CN102221615A - Double-antibody sandwich ELISA method based on Angiogenin detection - Google Patents
Double-antibody sandwich ELISA method based on Angiogenin detection Download PDFInfo
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Abstract
A double-antibody sandwich ELISA method based on the Angiogenin detection aims to provide a simple high-sensitivity method for detecting the ANG2 content. The main points of the technology provided by the invention comprise the steps of: firstly constructing ANG2 genes to prepare an ANG2 protein, expressing and purifying the ANG2 gene, taking the ANG2 gene as the antibody to prepare high-titer and high-specificity anti-human ANG2 monoclonal antibody and ANG2 polyclonal antibody, taking the anti-human ANG2 polyclonal antibody as the capture antibody, taking the biotin-labeled anti-human ANG2 monoclonal antibody as the detection antibody, and establishing the double-antibody sandwich ELISA method for detecting the soluble ANG2 content. The invention belongs to the technical field of bio-pharmacy detection; the double-antibody sandwich ELISA method based on the Angiogenin detection can be utilized for the diagnosis, curative effect observation and prognosis of Angiogenin related diseases such as tumour and autoimmune diseases, laying a foundation for the research of Angiogenin protein functions, tumour screening and diagnosis and the like.
Description
Technical field
The present invention discloses a kind of ELISA method, relates in particular to a kind of double antibodies sandwich ELISA method that detects based on Angiogenin, belongs to medicine bioengineering detection technique field.
Background technology
(Angiogenin is that a kind of molecular weight that is present in normal plasma and the entity tumor tissue is the protein of 14.1kD, pI 9.5 ANG) to angiogenine, and the assignment of genes gene mapping is in chromosome 14q11.ANG is a kind of stimulating factor that can promote effectively that new vessels forms, and has very strong short angiogenesis function, and ANG is considered to one of short topmost cell factor of angiogenesis.ANG separated from the nutrient solution of human colon cancer cell strain HT-29 in 1985 and gets, in human plasma and cow's milk, detect its existence subsequently, it belongs to the ribonuclease superfamily member, it is 35% identical that primary structure and people's pancreatic ribonuclease have, and has faint ribonuclease activity.ANG be by with the receptors bind on vascular endothelial cell surface, activate the second messenger in the born of the same parents and bring into play a series of biological actions (invading, organize the formation of newborn tubular structure etc.), thereby reach the effect that promotes that new vessels forms as mediated cell adhesion, inducing cell.
The Research Significance that ANG promotes new vessels to form is mainly reflected in three aspects: (1) improves local microcirculation, particularly plays an important role in the processes such as reconstruction offshoot circulation in the reparation of the relatively poor meniscal fibrocartilage of blood circulation and infraction or wound, burn equivalent damage tissue.(2) growth of entity tumor depends on the quick growth of inside tumor blood vessel, therefore can pass through to suppress the biologic activity of ANG, thereby stops the formation of new vessels to reach the purpose for the treatment of tumour.Studies show that (3) in the peripheral blood of patients serum such as cancer of pancreas, breast cancer, the level of ANG obviously raises, the level of serum ANG is remarkable positive correlation with the development of these tumours and transfer.In addition, research shows that also the ANG level more normally contrasts and obviously increases in cancer of the stomach, lung cancer, colorectal cancer and the patient with breast cancer's serum, therefore, can predict the generation and the development of tumour by the level that detects ANG in serum and the tumor tissues.
Summary of the invention
At the problems referred to above, the present invention discloses a kind of double antibodies sandwich ELISA method that detects based on Angiogenin and detects ANG content, is used for the clinical assistant diagnosis of Angiogenin relevant diseases such as tumour, autoimmune disease.
Technical scheme of the present invention is such: a kind of double antibodies sandwich ELISA method that detects based on Angiogenin, at first make up the ANG2 gene and prepare ANG2 albumen, express again and purifying ANG2 albumen, with ANG2 albumen is that the antigen preparation height is tired and high specific anti-people ANG2 monoclonal antibody and ANG2 polyclonal antibody, with anti-people ANG2 polyclonal antibody is capture antibody, with biotin labeled anti-people ANG2 monoclonal antibody serves as to detect antibody, sets up the double antibodies sandwich ELISA method that detects solubility ANG2 content.
Above-mentioned a kind of double antibodies sandwich ELISA method that detects based on Angiogenin, wherein, described structure ANG2 gene prepares ANG2 albumen and uses the RT-PCR technology and angle and get the ANG2 gene order, make up the pET28a/ANG2 recombinant expression plasmid, and change in the e. coli bl21 host strain, IPTG abduction delivering His-ANG2 fusion, nickel post affinitive layer purification ANG2 albumen adopts SDS-PAGE to analyze and expresses.
Above-mentioned a kind of double antibodies sandwich ELISA method that detects based on Angiogenin, wherein, the His-ANG2 fusion of described ANG2 MONOCLONAL ANTIBODIES SPECIFIC FOR process using purifying is as antigen, immunity BALB/c mouse and myeloma cell carry out Fusion of Cells, select to cultivate with the HAT nutrient culture media, indirect elisa method screens positive hole, the hybridoma behind the clone is inoculated into mouse peritoneal produced ascites, collects ascites and carries out purifying.
Above-mentioned a kind of double antibodies sandwich ELISA method that detects based on Angiogenin, wherein, described ANG2 Polyclonal Antibody Preparation technology is characterized in that, adopts the His-ANG2 immunizing rabbit with fusion protein of purifying, preparation and purifying polyclonal antibody.
Compared with prior art, it is simple that the present invention has detection method, highly sensitive, judging for the diagnosis of Angiogenin relevant diseases such as tumour and autoimmune disease, observation of curative effect and prognosis provides a kind of easy, economic detection method, for research Angiogenin protein function and lay the foundation at aspects such as tumor screening diagnosis.
Figure of description
Fig. 1 is a double antibodies sandwich ELISA typical curve;
Horizontal ordinate is the ANG2 antigen standard items of different dilute concentrations, and ordinate is corresponding OD450nm light absorption value.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, but do not constitute any limitation of the invention.
One, the structure of ANG2 gene, expression and purifying
1 method
1.1ANG2 angling of gene got
The ANG2 gene cDNA sequence
1 ?ATGTGGCAGA?TTGTTTTCTT?TACTCTGAGC?TGTGATCTTG?TCTTGGCCGCAGCCTATAAC?AACTTTCGGA?AGAGCATGGA?CAGCATAGGA?A
101?ATCAGGTCCA?GCATGGGTCC?TGCAGCTACA?CTTTCCTCCT?GCCAGAGATGGACAACTGCC?GCTCTTCCTC?CAGCCCCTAC?GTGTCCAATG?CTGTGCAGAG
201?GGACGCGCCG?CTCGAATACG?ATGACTCGGT?GCAGAGGCTG?CAAGTGCTGGAGAACATCAT?GGAAAACAAC?ACTCAGTGGC?TAATGAAGCT?TGAGAATTAT
301?ATCCAGGACA?ACATGAAGAA?AGAAATGGTA?GAGATACAGC?AGAATGCAGTAGAACCAG?ACGGCTGTGA?TGATAGAAAT?AGGGACAAAC?CTGTTGAACC
401?AAACAGCGGA?GCAAACGCGG?AAGTTAACTG?ATGTGGAAGC?CCAAGTATTAAATCAGACCA?CGAGACTTGA?ACTTCAGCTC?TTGGAACACT?CCCTCTCGAC
501?AAACAAATTG?GAAAAACAGA?TTTTGGACCA?GACCAGTGAA?ATAAACAAATTGCAAGATAA?GAACAGTTTC?CTAGAAAAGA?AGGTGCTAGC?TATGGAAGAC
601?AAGCACATCA?TCCAACTACA?GTCAATAAAA?GAAGAGAAAG?ATCAGCTACAGGTGTTAGTA?TCCAAGCAAA?ATTCCATCAT?TGAAGAACTA?GAAAAAAAAA
701?TAGTGACTGC?CACGGTGAAT?AATTCAGTTC?TTCAGAAGCA?GCAACATGATCTCATGGAGA?CAGTTAATAA?CTTACTGACT?ATGATGTCCA?CATCAAACTC
801?AGCTAAGGAC?CCCACTGTTG?CTAAAGAAGA?ACAAATCAGC?TTCAGAGACTGTGCTGAAGT?ATTCAAATCA?GGACACACCA?CGAATGGCAT?CTACACGTTA
901?ACATTCCCTA?ATTCTACAGA?AGAGATCAAG?GCCTACTGTG?ACATGGAAGCTGGAGGAGGC?GGGTGGACAA?TTATTCAGCG?ACGTGAGGAT?GGCAGCGTTG
1001?ATTTTCAGAG?GACTTGGAAA?GAATATAAAG?TGGGATTTGG?TAACCCTTCAGGAGAATATT?GGCTGGGAAA?TGAGTTTGTT?TCGCAACTGA?CTAATCAGCA
1101?ACGCTATGTG?CTTAAAATAC?ACCTTAAAGA?CTGGGAAGGG?AATGAGGCTTACTCATTGTA?TGAACATTTC?TATCTCTCAA?GTGAAGAACT?CAATTATAGG
1201?ATTCACCTTA?AAGGACTTAC?AGGGACAGCC?GGCAAAATAA?GCAGCATCAGCCAACCAGGA?AATGATTTTA?GCACAAAGGA?TGGAGACAAC?GACAAATGTA
1301?TTTGCAAATG?TTCACAAATG?CTAACAGGAG?GCTGGTGGTT?TGATGCATGTGGTCCTTCCA?ACTTGAACGG?AATGTACTAT?CCACAGAGGC?AGAACACAAA
1401?TAAGTTCAAC?GGCATTAAAT?GGTACTACTG?GAAAGGCTCA?GGCTATTCGCTCAAGGCCAC?AACCATGATG?ATCCGACCAG?CAGATTTCTA?A
According to the people ANG2mRNA sequence that is provided among the Genebank (the Genebank accession number: NM001147), design has the Auele Specific Primer of restriction enzyme digestion sites, and primer sequence is as follows:
ANG2-F:5 '-gcTCTAGAGAATTCatgtggcagattgttttcttac-3 ' (having EcoR I restriction enzyme site)
ANG2-R:5 '-tccCCCGGGAAGCTTgaaatctgctggtcggatca-3 ' (having Hind III restriction enzyme site)
The PCR response procedures:
94℃4min 1Cycle
72℃10min 1Cycle
After the PCR reaction finishes, detect with 1% agarose gel electrophoresis whether amplified reaction normally carries out or whether the clip size that increases is correct, reclaiming reaction product, be connected with carrier, conversion, positive colony identify, send order-checking, select to have the material of the clone of restriction enzyme site in the sequencing result as next step.
1.2pET28a/ANG2 construction of recombinant plasmid and evaluation
PCR product and prokaryotic expression carrier pET28a use EcoR I and Hind III double digestion respectively, gel reclaims the back by after 3.5: 1 mixed in molar ratio, 16 ℃ of connections under the effect of Ligation high enzyme, reaction overnight will connect product and transform the BL21 competent cell, screen according to the sign (anti-Kan) of recombinant vector, picking list spot, alkaline lysis is the extracting plasmid in a small amount, the reaction of EcoRI/Hind III double digestion, and 1% agarose gel electrophoresis detects to identify whether be recombinant plasmid.
1.3 the abduction delivering of recombinant protein
Picking contains the bacterium colony list spot of recombinant plasmid, be inoculated in the LB fluid nutrient medium of Chinese kalamycin resistance, 37 ℃ of shaken cultivation are spent the night, by 1: 50 dilution proportion bacterium that spends the night, generally the 1ml bacterium is joined in the 300ml culture flask that contains 50ml LB+Kan nutrient culture media, 37 ℃ of concussions are cultured to OD600 ≈ 0.4-1.0 (best 0.6, approximately need 3h).Get bacterium liquid 1ml as the control group of not inducing, remaining adding 100mmol/L IPTG derivant to final concentration 1mmol/L as experimental group, experimental group continues 37 ℃ of concussions cultivates, and gets bacterium liquid at 1.5h, 3h, 4.5h, 6h, 7.5h respectively and carries out the SDS-PAGE analysis.
1.4 the purifying of recombinant protein inclusion body
1.4.1 the processing of sample before the purifying
Method step: 600ml LB+Kan fluid nutrient medium adds 10ml bacterium liquid, and shaken cultivation to OD value is 0.3-0.5, adds 6ml 100mmol/L IPTG and induces 3h (37 ℃ of vibrations), and centrifugal (4 ℃, 10000rpm 20min), collects bacterium liquid.Ratio in the wet bacterium of 5ml/g adds outstanding bacterium liquid, and vibration suspends.-70 ℃ of freezing 30min, room temperature is melted, and repeats once.Ultrasonication cell (300W, ultrasonic 5s, 9s, 15min altogether at interval), centrifugal (4 ℃, 10000rpm, 15min) collecting precipitation.It is resuspended to add 9 volume rinsing damping fluids, centrifugal (4 ℃, 10000rpm, 15min) collecting precipitation repeats 2 times.Add the 30ml damping fluid, 4 ℃ are spent the night.Centrifugal (4 ℃, 7000g, 20min), supernatant was prepared against post with 0.45 μ m membrane filtration.
1.4.2Ni affinitive layer purification recombinant protein inclusion body
Method step: with level pad balance 2-5ml, flow velocity is 2ml/min; With the sample upper prop of 0.45 μ m membrane filtration, flow velocity is 1ml/min; Wash 2-5ml again with eluent 1, flow velocity is 2ml/min; Carry out wash-out with eluent 2, flow velocity is 2ml/min, the purification of samples when the collection eluting peak reaches mxm.; Detect the molecular weight size of the recombinant protein of purifying with SDS-PAGE.
2 results
2.1ANG2 angling of gene got
Go up the ANG2 gene order according to Genebank, the special primer of design ORF amplification carries out pcr amplification to its ANG2.Figure 1 shows that the electrophoretogram of ANG2 pcr amplification, the purpose fragment is about 1.5Kb.The PCR product is through 1% agarose gel electrophoresis, after Goldview dyeing, observes under the ultraviolet gel imaging system, and the PCR product reclaims, is connected with carrier, send order-checking after conversion and the evaluation of EcoR I/Hind III double digestion through glue.The contrast of the sequence that records and ANG2 gene order, consistance is 99.9% as a result, and contains specific restriction endonuclease sites, the amplification that our success is described is to having an ANG2 gene purpose fragment of restriction endonuclease sites.
2.2pET28a/ANG2 construction of recombinant plasmid and evaluation
To connect product transformed into escherichia coli BL21, screen according to the sign (anti-Kan) of recombinant vector, picking list spot, alkaline lysis is the extracting plasmid in a small amount, the reaction of EcoR I/Hind III double digestion, 1% agarose gel electrophoresis detects to be identified.From Fig. 2, we see that the 2nd swimming lane has 2 bands, and clip size is consistent with carrier and target gene fragment, ANG2 is connected with carrier pET28 successfully are described, for next step experiment is got ready.
2.3ANG2 the prokaryotic expression dynamic analysis
The pET28a/ANG2 recombinant plasmid is transferred to the e. coli bl21 competent cell, and at this moment, the prokaryotic expression engineering bacteria is finished equipment.What the abduction delivering of recombinant protein was adopted is single factor design, and induction time has been carried out gradient design.Shown in Figure 3, when inducing 1.5h, the destination protein band begins to occur, and along with the prolongation of induction time, the concentration of destination protein band increases gradually, illustrate our success with the recombinant protein abduction delivering.
2.4 the purifying of recombinant protein
The pET28a carrier has the His-tag label, it can with multiple transition metal ion Cu2+, Zn2+, Ni2+, special interaction takes place in Co2+, Fe3+, utilizes this principle being rich in the protein adsorption of this amino acid, thereby reaches the separation purpose.Use the AKTA prime protein purification system of Ni-NTA affinity column and GE company.Final concentration is that 1mmol/L IPTG induces about 4h at 37 ℃ recombinant protein, adopts multigelation method and ultrasound wave pair cell to carry out fragmentation.Broken back is centrifugal, collects supernatant and precipitation respectively, and precipitation is filtered with urea dissolving back.Supernatant and resolution of precipitate liquid are carried out the SDS-PAGE analysis, found that destination protein appears in the resolution of precipitate liquid, has promptly formed the albumen inclusion body.Selected to carry out the dialysis desalting renaturation after the affinity chromatography at this point with urea-denatured dissolving inclusion body.Carried out gradient elution with the elution buffer that contains 50mmol/L, 100mmol/L, 200mmol/L, 250mmol/L, 300mmol/L, 400mmol/L, 500mmol/L imidazoles respectively, the elute effect of the elution buffer of 250mmol/L imidazoles is better than other concentration as a result.Purifying through the protein chromatographic system to the resolution of precipitate liquid that contains many foreign proteins has formed the single band that only contains destination protein, sees Fig. 4 the 3rd swimming lane.
Two, MONOCLONAL ANTIBODIES SPECIFIC FOR and evaluation
1 method
1.1 the preparation of antibody
With the ANG2 fusion protein immunization BALB/c mouse in 6 age in week of purifying, totally three times, each 3w at interval, 200 μ g/ (inferior).Only merge first three day lumbar injection ANG2 300 μ g/, get mouse boosting cell and SP2/0 myeloma cell and merge under the 50%PEG effect, fused cell is put HAT and is selected 37 ℃ of nutrient solutions, CO
2Incubator is cultivated.When treating behind the 1w that cell grows to 1/3 hole, the Hybridoma Cell Culture supernatant is screened, positive hole hybridoma is carried out subclone with limiting dilution assay, reach 100% o'clock enlarged culture again with the positive rate of cloning the hole with indirect ELISA, freezing, preparation ascites.
1.2 ascites Purification of Monoclonal Antibodies
The silicon dioxide absorption method is carried out pre-service to ascites, adopt sad saturated ammonium sulphate method, ProteinA affinity chromatography to carry out purifying, SDS-PAGE purity assay, monoclonal anti body burden behind the ultraviolet spectrophotometer mensuration purifying, packing after the back filtration sterilization ,-70 ℃ of freezing preservations.
1.3ELISA measure (enzyme-linked immunosorbent assay)
Carry out the bag quilt of ANG2 antigen according to a conventional method, concentration is 1 μ g/ml, and other cell factor is also with same concentrations bag quilt, with PBV220/ANG/JM103 cellular lysate liquid eggs irrelevant contrast antigen in vain before inducing.
1.4 double diffusion test is surveyed the Ig subclass
The hybridoma culture supernatant bag filter of packing into puts 40%, is concentrated into 6 times among the PEG of molecular weight 8000, does two experiments of expanding with anti-mouse subclass serum.
1.5 immunoblotting
Cellular lysate liquid separated through SDS-PAGE with inducing afterwards before the ANG of purifying, PBV220/ANG/JM103 induced.In Bio-Rad electrotransfer system the gel protein band is transferred on the nitrocellulose membrane according to a conventional method, successively with 10% calf serum sealing, the hybridization supernatant is one anti-, and sheep anti mouse mark horseradish peroxidase IgG is two anti-, develops the color with the DAB system.
2 results
2.1 Fusion of Cells and screening
After immune mouse spleen cell and SP2/0 merge, cultivate respectively each 2 of 96 orifice plates and 24 orifice plates, there is the hybridoma growth in 60% hole behind the 1w, and with the ELISA screening, positive 3 holes of ANG have 2 strains can stably excreting antibody, called after C respectively through 3 subclones
12, C
3
2.2 ascites Purification of Monoclonal Antibodies
Ascites is carried out SDS-PAGE gel electrophoresis scanning behind ProteinA chromatographic column purifying, identify that its purity is 96.5%.Protein concentration is 16mg/ml behind the ultraviolet spectrophotometer mensuration purifying.
2.3 antibody classification and subclass
C
3, C
12The immunoglobulin class of hybridoma cell strain secretory antibody is respectively IgG1, IgM.
2.4 the specificity of monoclonal antibody is identified
Two strains anti-people ANG2 monoclonal antibody and various human cell factor, people and animal blood serum and induce before host's mycoprotein reactionless, and only with the ANG2 of purifying and the cellular lysate liquid eggs after inducing react in vain.The results are shown in Table 1, table 2.
The specific reaction of the anti-people ANG2 of table 1 monoclonal antibody
The reaction of the anti-people ANG2 of table 2 monoclonal antibody and different serum and thalline
2.5SDS-PAGE electrophoresis and immunoblotting result
Purification of recombinant human ANG2, SDS-PAGE result shows that its molecular weight is 14.1kD, induce the ANG relevant position of back mycoprotein and purifying precipitation line to occur, the immunoblotting result shows, the ANG2 of 2 antibody recognition 14.1kD and get along well and induce preceding cellular lysate liquid eggs to react in vain.
Three, Polyclonal Antibody Preparation
1 method
1.1 anti-ANG2 Polyclonal Antibody Preparation
2 of bull rabbit carry out immunity according to a conventional method.Consumption 1.5mg/ first of ANG2, later decrement is to 0.7mg/, and the 4th immunity back 3d begins the blood sampling survey and tires, and the 6th time immune back ELISA method was tired booster immunization 3d posterior vein intubate bloodletting once more 1: 12800.Separation of serum according to a conventional method is after getting part serum and doing dilution in 1: 1 with the PBS of 0.1mol/L PH 7.2, with twice of 50% saturated sulfuric acid salting out method purification, precipitation is dissolved with 0.1mol/L PBS, after carrying out protein quantification and antibody titer mensuration, add 50% glycerine ,-40 ℃ of preservations are standby.
1.2 anti-ANG polyclonal antibody titration
Wrap by 96 hole ELISA Plate with 100 μ g/L ANG2 albumen (being dissolved in the carbonate buffer solution of pH9.6), every hole 100 μ l, 37 ℃ of water-bath 2h, 4 ℃ are spent the night.The sealing of 1% bSA.With the dilution in 1: 800 of tested polyclonal antibody, doubling dilution according to a conventional method, adds ELIAS secondary antibody again, and H is used in PBS washing and the colour developing of OPD substrate
2SO
4Cessation reaction, the 490nm wavelength detects the OD value.Double diffusion according to a conventional method, center pit ANG is antigenic content 2mg/ml, the hole adds 1: 2～1: 64 different dilutability antibody on every side.
2 results
2 bull rabbit after 4 immunity from vein haemospasia, precipitation line appears in two expansions at 1: 16, and the ELISA method was tired 1: 6400, and the 6th time the ELISA method was tired 1: 12800, No. 2 rabbit is tired a little less than No. 1 rabbit, preferably tires between 1: 12800～1: 25600.It is 1: 32 that double-diffusion process detects antibody titer.
Four, the foundation of double antibodies sandwich ELISA method
1 method
1.1ANG2 the foundation of the double antibodies sandwich ELISA method standard curve that detects
To determine the mouse-anti people ANG2 monoclonal antibody coated elisa plate of concentration, 4 ℃ are spent the night, and wash 3 times; Sealing 2h adds each 100 μ l/ hole of standard antigen 100,50,25,10ng/ml and plasma sample to be measured of dilution, and adds sample diluting liquid as 0ng/ml.The standard items of every duplicate samples and variable concentrations are all established two parallel holes, and 37 ℃ of 1h wash 3 times; Add the anti-people ANG2 of the rabbit serum 100 μ l/ holes of dilution, 37 ℃ of 1h wash 3 times.Add and determine dilution HRP-goat anti-rabbit igg antibody 100 μ l/ holes, 37 ℃ of 1h wash 3 times; Add tmb substrate solution 100 μ l/ holes.37 ℃ of lucifuge reaction 10-15min, 2mol/L H
2SO
4Cessation reaction, microplate reader 450nm reads at the place OD value.With standard antigen (ng/ml) is horizontal ordinate, and corresponding OD value is an ordinate, subtracts zero hole OD value drawing standard curve with different dilutability standard antigen OD values.
1.2 the SABC of tumor in upper digestive tract sample ANG detects
The various tumor in upper digestive tract samples of having made a definite diagnosis, paraffin embedding, section according to a conventional method.Endogenous peroxydase is organized in dewaxing, aquation blocking-up, repairs antigen in the micro-wave oven.The dilution in 1: 1000 of the anti-people ANG2 of rabbit polyvalent serum, 37 ℃ of reaction 1h, 0.1mol/L pH 7.2PBS washing 4 times, add the biotin labeled goat anti-rabbit antibody of people according to a conventional method successively, streptavidin peroxidase, the DAB liquid that develops the color, mirror is control colour developing degree down, haematine speed transfect cell nuclear, dehydration, transparent, mounting, Nikon om observation.
2 results
2.1 the foundation of typical curve
As shown in Figure 1, the x+3 that presses blank calculates, the following 10.0ng/ml that is limited to that detects, double antibodies sandwich ELISA method is measured the typical curve of ANG2, in linear relation in the 10 μ g/ml scopes at 10ng/ml, the typical curve linear equation is y=0.0984x+0.0583, and linear regression coefficient is 0.9788, explanation is in this linear extent, and the result of mensuration is more accurate.
2.2 malignant tumor of digestive tract patients serum ANG content
With the double-antibodies sandwich ELISA of setting up, detect 25 parts of malignant tumor of digestive tract patients serum samples, comprise malignant tumours such as large intestine, small intestine, oesophagus, cancer of the stomach.ANG content is at 62.5～146.8ng/ml in its serum.
2.3 the SABC of ANG2 detects in the upper gastrointestinal malignant tumor tissue
The result adopts multivalent antibody or monoclonal antibody chromogenic reaction indifference in the immunohistochemical reaction of analyzing 30 routine upper gastrointestinal malignant tumor tissue ANG expression.Examine in the tumor specimen about 20%～30% and be the ANG positive reaction.The serous gland cell is strong positive reaction, is evenly distributed in cytoplasm; But the mucous gland cell is negative, and also has only serous gland to be positive in mixed gland.Cancer cell ANG is the weak positive, and a large amount of lymphocytic infiltrations are arranged on every side.
Claims (4)
1. double antibodies sandwich ELISA method that detects based on Angiogenin, it is characterized in that, at first make up the ANG2 gene and prepare ANG2 albumen, express again and purifying ANG2 albumen, with ANG2 albumen is that the antigen preparation height is tired and high specific anti-people ANG2 monoclonal antibody and ANG2 polyclonal antibody, with anti-people ANG2 polyclonal antibody is capture antibody, serves as to detect antibody with biotin labeled anti-people ANG2 monoclonal antibody, sets up the double antibodies sandwich ELISA method that detects solubility ANG2 content.
2. a kind of double antibodies sandwich ELISA method that detects based on Angiogenin according to claim 1, it is characterized in that, described structure ANG2 gene prepares ANG2 albumen and uses the RT-PCR technology and angle and get the ANG2 gene order, make up the pET28a/ANG2 recombinant expression plasmid, and change in the e. coli bl21 host strain, IPTG abduction delivering His-ANG2 fusion, nickel post affinitive layer purification ANG2 albumen adopts SDS-PAGE to analyze and expresses.
3. a kind of double antibodies sandwich ELISA method that detects based on Angiogenin according to claim 1, it is characterized in that, the His-ANG2 fusion of described ANG2 MONOCLONAL ANTIBODIES SPECIFIC FOR process using purifying is as antigen, immunity BALB/c mouse and myeloma cell carry out Fusion of Cells, select to cultivate with the HAT nutrient culture media, indirect elisa method screens positive hole, the hybridoma behind the clone is inoculated into mouse peritoneal produced ascites, collects ascites and carries out purifying.
4. a kind of double antibodies sandwich ELISA method that detects based on Angiogenin according to claim 1, it is characterized in that described ANG2 Polyclonal Antibody Preparation technology is characterized in that, adopt the His-ANG2 immunizing rabbit with fusion protein of purifying, preparation and purifying polyclonal antibody.
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| CN105264380A (en) * | 2013-05-14 | 2016-01-20 | 卫材R&D管理有限公司 | Biomarkers for predicting and assessing responsiveness of endometrial cancer subjects to lenvatinib compounds |
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| US11946938B2 (en) | 2017-12-13 | 2024-04-02 | Roche Diagnostics Operations, Inc. | Circulating Angiopoietin-2 (Ang-2) and insulin-like growth factor-binding protein 7 (IGFBP7) for the prediction of stroke |
| CN109307763A (en) * | 2018-11-11 | 2019-02-05 | 浙江理工大学 | A method of based on western blotting qualification Ancient Silk Textile |
| CN109307763B (en) * | 2018-11-11 | 2021-06-25 | 浙江理工大学 | Method for identifying ancient silk fabric based on immunoblotting |
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