CN107942055A - The ELISA method of transcript profile and protein science in a kind of liver cancer biological process - Google Patents
The ELISA method of transcript profile and protein science in a kind of liver cancer biological process Download PDFInfo
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- CN107942055A CN107942055A CN201711169087.5A CN201711169087A CN107942055A CN 107942055 A CN107942055 A CN 107942055A CN 201711169087 A CN201711169087 A CN 201711169087A CN 107942055 A CN107942055 A CN 107942055A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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Abstract
The invention discloses the ELISA method of transcript profile and protein science in a kind of liver cancer biological process, 1. 2. buffer solution stays overnight antibody dilution in the reacting hole of each polystyrene board；3. next day, discard solution in hole, washing buffer 4. add certain diluted measuring samples by reacting hole in, when after wash 5. in each reacting hole, 6. the enzyme labelled antibody for adding diluted fresh is incubated, washs the TMB 8. addition terminate liquid expression verifications that 9. the result judgement present invention uses clinical sample to carry out positioning and quantitative to it in each reacting hole that Extemporaneous is 7. added in each reacting hole, find its evidence with clinical correlation, clinical value is evaluated, new clue is provided for onset of liver cancer and liver cancer Mechanism Study.The liver cancer key molecule of problem screening will be to explore to establish Research foundation with early detection, classification, the evaluation relevant liver cancer marker of prognosis, and more efficient, the accurate liver cancer treatment target position of selection.
Description
Technical field
The present invention relates to genetic transcription group and proteomics field, transcribed in especially a kind of liver cancer biological process
The ELISA method of group and protein science.
Background technology
In biology and medical research, it is important that a field be structure to biosystem and life process, work(
The observation that can and regulate and control.But between the past centuries, biologist focuses on individual gene or protein in biology department always
Expression change and function in system, and the change of life system cannot be studied from global, overall angle.With medicine
It is progressive, it has been found that many diseases, the generation of particularly cancer is often multifactor, polygenes, multipath synergistic effect cause
's.This just need one can comprehensively, dynamic, the technology and means of systematic research life system, then " group is learned " is general
Thought is come into being[3].But with the completion of the Human Genome Project, it has been found that in only can not be complete from the angle of genomics
The shearing that occurs in total correctness predicted gene transcription, splicing and in translation the starting of open reading frame codon,
Various modification situations after final position and translation.
In gene expression research, extensive genetic analysis can be related to a physiological status either cell phenotype
Gene progress system monitoring, can utilize high throughput analysis to export and obtain the quick both sides advantage of data in data, to disease
Function candidate gene during disease is identified.The maturation of microarray technology, makes researcher be sequenced by transcript profile and studies,
Find marker gene interested.As oncogene expression is to the tissue in various sources and the correlation point of patient's survival outcome
Analysis example is the same, and the gene expression analysis research carried out by microarray technology will continue to play the part of in biomarker discovery procedure
Important function.
Although the analysis ability of microarray is very powerful, transcription group research platform only includes the change of those Adaptable growth conditions
The transcript of cell.In most cells and intercellular Biochemical processes can all be subject to protein-protein or other
The influence of protein-substrate interaction.The gene expression analysis of protein group level provides a quickly controllable life
The process of thing synthesis, wherein most are regulated and controled by transcription group platform.Meanwhile transcript profile passes through the protein of expression in itself
Other changes either under cellular biochemical state, carry out feedback control.
In other words, gene expression is not exclusively from transcript profile to the one-way flow of protein group, but both is mutual
Connection.Understanding to this function controlling is generally limited to special signal pathway, or metabolic pathway.It is to be understood that turn
Effect of Mutual Regulation between record group and protein group is, it is necessary to which the expression to RNA and protein carries out Integral synchronous monitoring.
The progress of transcription group, proteomics and bioinformatics investigative technique opens for research complex biological system
Brand-new approach, the reorganizing research that three is connected together can reveal that the hereditary information carried when disease occurs from gene turns
It is changed into distinguish the exception during the entire process of phenotype, its magnanimity information gathered is covered in disease incidence and disease mechanisms
Key function node, can be used to identify tumor-related gene and its protein of expression so that thousands of genes and egg
The analysis of white matter is possibly realized, for explore early detection, classification, evaluate prognosis tumor markers, and selection it is more efficient,
Accurate oncotherapy target position provides reliable guarantee.
Ion proton sequenators of new generation use the technology of semiconductor chips, and sequencing speed is fast, and has high extension
Property, by proprietary large-scale parallel semiconductor inductor, ion stream caused by DNA replication dna is realized and directly and is in real time examined
Survey.When reagent is entered in chip by integrated fluid passage, the reacting hole being clouded on chip immediately becomes up to a million a micro-
Reaction system.The technical combinations of this unique fluid system, the Machine Design of microbody system and semiconductor, enable researcher to exist
The 2 interior pinpoint accuracy sequences obtained more than from 10Mb to 1Gb when small.In addition, Ion Proton sequenators and Ion Reporter
Analysis software can complete the analysis of individual gene group in an independent server, break current data parsing bottleneck, greatly
Research cost is reduced greatly, improves the speed and accuracy of detection, in scientific research and clinically there is good application；To current
Untill, in the confluence analysis article delivered, most of LC-MS analyses are used in combination with stable isotope labeling, especially
It is iTRAQ reagents.Even with technology it is different, the confluence analysis published so far all indicate transcription group and
The importance of protein science.Transcription group or protein science usually only consider regulating system and the net effect of decomposition equilibrium state
Should, in fact, the inconsistency occurred is a kind of reflection synthesized with two kinds of replacement process of degraded, researcher was to changing
Mechanism in journey is interested；In addition, transcription group and proteomic assays to successful integration, it is necessary to efficiently and accurately phase
Mutually reference.Researcher needs flexibly to define the genome of oneself, it is also possible to needing to select to be directed to using predefined
The target figure of protein, when new genome, transcript profile and protein groups sequence occur, researcher needs timely register update,
And the information of deletion error.The development of bioinformatics technique is so that genetic transcription, expression during oncobiology are whole
Exception during a is disclosed, and clue is provided for tumour Mechanism Study.
This research is intended, using the sequencing of Ion Proton transcript profiles and LC-MALDI Discrepancy proteome analysis platforms, carrying out liver cancer
Transcript profile and proteomic assays in biological process.By building Rats With Hepatoma model, in relatively more normal and liver cancer tissue
Genetic transcription and protein expression difference, all occur to transcript profile in liver cancer and protein groups abnormal molecule carry out gene optimization,
Alternative splicing analysis, new gene or the screening of new transcript, expression analysis, Differential expression analysis, differential expression cluster analysis and
The processing of the bioinformatic analysis such as functional annotation, screens liver cancer key function node and tumor cells, and carry out clinic to it and test
Card and clinical value assessment.This research will provide new clue for onset of liver cancer and liver cancer Mechanism Study.
Transcript profile and the ELISA of protein science are constituting portion indispensable in the research in liver cancer biological process
Point, the technical solution of the ELISA method of transcript profile and protein science, domestic through retrieval in a kind of liver cancer biological process of the present invention
The same industry has no identical.
The content of the invention
The object of the present invention is to provide the ELISA method of transcript profile and protein science in a kind of liver cancer biological process.
The ELISA method of transcript profile and protein science in this liver cancer biological process,
Comprise the steps of：
It is 1~10 μ g/ml that 1. antibody is diluted to protein content with coating buffer solution；
2. adding 0.1ml in the reacting hole of each polystyrene board, 4 DEG C overnight；
3. next day, discards solution in hole, washed 3 times, every time 3 minutes with lavation buffer solution；
4. plus certain diluted measuring samples 0.1ml is in the above-mentioned reacting hole being coated with, put 37 DEG C be incubated 1 it is small when after wash
（Pay attention to leave some space hole, negative control hole and Positive control wells）；
5. in each reacting hole, enzyme labelled antibody (dilution factor after titration) 0.1ml of diluted fresh is added；
6. 37 DEG C of incubations 0.5~1 are small, washing；
7. the tmb substrate solution 0.1ml of Extemporaneous is added in each reacting hole, 37 DEG C 10~30 minutes；
8. terminate liquid 0.05ml is added in each reacting hole；
9. result judgement：On ELISA detectors, at 450nm, each hole OD values are surveyed after returning to zero with blank control wells, if more than
2.1 times of defined negative control OD value, are the positive.
Invention beneficial effect：
The present invention carries out it using clinical sample the expression verification of positioning and quantitative, finds its evidence with clinical correlation, comments
Valency clinical value, new clue is provided for onset of liver cancer and liver cancer Mechanism Study.Problem screening liver cancer key molecule will be
Explore and early detection, classification, the evaluation relevant liver cancer marker of prognosis, and more efficient, the accurate liver cancer treatment of selection
Target position establishes Research foundation.
Embodiment
Embodiment：
The ELISA method of transcript profile and protein science in this liver cancer biological process,
Comprise the steps of：
It is 1~10 μ g/ml that 1. antibody is diluted to protein content with coating buffer solution；
2. adding 0.1ml in the reacting hole of each polystyrene board, 4 DEG C overnight；
3. next day, discards solution in hole, washed 3 times, every time 3 minutes with lavation buffer solution；
4. plus certain diluted measuring samples 0.1ml is in the above-mentioned reacting hole being coated with, put 37 DEG C be incubated 1 it is small when after wash
（Pay attention to leave some space hole, negative control hole and Positive control wells）；
5. in each reacting hole, enzyme labelled antibody (dilution factor after titration) 0.1ml of diluted fresh is added；
6. 37 DEG C of incubations 0.5~1 are small, washing；
7. the tmb substrate solution 0.1ml of Extemporaneous is added in each reacting hole, 37 DEG C 10~30 minutes；
8. terminate liquid 0.05ml is added in each reacting hole；
9. result judgement：On ELISA detectors, at 450nm, each hole OD values are surveyed after returning to zero with blank control wells, if more than
2.1 times of defined negative control OD value, are the positive.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (1)
- A kind of 1. ELISA method of transcript profile and protein science in liver cancer biological process, it is characterised in that：By following steps group Into：It is 1~10 μ g/ml that 1. antibody is diluted to protein content with coating buffer solution；2. adding 0.1ml in the reacting hole of each polystyrene board, 4 DEG C overnight；3. next day, discards solution in hole, washed 3 times, every time 3 minutes with lavation buffer solution；4. plus certain diluted measuring samples 0.1ml is in the above-mentioned reacting hole being coated with, put 37 DEG C be incubated 1 it is small when after wash （Pay attention to leave some space hole, negative control hole and Positive control wells）；5. in each reacting hole, enzyme labelled antibody (dilution factor after titration) 0.1ml of diluted fresh is added；6. 37 DEG C of incubations 0.5~1 are small, washing；7. the tmb substrate solution 0.1ml of Extemporaneous is added in each reacting hole, 37 DEG C 10~30 minutes；8. terminate liquid 0.05ml is added in each reacting hole；9. result judgement：On ELISA detectors, at 450nm, each hole OD values are surveyed after returning to zero with blank control wells, if more than 2.1 times of defined negative control OD value, are the positive.
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|---|---|---|---|---|
| WO2008021163A2 (en) * | 2006-08-09 | 2008-02-21 | Saint Louis University | Kits and methods for determining risk for primary liver cancer |
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Application publication date: 20180420 |