CN102466729A - Method for screening tumor specificity target and targeting ligand based on tissue chip - Google Patents
Method for screening tumor specificity target and targeting ligand based on tissue chip Download PDFInfo
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Abstract
The invention which belongs to the biotechnological field concretely relates to a method for screening a tumor specificity target and a targeting ligand with a tissue chip. The invention which aims to overcome disadvantages of low medicine or targeting treatment versatility, large gap between a screening system and in-vivo living environment of a tumor, small screening throughout, and time and labor consumption existing in the prior art provides a method for screening the tumor specificity target and the targeting ligand with the tissue chip. The targeting ligand screened in the invention has the advantages of high specificity, realization of tumor cell penetrating, no immunization reaction causing, and high versatility. The screening method maintains the original state of the in-vivo grown tumor; and compared with tumor-bearing mouse screening systems, the screening method has the advantages of animal breeding step omitting, time, labor saving and cost saving, and strong experiment result comparability. Compared with traditional screening systems, the screening method of the invention allows the tumor specificity target analysis and the targeting research to be simultaneously carried out, and the verification process is simple.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of based on the tumour-specific target spot of organization chip and the screening technique of target part.
Background technology
Tumour has become one of difficult and complicated illness of serious threat human health, and surmounting angiocardiopathy becomes human first cause of death.The traditional treatment means of tumour mainly are operation, radiation and chemotherapy.These treatment meanss are except excision and destruction tumor tissues, because its no target property also produces than havoc normal tissue, cell.
Targeted therapy is the impressive progress in modern oncotherapy field; It is on the basis of tumour molecular cytobiology; With specificity structure molecule that tumor tissues or cell had as target spot; Use some can with the antibody of these target molecule specific bond, part etc., reach direct treatment or targeted therapy purpose.
Targeted therapy relates to two aspects, i.e. target spot and targeting vector.
The target spot of targeted therapy is normally expressed behind the cell carcinogenesis to be different from Normocellular biomolecule, mainly is each albuminoid.Tumour cell had both contained the expressed albumen of normal cell; The albumen that also can contain some normal cell does not simultaneously have perhaps lacks some normal cell expressed proteins, and the variation of these tumor cell specific expressed proteins just provides the foundation for the targeted therapy of tumour.
The carrier of targeted therapy be have necessarily specific; Can bioactivator optionally be transported to tumor locus; Be limited to the effect of medicine in specific target cell, tissue or the organ as far as possible, and do not influence one type of carrier of normal cell, tissue or organ dysfunction.
The micromolecule part is the ideal carrier of targeted therapy.The micromolecule part has been compared very big advantage with the antibody carrier of present use: can quick penetration tissue and cell; The blood clearance rate is high; External synthetic simple, Quality Control is easy.
Micromolecule ligand screening system commonly used has: the screening of (1) solid phase or liquid phase purifying antigen; (2) building is cell screening; (3) tumor bearing nude mice screening; (4) histotomy screening.Wherein the screening system of solid phase or liquid phase purifying antigen can only be used to screen the part that combines with known antigens, has limitation.Building is that the part that the cell screening screening system arrives can only be directed against a certain specific cell subsets, can't overcome the not high shortcoming of part versatility that screens that the heterogeneity of tumour is brought.Though the maintenance that the tumor bearing nude mice screening system is maximum the virgin state of target protein, the part versatility that screens is not high.And the histotomy screening system is because also there is the not high defective of part versatility that is screened in the heterogeneity in the same tumour with different interpatient differences.
Therefore, be badly in need of a kind of method that can screen high specific, high flux, can also can confirm tumor markers simultaneously in the existing triage techniques to the part of a certain tumor types.
Summary of the invention
The objective of the invention is to overcome the prior art Chinese traditional medicine or the targeted therapy versatility is not high, screening system and tumour are big in body living environment gap, the screening flux is little, the shortcoming that wastes time and energy, provide a kind of based on the tumour-specific target spot of organization chip and the screening technique of target part.
Of the present inventionly comprise the steps: based on the tumour-specific target spot of organization chip and the screening technique of target part
(a). after the normal tissues chip being carried out the pre-service of antigen retrieval, carry out endogenous enzyme sealing and handle, add the part storehouse then, the antigen of expressing on part and the normal tissues is combined;
(b). washing normal tissues chip, thus obtain a large amount of ligand mixtures that do not combine with normal tissues;
(c). the ligand mixture that obtains in the step (b) is joined through antigen retrieval and having sealed on the tumor tissues chip of endogenous enzyme, the specific antigen of expressing on part and the tumor tissues is combined;
(d). after removing in the step (c) not the part with the tumor tissues chips incorporate, thereby obtain the phage mixtures that combine with tumor tissues in a large number with eluent washing tumor tissues chip;
(e). the phage mixture that obtains in the step (d) is increased, purifying then, repeating step (a)-(d) is more than 3 times again; And improve eluotropic strength by wheel; To increase biological elutriation dynamics, obtain not combining, but specificity combines the ligand mixture of tumor tissues with normal tissues;
(f). combine the ligand mixture of tumor tissues to carry out sequential analysis the specificity that obtains in the step (e), select the high ligands specific of consensus sequence;
(g). verify on the used tumor tissues chip when the high ligands specific of consensus sequence that screens in the step (f) is turned back to elutriation, then resulting part is carried out specificity and flux distribution;
(h). utilize ligands specific to identify the antigen that it combines.
Pre-service in the said step (a) is that conventional chip is handled, and promptly paraffin organization chip carries out being immersed in respectively in absolute ethyl alcohol-90% ethanol-80% ethanol-distilled water and carrying out aquation step by step after xylene dewaxing/frozen tissue chip ices acetone fixed.
The endogenous enzyme purpose of sealing, eliminate in the said step (a) is to reduce non-specific binding.Wherein confining liquid can adopt conventional confining liquids such as serum, BSA.
Antigen retrieval in the said step (a) comprises any method of present use.Like negative pressure of vacuum antigen retrieval method, microwave radiation antigen retrieval method, high pressure antigen retrieval method, the hot antigen retrieval method of water proof, electric furnace coctoantigen repairing method, pepsin digestion method and trypsinization method.But be not limited to above-mentioned antigen retrieval method.
Part storehouse in the said step (a) comprises any part storehouse of present use, with hangar, PNA etc., but is not limited to the above part storehouse like phage random peptide library, phage antibody library, mRNA displayed polypeptide storehouse, ribosomal display peptide storehouse, RNA.
The donor kind source of the tumor tissues chip in normal tissues chip in the said step (b) and the step (c) comprises people, monkey, rat, mouse, pig, rabbit and cavy; Different tissue-derived lung, liver, stomach, esophagus, tongue, duodenum, small intestine, colon, rectum, thyroid gland, parathyroid gland, kidney, adrenal gland, pancreas, prostate, brain, cerebellum, skeletal muscle, cardiac muscle, smooth muscle, blood vessel, hypophysis, testis, ovary, epidermis, retina, bone, cartilage, eye, placenta, spleen, the parotid gland, lower jaw gland, the thymus gland etc. of comprising; The cell source comprises Hep-2 cell, granulocyte, blood platelet etc., but is not limited to above-mentioned said cell or tissue source.The matrix of above-mentioned organization chip comprises paraffin organization, frozen tissue and/or cultured cell.
Verification method in the said step (f) comprises immunohistochemistry, immunofluorescence, in situ hybridization etc., but is not limited to the above detection method.
The method of identifying antigen described in the said step (h) includes but not limited to methods such as immunoprecipitation, dielectrophoresis, affinity chromatography.
Can use any suitable eluent in said (a)-(h) step.Eluent can contain the surfactant of 0%-2% (w/v), and the sustainable reasonable time of wash-out was as 5-30 minute.
Part storehouse in said (a)-(h) step combines and can under any suitable temperature, carry out with organization chip, as 4 ℃-70 ℃.Binding time can be controlled in 2h to spending the night.
Compared with prior art, advantage of the present invention is:
1. the target part that screens has higher specificity, can pass through tumour cell, can not cause immune response; Versatility is high simultaneously, can be to the tumour of a certain type of most patients.
2. because tissue array technology is ripe, the present invention has had very widely and has quoted, and is that the screening system of cell is compared with pure antigen with building, and this screening system has kept tumour virgin state when bulk-growth; Compare with the tumor-bearing mice screening system, owing to need not carry out the raising of animal, the present invention saves time, and is laborsaving, practice thrift cost, and the experimental result comparability is strong.
3. compare with traditional screening system, the present invention can carry out the research of analysis of tumour-specific target spot and target part simultaneously.
4. verification method of the present invention is simple, can analyze specificity and the location of part in tumor tissues that screens as long as carry out SABC, immunofluorescence or in situ hybridization, and is simple to operate.
Description of drawings
Fig. 1 is that the phage clone that filters out returns the figure as a result that the squamous cell lung carcinoma organization chip carries out specificity combination checking
Fig. 2 is that the phage clone that filters out returns the figure as a result (wherein not seeing tangible pale brown look dyeing) that lung normal tissues chip carries out specificity combination checking
A: dye the part that combines the squamous cell lung carcinoma tissue for the specificity of pale brown look
Embodiment
I. material
Ph.D.-7
TMPhage Display Peptide Library Kit is available from New England Biolabs (Beijing) Co.Ltd; The CS04-03-002 that uses in squamous cell lung carcinoma (middle differentiation) organization chip CS04-03-001, lung normal tissues (a plurality of position) organization chip NC04-01-001 and the demonstration test is all available from Shaanxi Chaoying Biotechnology Co., Ltd..IPTG is the Merck packing, and X-gal is the BDX packing, and PEG-8000 is the Amersco packing.
The mensuration of phage random sequence is given birth to worker technology company by Shanghai and is accomplished; The HRP-Anti-M13 monoclonal antibody that uses in the SABC is available from GE, and sealing serum, DAB chromogenic reagent box, haematoxylin are redyed liquid all available from company of middle China fir Golden Bridge.
Conventional reagent of some of other uses and material can not done detailed description at this with reference to cited in " molecular cloning: lab guide ".
II. specific embodiment
1. the amplification of phage peptide library, purifying and titer determination thereof
A. the amplification of phage peptide library and purifying
(1) Ph.D.-7
TMER2738 bacterium among the Phage Display Peptide Library Kit inoculates line on the LB-Tet flat board, be inverted flat board, 37 ℃ of overnight incubation.
(2) picking ER2738 monoclonal bacterium is inoculated in the 20ml LB nutrient culture media, and 37 ℃ of shaken cultivation are to logarithmic phase.
(3) from Ph.D.-7
TMGet the 2ul bacteriophage among the Phage Display Peptide Library Kit, join and continue to cultivate 4.5h in the culture of (2).
(4) culture is under 4 ℃ of conditions, the centrifugal 10min of 10000rpm, and supernatant is proceeded same centrifugally operated.
(5) 80% of supernatant is shifted, add the PEG/NaCl of 1/6 volume therein, let 4 ℃ of depositions of bacteriophage spend the night.
(6) overnight culture is under 4 ℃ of conditions, and the centrifugal 15min of 10000rpm discards supernatant, repeats this operation then once.
(7) sediment is resuspended among the 1ml TBS, and under 4 ℃ of conditions, the centrifugal 5min of 10000rpm makes the residual cells deposition.
(8) supernatant precipitates with the PEG/NaCl of 1/6 volume again, and hatches 30min on ice.
(9) under 4 ℃ of conditions, the centrifugal 10min of 10000rpm abandons supernatant, repeats this operation once.
(10) sediment is resuspended in 200ul TBS, 0.02%NaN
3In, centrifugal 1min precipitates the insolubles of any remnants, and supernatant changes in the fresh tube, the eluate after this promptly increases.
B. phage peptide library titer determination
(1) the single bacterium colony of inoculation ER2738 is in 5-10ml LB nutrient culture media, and wave and culture is to mid-log phase (0D
600～0.5).
When (2) cell was grown, micro-wave oven melted top-layer agar, was divided into the 3ml equal portions in the sterilization test tube, was stored in 45 ℃ of water-baths subsequent use.
(3) 37 ℃ of preparatory temperature LB/IPTG/Xgal are dull and stereotyped, and it is subsequent use that each bacteriophage dilutability is got a flat board.
(4) will increase, the bacteriophage behind the purifying carries out gradient dilution.
(5) reach mid-log phase when the yeast culture thing, be divided into every part 200 μ l equal portions in microcentrifugal tube, each bacteriophage dilutability one pipe.
(6) every pipe adds the different dilution bacteriophages of 10 μ l, shakes mixing fast, room temperature incubation 5min.
(7) infection cell is added in the top-layer agar culture tubes of 45 ℃ of preparatory temperature, each pipe, mixing is poured on 37 ℃ of warm in advance LB/IPTG/Xgal flat boards immediately fast.Suitably tilt flat plate evenly spreads out top-layer agar.
(8) treat dull and stereotyped cooling 5min after, be inverted overnight incubation under 37 ℃ of conditions.
(9) inspection is dull and stereotyped, calculates and has an appointment 10
2Spot number on the flat board of individual plaque.Multiply by the plaque forming unit (pfu) that dilution gfactor promptly obtains per 10 μ l bacteriophages, i.e. titre with this number then.
2. bacteriophage elutriation
A. organization chip pre-service
Organization chip is heated in constant temperature oven, after carry out xylene dewaxing, carry out aquation step by step with absolute ethyl alcohol, 95% ethanol, 85% ethanol, 70% ethanol and deionized water respectively; The organization chip of handling well is carried out the elimination of endogenousization enzyme, promptly use 3% H
2O
2Soak 10min, to reduce unspecific staining.Organization chip carries out can carrying out follow-up elutriation process after the microwave thermal reparation with citrate buffer solution.
It should be understood that in this specific embodiment and just do not limit usable range of the present invention that hot, high pressure reparation, enzyme repairing method can be selected according to concrete experiment needs with the microwave thermal reparation.
B. elutriation
(1) on organization chip, delimit conversion zone with the SABC pen, seal up blocking solution, 4 ℃ are spent the night.
(2) get rid of the liquid of blockading on the organization chip, wash 6 times with the 0.05%TBST damping fluid.All rotation is got rid of the TBST damping fluid so that each position all washes at every turn.
(3) with bacteriophage to the suitable titre after the 0.05%TBST damping fluid dilution amplification, get 150ul then and be added on the organization chip NC04-01-001, the room temperature gentleness is shaken 60min.
(4) major part of the phage solution on the organization chip NC04-01-001 is transferred on the organization chip CS04-03-001, the room temperature gentleness is shaken 60min.Residue a small amount of (about 1 μ l) is used to measure titre.
(5) phage solution that stays on the organization chip CS04-03-001 is done titer determination usefulness.Wash organization chip CS04-03-001 10 times with the 0.05%TBST damping fluid then; Get the tenth time washing lotion and do the titre detection; Be used for tentatively judging that 0.05%TBST washes the plate effect, promptly each takes turns the specificity of elutriation, and this operation is equally applicable to organization chip NC04-01-001.
(6) with the non-specific damping fluid 0.2M of 150ul Glycine-HCl (pH2.2), 1mg/ml BSA separates the bacteriophage that has combined, and gentleness is shaken 10min, and eluent sucks in another clean microcentrifugal tube.And then with 22.5 μ l 1M Tris-HCl (pH9.1) the above-mentioned eluent that neutralizes.The method is applicable to same organization chip NC04-01-001 and the organization chip CS04-03-001 that takes turns in the elutriation.
(7) measure the titre of (about 1 μ l) eluate on a small quantity by above-mentioned conventional phage titre assay method.
(8) eluate be will remain and (thalline should be in logarithm in earlier stage) in the 20ml ER2738 culture, 37 ℃ of violent wave and culture 4.5h joined.
(9) culture is changed in the centrifuge tube, then 4 ℃ of centrifugal 10min of 10000rpm.Supernatant changes in another centrifuge tube, and is centrifugal again.
(10) top 80% with supernatant changes in the fresh tube, adds the PEG/NaCl of 1/6 volume.Let 4 ℃ of depositions of bacteriophage spend the night.
(11) the centrifugal PEG deposition of 10000rpm 15min under 4 ℃ of conditions.Outwell supernatant, of short duration more centrifugal, inhale and remove residual supernatant.
(12) sediment is resuspended among the 1ml TBS, and suspension changes in the microcentrifugal tube, and centrifugal 5min makes the residual cells deposition under 4 ℃ of conditions.
(13) supernatant changes another fresh microcentrifugal tube over to, precipitates with the PEG/NaCl of 1/6 volume again.Hatch 30min on ice.Centrifugal 10min under 4 ℃ of conditions abandons supernatant, and is of short duration more centrifugal, inhales with micropipettor and removes remaining supernatant.
(14) sediment is resuspended in 200 μ l TBS, 0.02%NaN
3In.Centrifugal 1min precipitates the insolubles of any remnants.Supernatant changes in the fresh tube.This is the eluate after the amplification.
(15) according to the eluate titre of above-mentioned conventional method after with the dull and stereotyped titration amplification of LB/IPTG/Xgal.The residue eluate is stored under 4 ℃ of conditions.
(16) confirm titre according to locus coeruleus number on the tally.Be worth the bacteriophage addition of calculating corresponding to initial with this.
(17) carry out second and take turns elutriation: be equivalent to phagocytosis scale of construction repeating step (1)-(16) of initial addition of the first round in the eluate with first round elutriation amplification, the concentration with Tween in cleaning step increases to 0.1% (w/v).
(18) titre after mensuration second on the LB/IPTG/Xgal flat board is taken turns the amplification of elutriation gained eluate.
(19) carry out the third round elutriation: take turns phagocytosis scale of construction repeating step (1)-(16) that are equivalent to initial addition of the first round in the eluate of elutriation amplification with second, in the cleaning step with the Tween of 0.5% (w/v).
Titre when (20) mensuration third round elutriation gained eluate does not increase on the LB/IPTG/Xgal flat board.The third round eluate needn't increase again, and the plaque that obtains during titer determination can be done order-checking usefulness, does not surpass 18 hours as long as note dull and stereotyped incubation time, the long easy appearance disappearance of incubation time.All the other eluates are stored under 4 ℃ of conditions.
Whole elutriation process adopts the deduction elutriator; Promptly earlier combine phage solution with normal tissues; Unconjugated solution goes to combine with lung squamous cell carcinoma cancers again; Just can obtain the specific phage polypeptide of lung squamous cancer, and adopt the method that progressively improves Tween-20 concentration among the TBST to remove the bacteriophage of non-specific binding.For whether gained bacteriophage after tentatively judging the three-wheel elutriation has specificity to combine to lung squamous cancer, we are provided with the different detection point, and testing result is seen table 1.
Table 1
| ? | The first round | Second takes turns | Third round |
| Input quantity | 3.35×10 20 | 3.63×10 10 | 4.05×10 11 |
| A | 5.19×10 14 | 4.73×10 6 | 3.16×10 4 |
| B | 1.94×10 14 | 4.60×10 6 | 3.06×10 4 |
| C | 1.07×10 3 | 1.67×10 2 | 0 |
| D | 2.97×10 3 | 2.13×10 2 | 87 |
| E | 14 | 25 | 0 |
| F | 15 | 18 | 0 |
As shown above: A is the bacteriophage that does not combine with organization chip NC04-01-001; B is the bacteriophage that does not combine with organization chip CS04-03-001; C is the bacteriophage that combines with organization chip NC04-01-001; D is the bacteriophage that combines with organization chip CS04-03-001; E is the tenth TBST eluate of organization chip NC04-01-001; F is the tenth TBST eluate of organization chip CS04-03-001;
It should be understood that elutriation number of times and elutriation pressure in this elutriation experiment do not limit usable range of the present invention, can select according to concrete requirement of experiment.
3. the purifying of plaque and amplification
(1) the ER2738 overnight culture was inoculated in the LB fluid nutrient medium of 1ml by dilution in 1: 100.
(2) choose a blue plaque in above-mentioned 1ml culture tube with the sterilization toothpick.Plaque in this step will be selected less than the flat board of 100 plaques from total amount, only contains a dna sequence dna so that guarantee each plaque of being chosen.
(3) shaking table is cultivated 4.5h under 37 ℃ of conditions.Culture changes in the microcentrifugal tube, centrifugal 30s.Supernatant changes in the fresh centrifuge tube, and is centrifugal again.Change 80% supernatant over to fresh centrifuge tube with pipettor, this is amplification bacteriophage storage liquid, under 4 ℃ of conditions, stores.
4. the fast purifying of sequencing template
(1) carry out plaque amplification as stated above, the supernatant 500 μ l that get after centrifugal change a fresh centrifuge tube over to.
(2) add 200 μ l PEG/NaCl, put upside down mixing, room temperature is placed 10min.
(3) centrifugal 10min abandons supernatant.Of short duration then centrifugal, inhale and remove remaining supernatant.
(4) sediment thoroughly is resuspended in the 100 μ l iodide damping fluids, adds 250 μ l ethanol, room temperature incubation 10min.The room temperature incubation of short time makes single stranded phage DNA deposition and most of bacteriophage albumen remains in the solution.
(5) centrifugal 10min abandons supernatant.Ethanol with 70% is washed deposition, of short duration vacuum drying.
(6) deposition is resuspended among the 30 μ l TE.
5. bacteriophage order-checking
(1) mensuration of phage random sequence is given birth to the completion of worker technology company by Shanghai.
(2) select the high bacteriophage of consensus sequence (sequence table is the amino acid sequence of the high bacteriophage of the consensus sequence selected) after comparing.
6. the monoclonal target adhesion of bacteriophage is identified
(1) chip is organized in pre-service, the organization chip pre-service among the method embodiment 2.
(2) pretreated organization chip carries out serum sealing, 37 ℃, 30min.
(3) 3%H
2O
2Eliminate endogenous peroxydase, 10min, PBS washes.
The bacteriophage monoclonal of (4) dilutions in 1: 100 is got and is added in right amount on the organization chip CS04-03-002, and 4 ℃ are spent the night.
(5) throw away unconjugated bacteriophage, PBS washes 10min, washes 3 times.
The HRP-Anti-M13 antibody of (6) dilutions in 1: 500,100 μ l are hatched 1h for 37 ℃, and PBS washes 10min or 5min, washes 3 times.
(7) DAB colour developing 8～12min, mirror is observed down, control dyeing.
(8) fully washing, haematoxylin is redyed, the acidic alcohol differentiation.
(9) conventional gummy mounting.
Material that uses in the specific embodiment of the invention and method all are as further illustration and explanation instrument of the present invention and means, and are not limitation of the present invention.And conventional operations such as microbe growth are that the state of the art personnel know.Only if definition separately, same meanings that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition, in addition, any and the method and the material of the similar content of putting down in writing or equalization all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Claims (11)
1. the method based on organization chip screening tumour-specific target spot and target part comprises organization chip pre-service, bacteriophage amplification and purifying, bacteriophage sequential analysis, antigen evaluation, it is characterized in that:
(a). after the normal tissues chip being carried out the pre-service of antigen retrieval, carry out endogenous enzyme sealing and handle, add the part storehouse then, the antigen of expressing on part and the normal tissues is combined;
(b). washing normal tissues chip, thus obtain a large amount of ligand mixtures that do not combine with normal tissues;
(c). the ligand mixture that obtains in the step (b) is joined through antigen retrieval and having sealed on the tumor tissues chip of endogenous enzyme, the specific antigen of expressing on part and the tumor tissues is combined;
(d). after removing in the step (c) not the part with the tumor tissues chips incorporate, thereby obtain the phage mixtures that combine with tumor tissues in a large number with eluent washing tumor tissues chip;
(e). the phage mixture that obtains in the step (d) is increased, purifying then, repeating step (a)-(d) more than 3 times again, and improve eluotropic strength by wheel, obtain not combining with normal tissues, but the ligand mixture of specificity combination tumor tissues;
(f). combine the ligand mixture of tumor tissues to carry out sequential analysis the specificity that obtains in the step (e), select the high ligands specific of consensus sequence;
(g). verify on the used tumor tissues chip when the high ligands specific of consensus sequence that screens in the step (f) is turned back to elutriation, then resulting part is carried out specificity and flux distribution;
(h). utilize ligands specific to identify the antigen that it combines.
2. the method based on organization chip screening tumour-specific target spot and target part according to claim 1; It is characterized in that: the pre-service in the said step (a) is that conventional chip is handled; Be that paraffin organization chip carries out after xylene dewaxing/frozen tissue chip ices acetone fixed, aquation step by step.
3. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: the confining liquid in the said step (a) comprises serum, BSA.
4. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: the antigen retrieval method in the said step (a) comprises negative pressure of vacuum antigen retrieval method, microwave radiation antigen retrieval method, high pressure antigen retrieval method, the hot antigen retrieval method of water proof, electric furnace coctoantigen repairing method, pepsin digestion method and trypsinization method.
5. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: the part storehouse in the said step (a) comprises that phage random peptide library, phage antibody library, mRNA displayed polypeptide storehouse, ribosomal display peptide storehouse, RNA are with hangar, PNA.
6. the method based on organization chip screening tumour-specific target spot and target part according to claim 1; It is characterized in that: the kind source of the donor of the tumor tissues chip in normal tissues chip in the said step (b) and the step (c) comprises people, monkey, rat, mouse, pig, rabbit and cavy; Different tissue-derived lung, liver, stomach, esophagus, tongue, duodenum, small intestine, colon, rectum, thyroid gland, parathyroid gland, kidney, adrenal gland, pancreas, prostate, brain, cerebellum, skeletal muscle, cardiac muscle, smooth muscle, blood vessel, hypophysis, testis, ovary, epidermis, retina, bone, cartilage, eye, placenta, spleen, the parotid gland, lower jaw gland, the thymus gland of comprising, the cell source comprises Hep-2 cell, granulocyte, blood platelet.
7. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: the matrix of said organization chip comprises paraffin organization, frozen tissue and/or cultured cell.
8. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: the verification method in the said step (f) comprises immunohistochemistry, immunofluorescence, in situ hybridization.
9. the method based on organization chip screening tumour-specific target spot and target part according to claim 1 is characterized in that: the method for identifying antigen in the said step (h) comprises immunoprecipitation, dielectrophoresis, affinity chromatography.
10. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: said eluent contains the surfactant of 0%-2% (w/v), and elution time continues 5-30 minute.
11. the method based on organization chip screening tumour-specific target spot and target part according to claim 1, it is characterized in that: said part storehouse is 4 ℃-70 ℃ with the temperature that combines of organization chip, and binding time is that 2h is to spending the night.
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| CN105601716A (en) * | 2014-03-24 | 2016-05-25 | 朱育盼 | Short peptide PCP11 specifically bound with prostatic cancer and application thereof |
| CN105938064A (en) * | 2014-09-26 | 2016-09-14 | 南通大学 | Application of reagent dose reducing histochemical pen in immunohistochemical detection of cell growing slides |
| CN106872243A (en) * | 2015-12-10 | 2017-06-20 | 深圳大学 | RNA- protein-DNA original positions multiple staining the method for organization chip |
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| CN1556413A (en) * | 2004-01-08 | 2004-12-22 | 中南大学湘雅医学院肿瘤研究所 | Tissue chip used for tumour early stage diagnosis and preparation device |
| CN101024856A (en) * | 2007-01-30 | 2007-08-29 | 华东理工大学 | Method for preparing small-molecular micro-array |
| CN101059515A (en) * | 2007-05-14 | 2007-10-24 | 西安交通大学 | Use of phage chip as protein chip or gene chip |
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2010
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| CN1556413A (en) * | 2004-01-08 | 2004-12-22 | 中南大学湘雅医学院肿瘤研究所 | Tissue chip used for tumour early stage diagnosis and preparation device |
| CN101024856A (en) * | 2007-01-30 | 2007-08-29 | 华东理工大学 | Method for preparing small-molecular micro-array |
| CN101059515A (en) * | 2007-05-14 | 2007-10-24 | 西安交通大学 | Use of phage chip as protein chip or gene chip |
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| TIANTOM JARUTAT ET AL.: "Selection of vimentin-specific antibodies for the HuCAL phage display library by substractive panning formalin-fixed, paraffin-embedded tissue", 《BIOL. CHEM.》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837592A (en) * | 2014-03-18 | 2014-06-04 | 中国计量科学研究院 | Method for in-situ measuring iron content in biological tissue sample by adopting isotopic dilution LA-ICP-MS (laser ablation inductively coupled plasma mass spectrometry) |
| CN105601716A (en) * | 2014-03-24 | 2016-05-25 | 朱育盼 | Short peptide PCP11 specifically bound with prostatic cancer and application thereof |
| CN105938064A (en) * | 2014-09-26 | 2016-09-14 | 南通大学 | Application of reagent dose reducing histochemical pen in immunohistochemical detection of cell growing slides |
| CN105938065A (en) * | 2014-09-26 | 2016-09-14 | 南通大学 | Application of Pap pen with fast operation speed in cell growing slide immunohistochemical detection |
| CN105938065B (en) * | 2014-09-26 | 2019-03-12 | 南通大学 | The fast group pen of service speed is applied in the detection of cell climbing sheet immunohistochemistry |
| CN105938064B (en) * | 2014-09-26 | 2019-03-19 | 南通大学 | The group pen for reducing reagent dosage is applied in the detection of cell climbing sheet immunohistochemistry |
| CN106872243A (en) * | 2015-12-10 | 2017-06-20 | 深圳大学 | RNA- protein-DNA original positions multiple staining the method for organization chip |
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| Publication number | Publication date |
|---|---|
| CN102466729B (en) | 2015-06-17 |
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