CN1316249C - Enzyme-linked assay kit and producing process thereof - Google Patents
Enzyme-linked assay kit and producing process thereof Download PDFInfo
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- CN1316249C CN1316249C CNB2004100569837A CN200410056983A CN1316249C CN 1316249 C CN1316249 C CN 1316249C CN B2004100569837 A CNB2004100569837 A CN B2004100569837A CN 200410056983 A CN200410056983 A CN 200410056983A CN 1316249 C CN1316249 C CN 1316249C
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Abstract
The present invention relates to an enzyme-linked assay reagent kit for detecting human vascular endothelial cell growth factors (VEGF). The inner part of a kit body of the reagent kit comprises an enzyme-linked reaction plate which is expressed and purified from insect cells and is coated by VEGF receptors, an enzyme bond which is marked by horseradish peroxidase (HRP) and configured by an antibody and a protective agent, VEGF proteic freeze-dried powder as positive control, proteic freeze-dried powder as negative control, sample dilute solution, PBST concentrated rinsing solution, a color developing substrate configured by 3, 3', 5, 5'-tetramethyl benzidine, 2MH2SO4 as stop solution and plating sealing gummed paper. The reagent kit can be used for the diagnosis and the prognosis of patients with cancer or cardiovascular diseases or diabetes or other diseases, and has the advantages of precision, specificity, sensitivity, stability, convenience, etc. Simultaneously, the present invention also provides a method for efficiently expressing foreign protein by the insect cells.
Description
Technical field
The invention belongs to the biological medicine technology field, relate in particular to the elisa kit for detecting of a kind of detection human vascular endothelial growth factor (VEGF).
The invention still further relates to the preparation method of mentioned reagent box.
Background technology
The development of tumour and transfer are processes multistage, complicated, that have high selectivity, relate to complicated relation between tumour cell and the host, and its mechanism is still very unclear.Progress about new angiogenesis and tumor development, transfer and prognosis relation is rapid in recent years, prove that new angiogenesis (Angiogenesis) is the necessary condition of tumor growth, transfer and metastasis growth, and vascular endothelial growth factor (Vascular Endothelial Growth Factor is to regulate the most important cell factor that neovascularity generates VEGF).
At first separation and purification goes out a kind of heparin binding growth factor (Ferrara etc. in Niu Chuiti folliculus sternzellen in vitro culture liquid, Biochem Biophys Res Commun, 1989,161 (2): 851-855), because of its can specific action in vascular endothelial cell, cause vascular endothelial cell proliferation, and induction of vascular generates in vivo, so be referred to as vascular endothelial growth factor (vascular endothelial growth factorVEGF), can increase microvascular permeability because of it again, be referred to as again the blood vessel permeability factor (VascularPermeability Factor, VPF).It is the strongest vascular permeability factor of finding at present, acts on stronger 50000 times than histamine.
In cell culture fluids such as mouse anterior pituitary tumor cell line AtT20, person monocytic cell, cavy knurl, mouse glioma cell line, also be purified into vegf protein subsequently.Confirmed that VEGF uniquely has specific important growth factor to vascularization, other growth factors such as fibroblast growth factor, platelet-derived property growth factor etc. can act on the various kinds of cell that comprises vascular endothelial cell, are that tool is not specific.Studies show that vascular endothelial growth factor in the growth of vascular system differentiation, have irreplaceable effect (Ferrara et al.Endocr. Rev.1997,18:4-25).
VEGF is a basic protein that molecular weight is the high glycosylation of 45KD, is formed by disulfide-bonded by two identical subunits, and isoelectric point is 8.5, and very strong heat-resisting and acidproof ability is arranged.It is a member of platelet derived growth factor (PDGF) family, can be produced and secretion by some normal cells, also can be produced by kinds of tumor cells.Human VEGF gene is positioned at chromosomal 6p21.3 (Circulation 93 for Vincenti, V.et al, 1493-1495,1996), total length 28kb, and the gene of coding VEGF is about 14kb, alternately is made of 8 extrons and 7 intrones.The hypotype that VEGF is different is formed by the different montage mode of this gene.Promptly montage became 5 kinds of different transcriptons (isomeride) through transcriptional level after human VEGF gene was encoded, was respectively VEGF206, VEGF189, VEGF165, VEGF145, VEGF121.Difference between the isomeride on the function shows as that they are different with heparin binding activity in cell surface and the extracellular matrix.Placenta growth factor (PLGF) is cloned into from people's placenta, with VEGF 53% homology is arranged on amino acid levels, also be considered to a member in the VEGF growth factor family, but PLGF only limits to express (Maglione D et al.PNAS USA in placenta, 1991,88:9267).
The VEGF high affinity combined sites only is positioned at 3 kinds of vegf receptors on the vascular endothelial cell, be VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4), these 3 kinds of acceptors mainly are distributed in the vascular endothelial cell surface, by the cell outskirt that contains 7 immunoglobulin (Ig) spline structures, film district and tyrosine kinase district form, all are transmembrane receptors, belong to RTK (receptor tyrosinekinase) III type, its common feature is to have tyrosine kinase to insert the district in the catalytic domain, the activity of this tyrosine kinase activates by acceptor and part combination, cause many enzymes and other reactions in the cell by receptor phosphorylation, in the growth of cell and differentiation, play an important role.Studies show that, vegf receptor external and VEGF have height affinity (Kendall et al.PNAS USA, 1993,90:10705-9).
VEGF is growth of cancers, the necessary a kind of factor of transfer.Studies show that at present in the various malignant tumours of the mankind (being cancer) disease, the secretion increase of VEGF has ubiquity and popularity, promptly when in the human body malignant growth being arranged, the VEGF secretion will increase greatly, and discharges into blood circulation.Lot of documents report is arranged, and VEGF is obviously different with concentration among the cancer patient the normal person, the very low or detection of the VEGF concentration among the normal person less than, and VEGF is dense in the cancer patient.Therefore, detect VEGF concentration in the human blood, can be used in the early stage generaI investigation diagnosis of cancer.On the other hand, the concentration of VEGF in blood of human body changes with the growth and decline situation of tumour.Lump is big, grow when fast, and the haemoconcentration of VEGF is just high; And when tumour during because of chemotherapy or operation " clinical cure ", the haemoconcentration of VEGF reduces.So the detection of VEGF can be used as the index of observation of curative effect and prognosis.VEGF is an important component part in the diagnosis of malignant tumor.In addition, blood VEGF level increases and also sees diabetes, vascular inflammation disease, some immunity disease and gestation etc.
(Clin.Chem.1992 38:71) reported ELISA method based on the detection VEGF of immunofluorescence, but its detection sensitivity is lower for Yeo et al..(J Biol.Chem.1992 267:26031) reported ELISA method based on the detection VEGF of colourimetry, but its detection sensitivity only is ng/ml to Houck et al..(Biosci.Biotechnol.Biochem.1995 59:1985) has reported the chemiluminescence ELISA that measures VEGF to Hanatani et al., and the Serum VEGF of measuring among the normal person is 8-36ng/l.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme joint inspection and survey the kit of human vascular endothelial growth factor (VEGF).This kit result is accurately stable, high specificity, highly sensitive, easy to use, be convenient to promote.
Another object of the present invention provides the production preparation and the using method of above-mentioned elisa kit for detecting.
For achieving the above object, kit provided by the invention consists of:
1, elisa plate; 2, positive control (freeze-drying); 3, negative control (freeze-drying); 4, sample diluting liquid; 5, enzyme connection thing; 6, concentrated cleaning solution; 7, developer A﹠amp; B; 8, distilled water; 9, stop buffer; 10, shrouding gummed paper.
The preparation method and the operation steps of mentioned reagent box provided by the invention are as follows:
A) with the enzyme-linked reaction plate be solid support, with the vegf receptor bag of gene engineering expression by on solid support;
B) seal with confining liquid
C) biological sample is contacted and is incubated with vegf receptor fixing and on the enzyme-linked reaction plate, the VEGF in the biological sample combines with vegf receptor specifically;
D) separating bio sample from the vegf receptor;
E) the VEGF polyclonal antibody with horseradish peroxidase (horseradish peroxidase HRP) mark detects VEGF, and used chromogenic substrate is 3,3 ', 5,5 '-tetramethyl benzidine (TMB).
The uniqueness of kit of the present invention is that it uses the vegf receptor bag by elisa plate, can guarantee that the VEGF of reduced levels can be detected by this kit, can have more accurately sample to measure.
VEGF in this kit detection of biological sample is preferably from cancer, angiosis patient or other diseases patient's sample.
Description of drawings
Fig. 1: kit box body of the present invention and composition.
Fig. 2: from the vegf protein polyacrylamide gel electrophoresis figure of expressed in insect cells purifying.
Fig. 3: from the vegf receptor protein polyacrylamide gel electrophoresis figure of expressed in insect cells purifying.
Embodiment
Following embodiment is for the more detailed the present invention of explanation, is confined to this but should not be construed as the present invention.
Embodiment 1:
Kit of the present invention can be following material for example:
1) the vegf receptor bag is by elisa plate, and
2) positive control (vegf protein freeze-dried powder) is 1;
3) negative control (animal blood serum freeze-dried powder) is 1;
4) sample diluting liquid: 0.05% Tween-20 (Tween-20), 1% bovine serum albumin(BSA) (BSA), 10mM disodium ethylene diamine tetraacetate (EDTA), 0.05% thimerosal, 0.4M NaCl, pH6.5,1 bottle;
5) enzyme connection thing (horseradish peroxidase-labeled VEGF polyclonal antibody) 2 bottles;
6) PBST concentrated cleaning solution (137mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 2mM KH
2PO
4, 0.05%Tween-20, pH 7.4) and 1 bottle;
7) developer A﹠amp; B, wherein A liquid is H
2O
2Solution, B liquid are 3,3 ', 5, each 1 bottle of 5 '-tetramethyl benzidine (TMB) solution;
8) distilled water is 1 bottle;
9) stop buffer (2M H
2SO
4) 1 bottle;
10) the shrouding gummed paper is 2;
The described various amounts of this example are that the example in 96 holes (12 8 holes or 8 12 holes) calculates with elisa plate all, if the hole count of elisa plate greater or less than 96 holes, then each amount of substance is also more or less corresponding.
This kit outward appearance can be consulted shown in Figure 1.
Its preparation method:
1) clone of VEGF gene and vegf receptor gene
With human placenta cDNA library is that template is carried out PCR (polymerase chainreaction, PCR) reaction.According to the report sequences Design primer of VEGF gene, the PCR reaction conditions: 94 ℃ of sex change 40 seconds, 52 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute, 30 back 72 ℃ of insulations of circulation 10 minutes.According to the report sequences Design primer of vegf receptor gene, the PCR reaction conditions: 94 ℃ of sex change 40 seconds, 52 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, 30 back 72 ℃ of insulations of circulation 10 minutes.
Reaction system: ddH
2O 20.0 μ l
10×Buffer 2.5μl
dNTP(10mM) 0.5μl
3’primer(100ng/μl) 0.5μl
5’primer(100ng/μl) 0.5μl
Taq enzyme (5U/ μ l) 0.5 μ l
Template DNA 0.5 μ l
Cumulative volume 25 μ l
The fragment of pcr amplification is reclaimed in gel electrophoresis, is cloned among the carrier pMT18-T, and transformed into escherichia coli DH5 α, the extraction recombinant plasmid carries out enzyme and cuts evaluation.The recombinant plasmid called after pMT18-1 of VEGF gene, the recombinant plasmid called after pMT18-2 of vegf receptor gene.Dna sequence analysis is finished by the rich inferior bioengineering in Shanghai company limited.
2) structure of insect expression vector
With SalI, BamHI difference digested plasmid pMT18-1 and pMT18-2, reclaim VEGF genetic fragment and vegf receptor genetic fragment respectively.Use SalI, BamHI digested plasmid pFastBac-HT simultaneously, reclaim the 4.7kb fragment.The VEGF genetic fragment is connected carrier construction pFastBac-1 and pFastBac-2 respectively with the vegf receptor genetic fragment with the 4.7kb fragment.Carry out enzyme with different restriction enzymes and cut evaluation, show that constructed carrier is correct.
Respectively carrier pFastBac-1 and pFastBac-2 are transformed the DH10Bac competent cell.Adopt the screening of blue hickie, after 24 hours hickie is drawn on the new flat board, confirm its whether hickie.Bacterium colony is chosen in the 2ml LB nutrient culture media, 37 ℃, 250rpm shaken cultivation 12 hours.
The plasmid method of extracting in the bacterium liquid of cultivating is as follows:
A1) 1.5ml bacterium liquid is changed in the centrifuge tube, 10, the centrifugal 30s of 000rpm abandons supernatant, and centrifuge tube is stood upside down on paper handkerchief, makes bacterial precipitation dry as far as possible;
B1) (10mM EDTA pH8.0), shakes mixing on oscillator for 50mM glucose, 25mM Tris-HCl to add 300 μ l solution I;
C1) add the solution II that 300 μ l now join (0.2N NaOH, 1%SDS), mixing, room temperature was placed 5 minutes;
D1) (5M KAc pH5.5), ices pre-5min to add the solution III of 300 μ l precoolings;
E1) 12, the centrifugal 15min of 000rpm;
F1) shift in the new centrifuge tube of supernatant to, add the absolute ethyl alcohol of 2 times of volumes, mixing;
G1) 10,000～12, the centrifugal 10min of 000rpm;
H1) with 70% and absolute ethanol washing precipitation;
I1) dry up, be dissolved in the TE (10mMTris-HCl that 50 μ l add RNase; 1mMEDTA, pH8.0) in, placed one hour in 37 ℃, place-20 ℃ standby.
The plasmid that extracts is carried out PCR identify, the PCR reaction is according to aforesaid method.
3) transfection insect cell (with the sf9 cell is example, but is not limited only to the sf9 cell, adopts sf21, sf158 or c127 cell line can obtain identical effect, in this not narration one by one).
The sf9 cellular incubation is added 15% hyclone, penicillin 50U/ml, streptomysin 50ug/ml in the Grace nutrient solution, transfection method is as follows:
A2) the previous day is assigned to 24 orifice plates with cell in transfection;
B2) transfection is preceding 2 hours, changes fresh serum-free, unparalleled anti-nutrient solution, washes once, adds the 500ul nutrient solution then;
C2) add 2ul liposome, the aforesaid recombinant plasmid of 5ul respectively in 50ul serum-free, unparalleled anti-nutrient solution, room temperature was placed 5 minutes, and plasmid is joined in the liposome, and room temperature was placed 20 minutes;
E2) previous step is rapid potpourri is added in the cell, 27 ℃ of cultivations;
F2) transfection sopped up nutrient solution after 4 hours, added 1ml and newly rarely had serum, two anti-Grace nutrient solutions are arranged, and cultivated 72 hours for 27 ℃;
G2) transfection is after 72 hours, and whether observe has malicious sign;
H2) will there be the baculoviral supernatant to infect new sf9 cell.
4) purifying of insect expressing protein (Ni column adsorption method)
A3)) preparation of Ni post
I) get 1ml Ni-NTA in a centrifuge tube, centrifugal 5 minutes of 1500g;
Ii) remove supernatant, with Ni-NTA with the lavation buffer solution of 1 times of volume (20mM Tris-HCl, 500mM KCl, 20mM miaow Cuo, the 2mM mercaptoethanol, 10% glycerine, pH8.5) resuspended;
Iii) with resuspended liquid to going in the post, with the wash buffer balance of 5-10 times of volume.
B3) preparation of cell extract
I) 500g collected the 50ml cell in centrifugal 5 minutes;
Ii) use lysis buffer (50mM Tris-HCl, 100mM KCl, 20mM miaow Cuo, 5mM mercaptoethanol, the stupid methanesulfonyl fluoride of 1mM, pH8.5) re-suspended cell;
Iii) 10, centrifugal 10 minutes of 000g transfers to supernatant in the one new pipe.
C3) protein purification
I) previous step is rapid supernatant is added in the good Ni post of balance;
Ii) use 10 times of volumes buffer A (20mM Tris-HCl, 500 mM KCl, 20mM miaow Cuo, the 5mM mercaptoethanol, 10% glycerine pH8.5) is washed post;
Iii) use 2 times of volumes buffer B (20mM Tris-HCl, 1M KCl, 20mM miaow Cuo, the 5mM mercaptoethanol, 10% glycerine pH8.5) is washed post;
Iv) the buffer A with 2 times of volumes washes post;
V) with 5-10 times of buffer C (20mM Tris-HCl, 100mM KCl, 100mM miaow Cuo, 5mM mercaptoethanol, 10% glycerine, pH8.5) wash-out, collection eluent.
Vi) detect the concentration and the purity of purified albumen with SDS-PAGE glue; Testing result is consulted Fig. 2 and shown in Figure 3.
5) VEGF Polyclonal Antibody Preparation, purifying and mark
As immune animal, stimulate animal with the injection of 5-10mg Bacille Calmette-Guerin with rabbit earlier.After one week, vegf protein is made the Fu Shi Freund's complete adjuvant, adopt subcutaneous multi-point injection to carry out the immunity first time.After 2 weeks, carry out the immunity second time.After 2 weeks, examination blood is also surveyed and is tired.As it is on the low side to tire, and then carries out booster immunization.
Gather tame rabbit whole blood, centrifuging and taking serum.Add saturated ammonium sulfate in serum, the volume ratio of serum and ammonium sulfate is 2: 1, makes the antibody precipitation, centrifuging, and antibody purity reaches more than 98%.
Adopt glutaraldehyde method with horseradish peroxidase (HRP) mark to the VEGF polyclonal antibody.
6) preparation of kit
The bag of elisa plate is used following steps: antigen is cushioned liquid dilution certain multiple with bag, and every hole adds 100ul.Bag was placed on ambient temperature overnight then by 2 hours in 37 ℃ of water-baths or the incubator.The PBST cleansing solution is filled it up with in every hole, places about 30 seconds, dries.Every hole adds the 120ul confining liquid, and room temperature was placed 2 hours.The confining liquid prescription mainly contains BSA, some salts etc.Get rid of confining liquid, pat and remove remaining confining liquid.Dry up on the super-clean bench.Or blow a few hours on the super-clean bench, room temperature is placed and is spent the night then.Pack with vacuum packing machine.
Embodiment 2: the kit using method
1. take out required lath from balance to the sealing bag of room temperature, other lath please seal puts back to 4 ℃.
2. staying a hole is blank well, and other each holes add sample diluting liquid 100 μ l (or 2).
3. except that blank well, add negative control 1 hole, positive control 2 holes, each 100 μ l of serum specimen to respective aperture, flick mixing.Seal reacting hole with the shrouding gummed paper, 37 ℃ 60 minutes.
4. wash plate 3 times: (1) automatic washer: get rid of liquid in the most hole, the cleansing solution that requires to inject is 350 μ l, injects and sucking-off 15～30 seconds at interval, washes plate 3 times.(2) wash plate by hand: get rid of liquid in the most hole, every hole adds cleansing solution 350 μ l, leaves standstill after 30 seconds and gets rid of most liquid, repeatedly pats dry on the thieving paper thick, washes 3 times.
5. except that blank well, the enzyme-added thing 200 μ l in every hole (or 4) seal plate hole, 37 ℃ 60 minutes.
6. wash plate 3 times.
7. chromogenic reaction: developer A, each 100 μ l (or 2) of B liquid are added in the reacting hole (comprising blank well) lucifuge color development at room temperature 25 minutes.
8. cessation reaction: every hole (comprising blank well) adds stop buffer 50 μ l (or 1), and mixing is measured the 450nmOD value.
The advantage that the present invention has:
1. the time is short, cost is low, preparation is simple.General encrusting substance uses monoclonal antibody, and the MONOCLONAL ANTIBODIES SPECIFIC FOR time is long, generally needs 9-12 month, needs 6-8 month at the soonest, and expense is higher, and MONOCLONAL ANTIBODIES SPECIFIC FOR is comparatively loaded down with trivial details; And among the present invention, acceptor is as encrusting substance, and the preparation of the genetic engineering acceptor by insect cell expression only needs about 3 months, and cost is low, prepares comparatively simple.
2. high specificity as a result.Acceptor has the specificity of height with combining of part, and encrusting substance is the acceptor of VEGF among the present invention, and checking matter (part) is VEGF.And a kind of monoclonal antibody of albumen is screened easy and other albumen generation cross reaction of not exclusively prepared monoclonal antibody owing at a certain antigenic determinant, and with respect to the receptor-ligand combination, specificity is lower.
3. highly sensitive.Detection sensitivity can reach the ng/L level.
4. stable.The present invention is in 4 ℃ of preservations, and the term of validity reaches 9-12 month
5. convenient.The present invention is easy and simple to handle, and required detecting instrument (microplate reader) generally uses at various big hospital and inspection center.
Claims (11)
1. elisa kit for detecting, it consists of:
Elisa plate, positive control, negative control, sample diluting liquid, enzyme connection thing, concentrated cleaning solution, developer A, developer B, distilled water, stop buffer and shrouding gummed paper;
Wherein:
Elisa plate be vascular endothelial growth factor can specificity the vegf receptor bag of combination by elisa plate;
Positive control is a vascular endothelial growth factor albumen freeze-dried powder;
Negative control is the animal blood serum freeze-dried powder;
Sample diluting liquid: 0.05% Tween-20,1% bovine serum albumin(BSA), 10mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4M NaCl, pH6.5;
Enzyme connection thing is a horseradish peroxidase-labeled vascular endothelial growth factor polyclonal antibody;
Concentrated cleaning solution: 137mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 2mMKH
2PO
4, 0.05% Tween-20, pH 7.4;
Developer A, developer B, wherein developer A is H
2O
2Solution, developer B are 3,3 ', 5,5 '-tetramethyl biphenyl amine aqueous solution;
Distilled water
Stop buffer is 2M H
2SO
4
2. according to the kit of claim 1, it is characterized in that each amount of substance is a standard with the hole count of elisa plate.
3. according to the kit of claim 1, it is characterized in that described elisa plate is 96 holes.
4. according to the preparation method of the described kit of claim 1, key step is:
(a) with the vascular endothelial growth factor receptor bag of gene engineering expression by on the solid support enzyme-linked reaction plate;
(b) seal with confining liquid;
(c) biological sample is joined in the enzyme-linked reaction plate reacting hole of pre-bag quilt, the vascular endothelial growth factor in the biological sample combines with vascular endothelial growth factor receptor specifically;
(d) detect vascular endothelial growth factor with the vascular endothelial growth factor polyclonal antibody:
(e) chromogenic substrate is 3,3 ', 5,5 '-tetramethyl benzidine.
5. according to the method for claim 4, it is characterized in that the described genetic engineering acceptor of step a insect cell expression.
6. according to the method for claim 5, it is characterized in that described insect cell is sf9, sf12, sf158 or c127 cell line.
7. according to the method for claim 5, it is characterized in that the genetic engineering acceptor of described insect cell expression Ni post purifying.
8. according to the method for claim 4, it is characterized in that the described biological sample of step c is cancer patient, angiosis patient's serum, blood plasma, cerebrospinal fluid, saliva, tissue extract or a urine.
9. according to the method for claim 4, it is characterized in that the vascular endothelial growth factor that the described vascular endothelial growth factor polyclonal antibody of steps d is used is with insect cell expression and use Ni post purifying.
10. according to the method for claim 4, it is characterized in that the polyclonal antibody described in the steps d is with vascular endothelial growth factor immunizing rabbit gained.
11. the method according to claim 4 is characterized in that, the horseradish peroxidase-labeled of polyclonal antibody described in the steps d.
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| CN102435743A (en) * | 2011-09-15 | 2012-05-02 | 北京华创远航科技有限公司 | ELISA detection kit and preparation method thereof |
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| CN100465642C (en) * | 2007-07-18 | 2009-03-04 | 清华大学 | Method for increasing anti matrix effect in immunity detection for environmental sample, and dedicated buffer solution |
| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
| CN102323421A (en) * | 2011-05-24 | 2012-01-18 | 北京健平九星生物医药科技有限公司 | Enzyme linked immunosorbent assay kit and preparation method thereof |
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| CN102426240B (en) * | 2011-09-19 | 2014-10-15 | 北京健平金星生物科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| CN102854322A (en) * | 2012-07-31 | 2013-01-02 | 吴克 | Vascular endothelial growth factor (VEGF) receptor enzyme linked diagnosis kit and preparation method thereof |
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| CN105353136A (en) * | 2015-12-01 | 2016-02-24 | 北京健平九星生物医药科技有限公司 | Kit for detecting concentration of VEGF in human serum on basis of quantum dot CdSe and using method thereof |
| CN108957001B (en) * | 2018-06-27 | 2019-11-12 | 中国科学院化学研究所 | Aptamer elisa kit for detecting and preparation method and applications |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1260492A (en) * | 1999-11-09 | 2000-07-19 | 赵永同 | Tumor prognosis evaluation reagent kit and its preparation technology |
| WO2000064946A2 (en) * | 1999-04-28 | 2000-11-02 | Board Of Regents, The University Of Texas System | Compositions and methods for cancer treatment by selectively inhibiting vegf |
| CN1420987A (en) * | 1999-11-16 | 2003-05-28 | 杰南技术公司 | ELISA for VEGF |
-
2004
- 2004-08-25 CN CNB2004100569837A patent/CN1316249C/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000064946A2 (en) * | 1999-04-28 | 2000-11-02 | Board Of Regents, The University Of Texas System | Compositions and methods for cancer treatment by selectively inhibiting vegf |
| CN1260492A (en) * | 1999-11-09 | 2000-07-19 | 赵永同 | Tumor prognosis evaluation reagent kit and its preparation technology |
| CN1420987A (en) * | 1999-11-16 | 2003-05-28 | 杰南技术公司 | ELISA for VEGF |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435743A (en) * | 2011-09-15 | 2012-05-02 | 北京华创远航科技有限公司 | ELISA detection kit and preparation method thereof |
| CN102435743B (en) * | 2011-09-15 | 2014-10-15 | 北京健平九星生物医药科技有限公司 | ELISA detection kit and preparation method thereof |
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