time, but long-term relatively low temperature also can be used successfully.Another key that analyte is recovered is the pH value of heated solution.
Known low ph value can provide optimal immunostaining, but also often result in and need to use minor microstructure fragment as negative control
Background.Composition regardless of buffer solution, most powerful advantage (increase immunostaining is without increasing background) is usually using high pH
Solution obtains.Heating, time, pH and buffer solution is used to form as process optimization for the analyte recovery process of particular target
Parameter, empirically to optimize.Microwave analysis thing restoration methods allow different targets and antibody reagent continuous dyeing.However,
Time between staining procedure needed for completion antibody-multienzyme complex, which represents, can also reduce cell membrane analyte.Microwave heating side
Method further improves in situ hybridization (in situ hybridization).
Recover process to start analyte, wax is taken out first from fragment and is dehydrated.Then glass slide is immersed
In the 10mM sodium citrate buffer solutions of pH6.0 in disk or in bottle.In typical method, glass will be carried using the microwave of 1100W
Piece heats 2 minutes in the micro-wave oven of 100% power, and after the lid for confirming glass slide still covers in the solution,
Glass slide is heated to micro-wave oven 18 minutes with 20% power.Then glass slide is cooled down in non-covered container and is rushed with distilled water
Wash.In order to improve the reactivity of target and immuno-chemical reagent, HIER can degrade with enzyme and be used in combination.
The enzyme decomposing scheme uses Proteinase K (proteinase K).The EDTA of Tris salt, 1mM in 50mM, 0.5%
Triton X-100, pH 8.0 buffer solution in prepare concentration be 20 μ g/ml Proteinase K.The process includes passing through first
Replace secondary, 5 minutes removing fragments every time wax with dimethylbenzene.Hereafter, sample is respectively with 100% hydrous ethanol 3 minutes, and 95%
1 minute each with 80% ethanol, and use distilled water flushing.Fragment is covered with Proteinase K, and in moist room at a temperature of 37 DEG C
Cultivate 10~20 minutes (optimal incubation time can change according to tissue morphology and immobilization degree).By the fragment in room temperature
Lower cooling 10 minutes, is then rinsed 2 minutes 2 times with PBS polysorbas20s.If desired, fragment can be blocked to eliminate endogenous
The potential interference of compound and enzyme.Then when by above-mentioned fragment, appropriate dilution 1 is small at normal temperatures in first antibody dilution buffer
Or stay overnight at 4 DEG C and together cultivated with first antibody.Above-mentioned fragment is rinsed 2 minutes 2 times with PBS polysorbas20s.If necessary to special
Using, then carry out extra blocking, then again with PBS Tween 20 rinse 3 times, continue 2 minutes, finally complete immunostaining
Scheme.
Being also demonstrated that at room temperature with 1% SDS simple process improves immunohistochemical staining.Analyte restoration methods
It can be applied to the fragment of the fragment and glass slide covering flowed freely.Another therapeutic choice is to immerse glass slide to contain
PH is in 6.0 citric acid and the container of 0.1Nonidet P40 and is heated to 95 DEG C.Then washed with buffer solution such as PBS above-mentioned
Glass slide.
Dyed for the immunology of tissue, by the way that fragment is immersed protein solution such as serum or skimmed milk power (non-fat
Dry milk) in, it is possible to it is useful for blocking the non-specific binding of tissue protein and antibody.
Barrier reaction (blocking reaction) reduces the water of Endogenous Biotin (endogenous biotin)
It is flat；Eliminate endogenous charge effect (endogenous charge effects)；It may need inactivating endogenous nuclease
(endogenous nucleases) and/or inactivating endogenous enzyme such as peroxidase (peroxidase) and alkaline phosphatase
(alkaline phosphatase).Endogenous nucleic acid enzyme, by heat treatment, can be used by using the degraded of Proteinase K
Such as the chelating agent of EDTA or EGTA, by introducing carrier DNA or ribonucleic acid, urea (urea), thiocarbamide
(thiourea), guanidine hydrochloride (guanidine hydrochloride), guanidine thiocyanate (guanidine thiocyanate) are high
The chaotropic agent of lithium chlorate (lithium perchlorate) etc. or pyrocarbonic acid diethyl ester (diethyl pyrocarbonate) etc.
(chaotrope) handled, so as to inactivate.Alkaline phosphatase can be handled 5 minutes at room temperature by using the HCl of 0.1N
Or handled by using 1mM levamisols (levamisole), so as to inactivate.Peroxidase activity can be by using
0.03% hydrogen peroxide (hydrogen peroxide) is handled to remove.By the way that glass slide or fragment are immersed avidin 9
(streptavidin (streptavidin), neutravidin (neutravidin) can be with for (avidin) solution in vain
Alternatively) at least 15 minutes block endogenous nucleic acid enzyme at room temperature.Then above-mentioned glass slide or fragment are washed with buffer solution
Wash at least 10 minutes.Washing can repeat at least 3 times.Then glass slide or fragment are immersed into biotin solution 10 minutes.Every time
It can be repeated at least 3 times using new biotin solution.Repeat buffer solution cleaning procedure.Should minimally use blocking side
Case to prevent infringement cell or tissue structure or target interested, but can with one or the slow dissociation rate core more than it
Before sour aptamer is reacted, it can be combined with " stop " glass slide or fragment (with reference to Basic Medical
Histology:The Biology of Cells, Tissues and Organs, authored by Richard
G.Kessel, Oxford University Press, 1998).
Utilize the measure of the biomarker values of mass spectrometric analysis method
The mass spectrograph of various structures can be used for detecting biomarker values.Polytype mass spectrograph can be used, or
It can produce with various structures.In general, mass spectrograph has following primary structure key element：Sample inflow entrance (inlet), ion
Supply source (ion source), mass spectrograph (mass analyzer), detector (detector), vacuum system (vacuum
Syste m) and instrument-control system (instrument-control system) and data system (data s ystem).It is logical
Often, the difference between sample inflow entrance, ion supply source and mass spectrograph determines the form and characteristic of instrument.For example, above-mentioned stream
Entrance can be capillary column liquid chromatogram source (c apillary-column liquid chromatography source)
Or the direct probe (direct used in substance assistant laser desorpted (m atrix-assisted laser desorption)
) or sucker (stage) probe.Common ion supply source is e.g., including nano-spray (nanospray) and micro- spraying
(microspray) electron spray or substance assistant laser desorpted.Typical mass spectrograph includes quadrupole mass filter (quadrupole
mass filter)；Ion trap mass analyzer (ion t rap mass analyzer) and time-of-flight analyser
(time-of-flight mass analy zer).Mass spectrographic other methods are known (reference in the art
Burlingame et al., Anal.Chem.70:647R-716R(1998)；Kinter and Sherman, Ne w York
(2000))。
Protein biomarkers and biomarker values any can be detected and are measured by following：Electronic spraying
Ionization mass spectrometry (electrospray ionization mass spectrometry, ESI-MS), ESI-MS/MS, ESI-MS/
(MS) n, substance assistant laser desorpted ionization time of flight mass mass analysis (matrix-assisted laser
Desorption ionization time-of-flight mass spectrometry, MALDI-TOF-MS), surface enhanced
Mastrix-assisted laser desorption ionization time of flight mass spectrography (surface-enhanced laser desorption/ionization
Time-of-flight mass spectrometry, SELDI-TOF-MS), desorption/ionization (desorption/ on silicon
Ionization on silicon, DIOS), secondary ion mass spectrometry (secondary ion mass spectroscopy,
SIMS), quadrupole flight time (quadrupole time-of-flight；Q-TOF), it is referred to as ultraflexIII TOF/
Series connection flight time (tandem time-of-flight) (TOF/TOF) technology, the atmospheric pressure chemical ionization mass spectrography of TOF
(atmospheric pressure chemical ionization mass spectrometry；APCI-MS), APCI-MS/
MS, APCI- (MS)N, atmospheric pressure photoion mass spectrography (atmospheric pressure photoionization mass
spectrometry；APPI-MS), APPI-MS/MS and APPI- (MS)N, quadrupole mass analysis (quadrupole mass
Spectrometry), Fourier transform mass spectrometry (Fourier transform mass spectrometry；FTMS)、
Quantitative mass analysis (quantitative mass spectrometry) and ion trap mass spectrometry (ion trap mass
spectrometry)。
Sample preparation strategy is used to before mass spectral characteristi protein biomarkers and detection biomarker values mark
With quantitative sample.Labeling method is for opposite and absolute quantitation isobaric mark (isobaric tag) (isobaric tag
for relative and absolute quantitation；ITRAQ) and with the amino acid stable isotope in cell culture
Mark (stable isotope lab eling with amino acid in cell culture；SILAC), but it is not limited to
This.Before mass spectral analysis, the capture agent for the sample of selective enrichment candidate biomarker albumen is fitted including nucleic acid
Body, antibody, nucleic acid probe, chimera, small molecule, F (ab')2Fragment, single chain antibody fragments (single chain antibody
Fragment), Fv fragments (Fv fragm ent), Single-Chain Fv Fragment of Murine (single chain Fv fragment), nucleic acid
(nucleic aci d), agglutinin (lectin), ligand binding receptor (ligand-binding receptor), affine body
(affybodies), nano antibody (nanobodies), ankyrin class (ankyrins), domain antibodies (domain
Antibodies), alternative antibody scaffold (alternative antibody sc affolds) is (for example, bifunctional antibody
(diabodies)), imprinted polymer (imprint ed polymer), Avimers (avimers), the simulating peptide such as
(peptidomimetic s), class peptides (peptoids), peptide nucleic acid (peptide nucleic acid), threose nucleic acid
(threose nucleic acid), hormone receptor (hormone receptor), cytokine receptor (cytokine
Receptor) and synthesis of receptor (synthetic receptors) and their change and fragment, but it is not limited to this.
Utilize the measure of the biomarker values of neighbouring binding assay
Proximity ligation assay (proximity ligation assay) can be used for the measure of biomarker values.Letter and
Yan Zhi, sample is contacted with a pair of of affinity probe (affinity probe), the affinity probe can be a pair of of antibody or one
To aptamer, and the pair of each constituent extends to oligonucleotides.The target of a pair of of affinity probe can be one
Each determinant in two different determinants or two different proteins on a protein, it can be used as same
Source polymer (homomultimeric) or heteromultimers (heteromultimeric) complex exist.When probe and target
When determinant combines, the free end (free end) of oligonucleotides extension (oligonucleoti de extension) is mobile
It is near so that the degree hybridized each other enough.When oligonucleotides extended position sufficiently closes to, oligonucleotides extension is easy to pass through
Connect their common connection oligonucleotides (common connector oligonucleotide) hybridization.Once the widow of probe
Nucleotide extension is hybridized, and the end of extension is combined by enzymatic DNA to be connected.
Each oligonucleotides extension is containing the primer sites for being useful for PCR amplification.Once oligonucleotides extension is bonded to each other, few
The identity (i dentity) and quantity (amount) of target protein will be not only presented in nucleotide by PCR amplification, but also works as target
When determinant is on two kinds of different albumen, the continuous DNA sequence for representing protein interaction information is formed.Close to combination
By real-time PCR high sensitivity and specific detection can be provided for real-time protein concentration and interaction information.Not with feeling emerging
The probe that is combined of determinant of interest will not make corresponding oligonucleotides extend close to it is mobile, and any combination cannot be carried out
Or PCR amplification, any signal is not as a result produced.
Pass through said determination so that can detect can be usefully used for the biomarker values of diagnosing non-small cell lung cancer,
It includes the following steps：The biomarker values detection at least N of the group of the biomarker composition provided in Free Surface 2 is selected in detection
A corresponding biomarker values.As described below, show above-mentioned individual whether with non-small thin using the classification of bi upsilonmtirkcr values
Born of the same parents' lung cancer.Although the particular organisms marker of the non-small cell lung cancer biomarker individually described can be used for detect and diagnose non-
Small Cell Lung Cancer, but can be used as multiple non-small cell lung cancers biology mark of the group comprising three or more biomarkers respectively
The subset of will thing, will be explained how to determine in the present invention.Therefore, various embodiments of the present invention are provided gives birth to comprising N kinds
The combination of thing marker, wherein N are at least three kinds of biomarkers.In another embodiment, N is arbitrarily selected from 2 to 59 kinds
Biomarker.N can be selected arbitrarily within the above range, but can include similar but higher level scope with selected as.Root
According to method described herein, biomarker values individually can be detected and classified, such as can be in the form of many measure
Detected and classified by collective.
According to another embodiment, there is provided for checking the method being not present of non-small cell lung cancer.Above method bag
Include individual biological specimen in select Free Surface 2 provide biomarker group selection it is corresponding with biomarker respectively
At least N number of be put into the step of biomarker values are detected.Such as described further below, point of biomarker values is utilized
Class represents that non-small cell lung cancer is not present in above-mentioned individual.The specific life in described non-small cell lung cancer biomarker
Thing marker independence to detection and diagnosing non-small cell lung cancer there is no useful, but in the present invention to as three or
Biomarker group more than it is used for the method for more subsets of the useful non-small cell lung cancer biomarker of classification difference
Illustrate.Therefore, various embodiments of the invention provide the combination for including N number of biomarker, wherein, N at least three
Biomarker.In another embodiment, arbitrarily selected in biomarkers of the N from 2 to 59.N is not only remembered above-mentioned
Arbitrarily selected in the scope of load, although and may be selected to similar to the scope that can include higher.Can middle institute according to the present invention
The method of record, is detected individually and biomarker values of classifying, or for example, such as a variety of many measure forms, in a manner of set
Detect and classify.
The classification of biomarker and the calculating of Disease Score
The biomarker " signature (signature) " of diagnostic test for being provided includes a series of markers, respectively
A marker has mutually different level in concern is gathered.Wherein, mutually different level refers to more than two or its
The individual different marker levels of group are averaged, or two or organize different changes or their two kinds wholes more than it
Combination.For the diagnostic test of simplest form, individual is being distributed to normal group as one of two groups or disease group
Unknown sample when, their marker can be used.A kind of distribution sample in more than two groups will be known as classifying, will be
Method used in this sample distribution is known as grader (classifier) or sorting technique (classification
method).In addition, sorting technique is also known as point system (scoring method).In order to from a series of biomarker
Value forms diagnostic classification device (diagnostic classifier) using more sorting techniques.In general, in be distinguished two
A (or in order to which various classification states are the data set more than it) mutually different group of interior sample by using from individual acquirement,
To utilize the supervised learning technology (supervised learning techniques) for collecting data set, then it is easiest to hold
Row sorting technique.Species (group or set) belonging to known each sample, therefore, can be can provide in above-mentioned sorting technique
The mode of required classification reaction gives training.Unsupervised learning technology also can be used in order to produce diagnostic classification device
(unsupervised learning techniques)。
Include for developing the conventional of diagnostic classification device close to method：Decision chart (decision tre e)；Bagging
(bagging)+promote (boosting)+forest (forests)；Rule deduction based on study；Parzen windows (Parzen
Window)；Linear model (linear mo dels)；Symbolic logic (logistic)；Neutral net (neural
Network) method；Unsupervised clustering (unsupervised clustering)；K- is average (K-means)；It is layered ascending order/drop
Sequence (hierarchical ascending/descending)；Semi-supervised learning (semi-supe rvised learning)；
Prototype method (prototype methods)；Neighbour samples (neares t neighbor)；Density Estimator (kernel
density estimation)；Support vector machines (support vector machines)；Hidden Markov model (hidden
Markov mode ls)；Boltzmann learns (Boltzmann Learning), and grader can simply combine or can be with by spy
The mode for determining purpose function (objective function) minimum combines.Pattern Classification are can refer to,
R.O.Duda, et al., editors, John Wiley＆S ons, 2nd edition, 2001 and The Elements of
Statistical Learning-Dat a Mining, Inference, and Prediction, T.Hastie, et al.,
Editors, Spri nger Science+Business Media, LLC, 2nd edition, 2009, they each is logical
Reference is crossed to be integrally incorporated.
In order to generate grader by using supervised learning technology, a series of sample for being referred to as training data is obtained.
In diagnostic test, training data includes the sample from the group (classification) mutually distinguished for distributing unknown sample later.Example
Such as, from control group individual and specified disease group individual collect sample may make up in order to develop be used for using disease whether
The training data used in the presence of the grader classified to unknown sample (or in more detail, obtaining the individual of sample).
It is also known as giveing grader training from training data exploitation grader.Usually to the special details of grader training
It is the characteristic of supervised learning technology.In order to illustrate following, to training Naive Bayes Classifier (naive Bayesian
Classifier example) illustrate (for example, referring to Pattern Classification, R.O.Duda, et al.,
Editor s, John Wiley＆Sons, 2nd edition, 2001；Also, with reference to The Elemen ts of
Statistical Learning-Data Mining, Inference, and prediction, T.Hastie, et al.,
Editors, Springer Science+Business Media, LLC, 2nd edition, 2009).
Generally, due to the potentiality biomarker values that there is the sample more than training set, thus pay attention to avoiding excessively
It is fitted (over-fitting).In the case where statistical models substitute relation radix random error or the noise of radix, occur
Overfitting.For example, overfitting can be avoided by following a variety of methods：Limitation is suitable for the number of the marker of grader exploitation
Amount, assume that marker reaction is separate, the complexity of elementary statistics model used in limitation, and ensures basic statistical
Learn model be based on data etc..
The example for developing the diagnostic test of a series of biomarker is included to Naive Bayes Classifier, biology mark
Will thing is strictly independently handled, and the simple probability grader based on Bayes principle is applicable.Biomarker passes through
The RFU values that are measured in various species or with the relevant species of log RFU values (Relative fluorescence units)-dependence density function
(probabi lity density function；Pdf) described.In a group, it is relevant with a series of marker
Joint probability density function (joint pdfs) is assumed to be the production with the relevant group of individuals dependence pdf of each biomarker
Thing.It is related to this, Naive Bayes Classifier is giveed training for equivalent to screening be used for mentioned kind-dependence pdf into
The parameter (" parametrization (parameterization) ") that row characterizes.The basic mould based on species-dependence pdf can be used
Type, still, above-mentioned model need and the Data Matching usually what is observed in training set.
Specifically, measure and species-dependence probability of the relevant value xi of biomarker i of kinds of Diseases are recited as
P (xi | d), observable have total naive Bayesian of n marker of { tilde over (x) }=(x1, x2 ... xn) values general
Rate is recited as p ({ tilde over (x) } | d)=Π i=1np (xi | d), wherein, individual xi is utilizes RFU or logRF U
The biomarker level being measured.With it is unknown it is relevant it is classification-designated for, for same measured value, by with without
Possibility (control group) p (c | { tilde over (x) }) of disease is compared, can be with measured value { tilde to calculate
Over (x) } disease possibility p (d | { tilde over (x) }), so as to become easy.The ratio of above-mentioned possibility can lead to
Cross and be applicable in Bayes principle, to be calculated from species-dependence pdf, wherein, p (d) is in the set for being suitable for test
The ill rate of disease.If the logarithm of the both sides ratio by taking above-mentioned ratio, substitute into from above-mentioned naive Bayesian species-according to
Rely property probability, then it is as follows.
It is known that above-mentioned form is log-likelihood ratio (log likelihood ratio), refer to simply deposit with disease
Relevant log-likelihood (log likelihood) is being not present with disease, disposably, by N number of each biomarker
The conjunction of log-likelihood ratio is formed.In the simplest form for this, (or in more detail, obtained above-mentioned in unknown sample
The individual of sample) above-mentioned ratio be more than 0 in the case of, be categorized as no disease, above-mentioned ratio be less than 0 in the case of be categorized as
With disease.
In an exemplary embodiments, species dependence biomarker pdfsp (xi | c) and p (xi | d) it is assumed to be and is surveyed
Fixed RFU values xi, i.e. to the P (xi | d) using μ d and σ d, be assumed to be in the RFU values with similar formula normal distribution or
Log-normal distribution.In order to above-mentioned model parametrization, it is necessary to from training data estimate it is related to various species-dependence pdf
Two parameters, average μ and scattered σ 2.For example, this is can be by maximal possibility estimation, skill belonging to least square method and the present invention
Multiple methods of arbitrary method known to the those of ordinary skill in art field are realized.If will be with μ and σ relevant normal distribution generations
Enter the log likelihood ratios defined in above-mentioned, then it is as follows.
If as a series of μ and σ 2 from the ill rate of disease of training data and set to each pdf of various species into
Row definition, then determine above-mentioned Bayes classifier completely, so as to be used as measured value { tilde over (x) } by above-mentioned, coming can profit
For classifying to unknown sample.
The performance of Naive Bayes Classifier with composition and classification device and the quantity of the biomarker for giveing training and
Characteristic is related.Such as it is defined in following example 3, single creature marker is according to the KS- distances of above-mentioned biomarker
(Kolmogorov-Smirnov) work.If classifier performance index (classifier performance metric)
It is defined as the bottom area (AUC) of Receiver operating curve, then complete grader has 1 fraction, random assortment
Device fifty-fifty has 0.5 fraction.The KS- distance definitions that size is n between two the set A and B of m are Dn, m=supx |
FA, n (x) FB, m (x) | value, this is two empirical cumulative distribution function (empirical cumulative
Distribution function, cdf) between maximum different value.With the relevant empirical cumulatives of set A of the observation Xi of No. n
Distribution function is defined as foloows, wherein, in Xi<In the case of x, IXix is identical with 1, is otherwise then the indicator function identical with 0
(indicator function).Above-mentioned value is between 0 and 1 in the sense, wherein, KS- distances 1 represent not repeat experience point
Cloth.
Addition with outstanding KS distances (for example,>0.3) in the case of succeeding designations thing, if that is added is above-mentioned
Marker is independent to the first marker, then usually improves classification performance.If use Receiver Operating Characteristics as grader fraction
Area under the curve (AUC), then can easily generate more high scores of the change with greedy algorithm (greedy algorithm)
Grader.(greedy algorithm is to be held in each step with the possibility for finding global optimum (global optimum)
Row solves the arbitrary algorithm of the meta-heuristic method (metaheuristic) of selection locality optimal value).
From table generation all single analyte graders, and make an addition to catalogue of potentiality biomarker.Then, to the greatest extent may be used
All the second analytes can be made an addition to the single analyte grader of each storage, store predetermined quantity is most in new directory
High score is to (pair), i.e. storage 1000.By using the relevant above-mentioned new directory of 2- marker graders on most, come
The whole possible three marker graders of investigation, 1000 be again stored in them on most.If above-mentioned score reaches stable
State (plateau) continues the above process because adding when the marker of additivity starts to reduce.In order to the purposes being intended to
Preferable performance, after convergence, it can be estimated that remaining high scores classification device.For example, one it is diagnostic be applicable in, there is Gao Ling
Sensitivity and appropriate specific grader are preferably in the grader with appropriate sensitivity and high specific.Examined another
During disconnected property is applicable in, it is preferable to be the grader with high specific and appropriate sensitivity.It is another it is diagnostic be applicable in, have
May more preferably have the grader of high specific and appropriate sensitivity.Usually as preferable performance level, base
Selected in shifting (trade-off) when being applicable in particular diagnosis between admissible false positive number and false negative number
Select.In general, this shifting Medical result for only relying upon one of false positive or false negative.
In the art, well-known various other technologies, from the biological marker for being applicable in Naive Bayes Classifier
The catalogue of thing can be used in the more potentiality grader of generation.In one embodiment, so-called genetic algorithm (genetic
Algorithms) can be used in by using the appropriate fraction defined in above-mentioned, to carry out group to mutually different marker
Close.Especially, genetic algorithm is suitable for finding out the potentiality grader of very big and various set.In addition, in another embodiment
In, so-called ant colony optimization algorithm (ant colony optimization) can be used in a series of grader is generated.Example
Such as, not using only the method for other evolution, but also it can be used and include simulated annealing simulated annealing (simulated
Annealing) and other stochastic search methods (s tochastic search methods) it is known in the art
Other methods.For example, as meta-heuristic method, such as harmonic search algorithm (harmony search) can be used.
Clinical sample obtains
In order to select biomarker, historically patient's group and control group are have collected in the research and design of Fig. 1 and table 1.
Patient's group is with the Asian with non-small cell lung cancer that stadium is 1 phase to 4 phases.Control group be by health ordinary people and
Patient with non-malignant Lung neoplasm is formed.Each sample is extracted by venipuncture, and serum is obtained according to common scheme.
Under below -80 DEG C, each serum is freezed and is stored.
The measure of candidate biomarker
In order to measure candidate markers, implement a variety of identification methods based on aptamer as shown in Figure 2.By using light
Biotin fragment is connected by the connection peptide cut with each aptamer respectively.Briefly, modification is mixed with serum
Aptamer mixture, and 3 hours are cultivated to implement balance coupling at a temperature of 37 DEG C.Afterwards, above-mentioned mixed liquor is transferred
In the tablet using Streptavidin (streptavidin) coding, and in order to by biotin label by aptamer-albumen
On matter complex capture aptamer, cultivate 30 minutes.By implementing a series of cleaning step, being gone from sample unless
Conjugated protein.Then, in order to mark biotin on the protein of capture, amine reactivity biotin reagent has been cultivated.Then,
Ultraviolet is irradiated, aptamer-protein complex is released by light cleavage reaction.Supernatant is transferred to utilization strepto-
On the new tablet of antibiotin (streptavidin) coding, and in order to which complex is being trapped in albumen by biotin fragment
Cultivated in matter.By implementing a series of cleaning, make a return journey unless with reference to aptamer.Afterwards, it is slow by eluting
Fliud flushing releases captured aptamer using high pH, and is neutralized.Eluent and detection probe, Lu Mingkesi (L
Uminex)-microballon integrating capture probe carries out dehydration.Lu Mingkesi microballons mixture is by cleaning, to use road
200 utensils of Ming Kesi are measured, and by using 3.1 software analysis datas of Lu Mingkesi.
Biomarker values adjust (calibration)
In order to which calibrating change is minimized, 3 point QC samples are together measured with sample.3 points in the detection range of identification method
Represent high-level, the middle horizontal and low-level of protein concentration.QC samples are by serum and spike protein (spiked protein)
Formed, and confirmed in the item value tested before.When carrying out each calibrating, by testing QC samples, with item
Value compares the Protein scores measured in QC samples, and generates Dynamic gene to each protein.Dynamic gene tune is utilized afterwards
Whole verification result.
Biomarker for non-small cell lung cancer is selected
In order to select thief to be used for the small-scale biomarker set of non-small cell lung cancer, compare patient's group and control group
The MFI values being adjusted, and export cumulative distribution chart and with the relevant Receiver operating curve of each aptamer
(ROC) (Fig. 4 a to 4n).Area under test curve for each marker, and be compared.
The establishment of Naive Bayes Classifier
In order to which multiple variable quantities to be prepared into single fraction, Naive Bayes Classifier is generated.From for non-small cell
7 markers are selected to form Naive Bayes Classifier in the biomarker catalogue of lung cancer.Below to for measuring single egg
The Naive Bayes Classifier of white matter illustrates.In the case where the measured value of protein is x, the probability with disease is P
(d|x).Also, in the case where the measured value of protein is x, the probability without disease is P (c | x).If P (d | x)>P(c|
X), then protein level x can be categorized as disease.According to Bayes' theorem,
Posterior probability (posterior)=likelihood (likelihood) × prior probability (prior)/evidence (evidence)
Species-dependence probability density function, p (xi | c) and p (xi | d) it is modeled to be defined by average u and deviation s2
Log-normal distribution function, wherein, xi be with the relevant adjustment MFI values of biomarker i to numerical value.Record and use in table 3
In the variable of the pdf of above-mentioned 7 biomarkers, and it is suitable for regular pdf models and together shows the one of non-processing data in Fig. 3
Example.Naive Bayes Classification for this model is represented with following mathematical expression, wherein, p (d) is the ill rate of disease of set.
Likelihood ratio (likelihood ratio)=P (d | x)/P (c | x)=p (d)/(1-p (d) xPi (p (x | d)/p (x
|c))
Log-likelihood ratio is used as being used for the single fraction for distinguishing morbid state.
Embodiment
This is provided for illustrative purpose to following embodiments, is not intended to limit the sheet defined by inventing claimed scope
The scope of invention.Described whole examples utilize the known mark of general technical staff of the technical field of the invention in the present invention
Note technology is implemented.The common Protocols in Molecular Biology illustrated in the examples below can be recorded according to original
Laboratory manual is marked to implement, such as Sambrook et al., Molecular Cloning:A Laboratory Manual,
3rdEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001) etc..
Embodiment 1
Sample preparation
In order to prepare sample, it is necessary to which whole blood is stored in red push pipe (red top tube).After gathering whole blood, to be blood
Solidification 30 minutes, under normal temperature condition, intactly cultivates testing tube.Afterwards, in centrifugal separator is refrigerated, with 1000 to
The centrifugation that 2000xg is carried out 10 minutes separates serum and thrombus., it is necessary to be moved in time to clean light after centrifugation
The serum of dynamic upper layer part.Sample needs to keep 2 DEG C to 8 DEG C of temperature in processing procedure.If not analyzing serum at the scene, need
Above-mentioned serum to be inoculated with a small amount of aliquots (aliquot) in time, in subzero less than 20 DEG C lower storages.Freezing-
Thawing cycle has mortality to many serum compositions, thus avoids this point important.Hemolytic, bilious or hyperlipidemic sample
Product can make fc-specific test FC ineffective treatment.
Embodiment 2
Sample analysis based on multiple nucleic acids aptamer identification method
The present embodiment records multiple nucleic acids aptamer identification method, and above-mentioned multiple nucleic acids aptamer identification method is in order to for differentiating table
The sample of the biomarker for the non-small cell lung cancer listed in 2 is analyzed and used.Add every time in these methods molten
Suction nozzle is replaced during liquid.
Also, if without being otherwise noted, most solutions transfer and the addition of cleaning solution are clear using BioTek EL 406
Wash device and distributor.If not otherwise stated, the micro dispensers of 8- passages P200 are used in the step of manual liquid relief
(Pipetteman) (Rainin Instruments, LLC, Oakland, Calif.).In inside, preparation is referred to as determining for SB17
Buffer solution processed, and by 40mM hydroxyethyl piperazine second thiosulfonic acids (HEPES), 101mM NaCl, 5mM KCL, 5mM MgCl2, and
pH7.51mM EDTA are formed.Unless otherwise stated, all steps carry out under normal temperature condition.
1. prepare aptamer stoste (aptamer stock solution)
Under 0.05% Tween-20, the customization protokaryon for 2%, 0.1% and 0.01% serum is prepared with 2 × concentration
Sour aptamer solutions.
These solution storages are at -20 DEG C until using.On the day of calibrating, every kind of nucleic acid mixed liquor is thawed 10 at 37 DEG C
Minute, placed 10 minutes in boiling water groove, be cooled to 25 DEG C 20 minutes, and carry out active mixing between each step.
It is after heating-cooling, each 2x nucleic acid mixed liquor of 55 μ l is flat to 96 hole PCRs (PCR) using liquid is aspirated with hand
In plate, and it is sealed with a foil plate.
2. examine and determine sample preparation
The serum aliquot for the freezing 100% for being stored in subzero 80 ° is put into 25 DEG C of water-bath 5 minutes.It will thaw
Sample be put on ice for and slowly produce vortex, be then positioned over again on ice.
8- channel pipettors are put into by using 20ul, the sample of 3ul is moved to the PCR of 96- holes puts down
Plate, so that the dilution of 72ul (1 × SB17,0.02% Tween-20) titration is contained in each hole under 4C, to have prepared 4% sample
Product liquid (final 2x).By carrying out multiple liquid relief come after biased sample, by 4% sample solution of mobile 4ul, with 76ul's
Sample diluting liquid mixing is titrated, and obtains 0.2% sample liquid (final 2x).Finally, 0.2% sample of mobile 6ul is passed through
Liquid, mixes with the sample diluting liquid of 54ul, so as to achieve 0.02% sample liquid (final 2x).Prepare sample diluting liquid
Afterwards, moved to balance with reference to by each sample 55ul to new PCR tablet.
3. parallel combination
Three heating-cooling aptamer solution move to the titration sample diluting liquid that capacity is 55ul.Pass through pipette mixing
Sample and aptamer solution, and cover foil lid.Then in 37 DEG C of incubator by above-mentioned tablet place 3 it is small when.
4. catch 1 (Catch 1)
After balance combines step, the nucleic acid of 100 μ l-sample mixed liquor is transferred to coated with streptavidin
On new tablet, and above-mentioned plate is placed into hot mixing device (thermomixer) and mixes 30 minutes (800rpm), at a temperature of 37 DEG C
Cultivated.In order to avoid light, above-mentioned tablet is covered in 1 step is entirely caught.
5. automate step 1:Cleaning, mark
After catching 1 step, above-mentioned tablet is positioned over EL406 washers and and dispenser.Above-mentioned EL406 washers and point
Liquid device program turns to carry out following steps：Unabsorbed material is removed by aspirating, and is supplemented with 1mM's using 300ul
The buffer solution PB1 of the biotin of dextran sulfate and 500uM cleans in hole 4 times.Then using the PB1 buffer solutions of 300 μ l by hole
Clean again 3 times.
The NHS-PEG4- biotin solutions of the 1mM of the 150 μ L newly prepared are made an addition into hole in buffer solution PB1, to vibrate
5 minutes and cultivated.Liquid is inhaled into, and is cleaned in hole 8 times using the 300ul buffer solutions PB1 for being supplemented with 10mM glycerine.Addition
It is supplemented with the 100ul buffer solutions PB1 of 1mM dextran sulfates.
6. dynamic induces (kinetic challenge), light cutting and seizure 2
Take out tablet from EL406 cleaners and separator, and (LED is ultraviolet in the ultraviolet light source of the distance with 5cm
Line source) under the Hot mixer that configures place 20 minutes.Above-mentioned Hot mixer is set in 800rpm and RT.Irradiation after five minutes, will
Sample is transferred on the new tablet coated with Streptavidin manually.2 steps are caught in the hot mixing device of 800rpm and room temperature
Carry out 10 minutes.
7. automate step 2:Cleaning, elution
After catching 2 steps, above-mentioned tablet is positioned over EL406 washers and dispenser.Above-mentioned EL406 washers and liquid separation
Device program turns to carry out following steps：Pumping liquid, and it is clear using the buffer solution PB1 of the propane diols for being supplemented with 25% of 300ul
Wash 8 times.Using 5 secondary aperture of P B1 buffer solution for cleaning of 300 μ l, and aspirate final cleaning solution.The CAPSO elutions for adding 100ul are slow
Fliud flushing, and shake up 5 minutes and eluted.
8. elution and neutralisation
After these automation steps, by tablet from the deck of tablet cleaner, and the sample of 90ul can be divided
In the hole for the PCR tablet that amount is transferred to the neutralisation buffer solution for filling 10uL manually.
9. Lu Mingkesi is read
In order to float microsphere, vortex is produced 60 seconds in microsphere stoste, and carry out ultrasonication.Floating
Microsphere is diluted to the microsphere of every reaction 2000 in 1.5x tetramethyl ammonium chlorides (TMAC) hybridization solution, and passes through vortex
And ultrasonication is mixed.The microballon mixed liquor of 33uL is transferred to 96 hole PCRs in each reaction to put down
Plate.In 1x TE buffer solutions, go forward side by side in each 15nM for reacting addition 7ul through biotinylated detection oligonucleotides stock
Row mixing.The calibrating sample of the neutralisation of 10ul is added, and is sealed using silicon cap pad line.First, by above-mentioned tablet in 96C
At a temperature of cultivate 5 minutes, and cultivated in gene-amplificative instrament without stirring under 50C.Advance with and be supplemented with 0.5%
0.005 × tetramethyl ammonium chloride (TMAC) hybridization solution of bovine serum albumin(BSA) (BSA) get filter tablet wet.From above-mentioned miscellaneous
Reaction is handed over, whole sample is transferred to filter tablet.Utilize 0.005 × tetramethyl of the bovine serum albumin(BSA) for being supplemented with 0.5%
Ammonium chloride hybridization solution cleans above-mentioned hybridization tablet, and residue is transferred to filter tablet.In slow vacuum shape
Sample is filtered under state.0.005 × tetramethyl ammonium chloride using the bovine serum albumin(BSA) for being supplemented with 0.5% of 75ul is miscellaneous
Solution is handed over to clean above-mentioned hybridization tablet, the microsphere for being present in filter tablet, carries out one again in sample buffer
Secondary floating processing.For filter tablet, shading is carried out, is cultivated 5 minutes in hot mixing device with 1000rpm.Utilize afterwards
The molten glue soluting cleaning filter tablet of 0.005 × tetramethyl ammonium chloride comprising 0.5% bovine serum albumin.Including 0.5% ox
In the SAPE of each 10ug/ml for reacting addition 075ul in 0.005 × tetramethyl ammonium chloride solution of haemocyanin, and with
1000rpm cultivated in 25 DEG C of hot mixing device 1 it is small when.Utilize the 0.005 × tetramethyl for including 0.5% bovine serum albumin
Ammonium chloride solution cleans filter plate twice, and the chlorination of 0.005 × tetramethyl is utilized to the microsphere for being present in filter tablet
Ammonium salt solution cleans twice, and 0.005 × tetramethyl ammonium chloride hybridization solution of the bovine serum albumin comprising .5% using 75ul
Floating processing is carried out again, and is analyzed on 200 utensils of Lu Mingkesi for starting 3.0 softwares of XPonent.7500 to
Under 18000 PMT adjustment and the setting of double type arbiter, the microsphere of at least 100 is counted in each bead type.
Embodiment 3
The discriminating of biomarker
In order to which calibrating change is minimized, 3 point QC samples are together measured with sample.3 points in the detection range of identification method
Represent high-level, the middle horizontal and low-level of protein concentration.QC samples are formed by serum and bur protein, and are confirmed at it
The item value of preceding test.When carrying out each calibrating, by testing QC samples, come with item be worth compared with QC samples
The Protein scores of measure, and Dynamic gene is generated to each protein.Dynamic gene be can refer into referred to as item value/test value.It
Afterwards, verification result is adjusted using Dynamic gene.
In order to select the small-scale biomarker set for non-small cell lung cancer, compare patient's group and control group
The MFI values being adjusted, and export and the relevant Receiver Operating Characteristics of each aptamer (ROC) curve (Fig. 4 a to figure
4n).Test curve area below and it is compared for each marker.
Embodiment 4
Naive Bayes Classifier training for non-small cell lung cancer
In order to which multiple variable quantities to be prepared into single fraction, Naive Bayes Classifier is generated.From for non-small cell
7 markers are selected to form Naive Bayes Classifier in the biomarker catalogue of lung cancer.Below to for measuring single egg
The Naive Bayes Classifier of white matter illustrates.In the case where the measured value of protein is x, the probability with disease is P
(d|x).Also, in the case where the measured value of protein is x, the probability without disease is P (c | x).If P (d | x)>P(c|
X), then protein level x can be categorized as disease.According to Bayes' theorem,
Posterior probability (posterior)=likelihood (likelihood) × prior probability (prior)/evidence (evidence)
Species-dependence probability density function, p (xi | c) and p (xi | d) it is modeled to be defined by average u and deviation s2
Log-normal distribution function, wherein, xi be with the relevant adjustment MFI values of biomarker i to numerical value.Record and use in table 3
In the variable of the pdf of above-mentioned 7 biomarkers, and an example of the non-processing data of the model with being suitable for regular pdf is together
Shown in Fig. 5 a to 5c.Naive Bayes Classification for this model is represented with following mathematical expression, wherein, p (d) is set
The ill rate of disease.Likelihood P (d | x)/P (c | x) of disease can be expressed as below.
P (d | x)/P (c | x)=p (d)/(1-p (d) x Pi (p (x | d)/p (x | c))
Log-likelihood ratio (P (d | x)/P (c | x)) be used as being used for the single fraction for distinguishing morbid state.
As shown in table 4, the variable for 7- marker graders has been calculated.And as shown in Fig. 5 a to 5c and Fig. 7, warp
Trained grader shows that disease distinguishes performance.
Embodiment 5
In order to optimize the minimum number of the biomarker needed for group without losing diagnosis performance, greedy superseded calculation has been used
Method (greedy backward elimination algorithm) in brief, is classified although foring 7 6- markers
Device, but compared with above-mentioned 7- markers model, hydraulic performance decline (Fig. 6 a to 6g and table 6).In order to simplify for diagnosing
Group, selects 7 final markup models to provide the optimum performance of minimum protein.
Embodiment 6
The verification of 7- marker graders
In order to verify that the disease of grader distinguishes performance, the sample do not tested is utilized based on the multiple of aptamer
Detection method is determined.The measured value of 7 markers is suitable for 7- marker graders, and show with they
The similar Receiver operating curve of training set (ROC) (Fig. 5 a to 5c and Fig. 7, table 5).
Embodiment 7
Compared with the performance of existing blood testing, Cyfra21-1
In order to compare the performance of 7- marker graders, all samples are utilized into Cyfra 21-1 enzyme linked immunosorbent assay (ELISA)s
Kit is tested.Enzyme-test Cyfra 21-1 (DRG I nstruments GmbH, Germany) is based on common
Interlayer scheme is come the enzyme immunoassay technology that carries out.The concentration of Cyfra 21-1 is calculated by standard curve.Detection method it is sensitive
Spend for 0.15ng/mL.The Receiver operating curve (ROC) for producing Cyfra 21-1 comes and 7- marker grader foods
Receiver operating curve (ROC) compares (Fig. 7 a and 7b).
For all non-small cell lung cancer cases, the area under the curve (AUC) of above-mentioned grader is respectively 0.88, and from
Early stage of lung cancer patient (1 phase and 2 phases) observes 0.83, otherwise area under the curve of the C yfra 21-1 in all cases is
0.72, observe that area under the curve is 0.63 (Fig. 7 a and 7b) in early stage of lung cancer patient.
Table 1
[table 2]
[table 3]
[table 4]
[table 5]
[table 6]
Claims (28)
1. a kind of method being used for from human diagnostic's lung cancer, wherein,
Including：
There is provided and be at least 2 comprising N number of biomarker group in the multiple biomarker protein matter listed in table 2, above-mentioned N
Integer the step of；And
In order to assign N number of corresponding biomarker values of biomarker protein matter with above-mentioned biomarker group respectively,
The step of multiple above-mentioned biomarker protein matter are detected from from the biological specimen of the mankind,
Above-mentioned lung cancer is diagnosed based on multiple above-mentioned biomarker values.
2. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, detect multiple above-mentioned biomarkers
The step of the step of value, includes carrying out biological vivo detection.
3. according to claim 2 be used for from the method for human diagnostic's lung cancer, wherein, above-mentioned biology vivo detection includes pair
Should be above-mentioned to be used to also wrap from the method for human diagnostic's lung cancer in a capture agent of each above-mentioned biomarker protein matter
Include the step of at least one capture agent is selected in the group being made of aptamer, antibody and nucleic acid probe.
4. the method according to claim 3 being used for from human diagnostic's lung cancer, wherein, above-mentioned at least one capture agent is
Aptamer.
5. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, above-mentioned biological specimen is selected from by complete
In the group of blood, blood plasma and serum composition.
6. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, above-mentioned biological specimen is serum.
7. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, the above-mentioned mankind are Asian.
8. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, the above-mentioned mankind are smoker.
9. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, the above-mentioned mankind have malign lung knot
Section.
10. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, N is at least 3.
11. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, N is at least 4.
12. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, N is at least 5.
13. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, N is at least 6.
14. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, N is at least 7.
15. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, multiple above-mentioned biomarker values
With complement component C9, carbonic anhydrase 6, C reactive protein, ErbB1, matrix metalloproteinase 7, the anti-eggs of α 1-
The measured value of white enzyme and stem cell factor acceptor.
16. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, multiple above-mentioned biomarker values
It is measured by the method in the group that is made of real-time polymerase chain reaction, microarray and Lu Mingkesi microballoon detection methods.
17. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, by statistical method to above-mentioned lung
Cancer is diagnosed.
18. the method according to claim 1 being used for from human diagnostic's lung cancer, wherein, above-mentioned statistical method is selected from by line
Property discriminant analysis, logistic regression analysis, Naive Bayes Classification, support vector machines and random forest composition group in.
19. a kind of protein biomarkers group being used for from human diagnostic's non-small cell lung cancer, wherein,
It is above-mentioned to be used to contain stem cell factor from the protein biomarkers group of human diagnostic's non-small cell lung cancer
N number of biomarker protein matter in the multiple biomarker protein matter listed in the table 2 of acceptor,
N is at least 2.
20. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, N is at least 3.
21. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, N is at least 4.
22. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, N is at least 5.
23. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, N is at least 6.
24. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, N is at least 7.
25. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, it is above-mentioned to be used for that to there is complement component C9, carbonic anhydrase from the protein biomarkers group of human diagnostic's non-small cell lung cancer
6th, C reactive protein, ErbB1, matrix metalloproteinase 7, α 1- antiproteases and stem cell factor by
The measured value of body.
26. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, the above-mentioned mankind are Asian.
27. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, the above-mentioned mankind are smoker.
28. the protein biomarkers group according to claim 19 being used for from human diagnostic's non-small cell lung cancer, its
In, the above-mentioned mankind have malign lung nodules.
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| CN110333341A (en) * | 2019-07-04 | 2019-10-15 | 浙江理工大学 | A method of silk fabric cultural relics sample is identified based on protein biochip technology |
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