CN102426240B - Enzyme-linked detection kit and preparation method thereof - Google Patents
Enzyme-linked detection kit and preparation method thereof Download PDFInfo
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- CN102426240B CN102426240B CN201110277681.2A CN201110277681A CN102426240B CN 102426240 B CN102426240 B CN 102426240B CN 201110277681 A CN201110277681 A CN 201110277681A CN 102426240 B CN102426240 B CN 102426240B
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Abstract
An enzyme-linked detection kit for detecting VEGF. A kit body contains an enzyme-linked reaction plate coated by a VEGF acceptor and a monoclonal antibody, an enzyme conjugate labelled by HRP and configured by antibody and a protective agent, a freeze-drying powder of the VEGF protein as a positive contrast, a protein freeze-drying powder as a negative contrast, a sample diluent, a PBST concentrated lotion, a chromogenic substrate configured by 3,3',5,5'-tetramethyl benzidine, 2MH2SO4 as a stopping solution and a seal plate gummed paper. The kit can be used in diagnosis and prognosis of cancer, angiopathy, diabetes, retinopathy and rheumatoid arthritis, and has advantages of accuracy, specificity, sensitivity, stability and convenience, etc.
Description
Technical field
The invention belongs to biological medicine technology field, relate in particular to a kind of elisa kit for detecting that detects 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 host, and its mechanism is still very unclear.Rapid about the progress of new Angiogenesis and tumor development, transfer and Prognostic significance in recent years, prove that new Angiogenesis is the necessary condition of tumour generation, transfer and metastasis growth, vascular endothelial growth factor (VEGF) is the most important cell factor that regulates new Angiogenesis.Research shows that vascular endothelial growth factor (VEGF) has irreplaceable effect (Ferrara et al. Endocr. Rev.1997,18:4-25) in the Development And Differentiation of vascular system.
VEGF is the basic protein of the molecular weight high glycosylation that is 45KD, has two identical subunits to form by disulfide-bonded, and isoelectric point is 8.5, has very strong heat-resisting and acid-fast ability.Human VEGF gene has 5 kinds of different isomeride, is respectively VEGF206, VEGF189, VEGF165, VEGF145, VEGF121.
VEGF high affinity combined sites is only positioned at 3 kinds of vegf receptors on vascular endothelial cell, i.e. VEGFR-1, VEGFR-2, VEGFR-3, and these 3 kinds of acceptors are mainly distributed in Surface of Vascular Endothelial Cells, are all transmembrane receptors.Research shows, vegf receptor has the affinity (Kendall et al. PNAS USA, 1993,90:10705-9) of height in vitro with VEGF.
VEGF is growth of cancers, shift necessary a kind of factor.Study and show at present, in the various malignant tumours of the mankind (being cancer) disease, the secretion increase of VEGF has ubiquity and popularity.Have lot of documents report, the concentration of VEGF in normal person and cancer patient is obviously different, and VEGF concentration in normal person is very low or can't detect, and VEGF is dense in cancer patient.Therefore, detect the VEGF concentration in 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 large, grow when fast, and the haemoconcentration of VEGF is just high; And when tumour is during because of chemotherapy or operation " clinical cure ", the haemoconcentration of VEGF reduces.Therefore VEGF must detect the index that can be used as observation of curative effect and prognosis.VEGF is an important component part in diagnosis of malignant tumor.In addition, blood VEGF level increases and also sees diabetes, vascular inflammation disease, some immunity disease and gestation etc.
Yeo et al. (Clin. Chem. 1992,38:71) has reported the ELISA method taking immunofluorescence as basic detection VEGF, but its detection sensitivity is lower.Houck et al. (J Biol. Chem. 1992,267:26031) has reported with colorimetric and has sent out the ELISA method into basic detection VEGF, but its detection sensitivity is only ng/ml.Hanatani et al. (Biosci. Biotechnol. Biochem. 1995,59:1985) has reported the chemiluminescence ELISA that measures VEGF, and the Serum VEGF of measuring normal person is 8-36 ng/l.
Summary of the invention
The object of this invention is to provide the detection kit of a kind of enzyme linked immunosorbent detection human vascular endothelial growth factor (VEGF).This kit result accurate stable, high specificity, highly sensitive, easy to use, be convenient to promote.
Another object of the present invention is to provide manufacture 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 & B; 8. stop buffer; 9. shrouding gummed paper.
It is as follows that the preparation method of mentioned reagent box provided by the invention and behaviour do step:
(a), taking enzyme-linked reaction plate as solid support, the vegf receptor of gene engineering expression and monoclonal antibody are coated on solid support;
(b) seal with confining liquid;
(c) biological sample is contacted and is incubated with the vegf receptor and the monoclonal antibody that are fixed on elisa plate, the VEGF in biological sample is combined with vegf receptor and monoclonal antibody specifically;
(d) separating bio sample from vegf receptor and monoclonal antibody;
(e) detect VEGF by the VEGF monoclonal antibody of horseradish peroxidase (HRP) mark, chromogenic substrate used is 3,3 ＇, 5,5 ＇-tetramethyl benzidine (TMB).
The uniqueness of kit of the present invention is, its uses vegf receptor and monoclonal antibody combined packet by elisa plate, can guarantee that the VEGF of reduced levels can be detected by this kit, can sample be had more accurately and be measured.
VEGF in this kit detection of biological sample, preferably from cancer, angiosis patient or other diseases patient's sample.
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) vegf receptor and monoclonal antibody combined packet are by elisa plate;
(2) 1 of positive control (vegf protein freeze-dried powder);
(3) 1 of negative control (animal blood serum freeze-dried powder);
(4) sample diluting liquid: 0.05% Tween-20,1% bovine serum albumin(BSA), 10mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4M NaCl, pH6.5,1 bottle;
(5) enzyme connection thing: horseradish peroxidase-labeled vascular endothelial growth factor monoclonal antibody, 2 bottles;
(6) concentrated cleaning solution is: 137mM NaCl, 2.7 mM KCl, 10mM Na2HPO4,2 mM KH2PO4,0.05% Tween-20, Ph7.4,1 bottle;
(7) developer A, B, wherein A liquid level H2O2 solution, B liquid is 3,3 ＇, 5,5 ＇-tetramethyl biphenyl amine aqueous solution, each 1 bottle;
(8) stop buffer is 2MH2SO4,1 bottle;
(9) 2 of shrouding gummed papers.
All taking elisa plate as 96 holes, the example in (12 8 holes or 8 12 holes) calculates the described various amounts of this example, if the hole count of elisa plate greater or less than 96 holes, each amount is also more or less corresponding.
Its preparation method:
(1) clone of VEGF gene and vegf receptor gene
Carry out PCR (PCR) taking human placenta cDNA library as template.According to the report primers of VEGF gene, PCR reaction conditions: 94 DEG C of sex change 40 seconds, 52 DEG C of annealing 1 minute, 72 DEG C are extended 1 minute, 30 rear 72 DEG C of insulations of circulation 10 minutes.According to the report primers of vegf receptor gene, PCR reaction conditions: 94 DEG C of sex change 40 seconds, 52 DEG C of annealing 1 minute, 72 DEG C are extended 2 minutes, 30 rear 72 DEG C of insulations of circulation 10 minutes.
Reaction system:
ddH2O 20.0μl
10×Buffer 2.5μl
dNTP(10mM) 0.5μl
3＇primer(100ng/μl) 0.5μl
5＇primer(100ng/μl) 0.5μl
(5U/ μ is 0.5 μ l l) for Taq enzyme
Template NDA 0.5 μ l
Cumulative volume 25 μ l
The fragment of pcr amplification is reclaimed in gel electrophoresis, is cloned in carrier pMT18-T, transforms bacillus coli DH 5 alpha, and extraction recombinant plasmid carries out enzyme and cuts qualification.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 completed by Shanghai Bo Ya bioengineering company limited.
(2) structure of insect expression vector
With Sal I, BamH I digested plasmid pMT18-1 and pMT18-2 respectively, reclaim respectively VEGF genetic fragment and vegf receptor genetic fragment.Use Sal I, BamH I digested plasmid pFastBac-HT simultaneously, reclaim 4.7kb fragment.VEGF genetic fragment is connected respectively to carrier construction pFastBac-1 and pFastBac-2 with 4.7kb fragment with vegf receptor genetic fragment.Carry out enzyme with different restriction enzymes and cut qualification, show that constructed carrier is correct.
Respectively carrier pFastBac-1 and pFastBac-2 are transformed to DH10Bac competent cell.Adopt the screening of blue hickie, after 24 hours, hickie is drawn on new flat board, confirm its whether hickie.Bacterium colony is chosen in 2ml LB nutrient culture media, 37 DEG C, 250rpm shaken cultivation 12 hours.
Plasmid method in the bacterium liquid that extraction is cultivated is as follows:
(a) 1.5ml bacterium liquid is proceeded in centrifuge tube, the centrifugal 30s of 10,000rpm, abandons supernatant, and centrifuge tube is stood upside down on paper handkerchief, makes bacterial precipitation dry as far as possible;
(b) add 300 μ l solution I (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH8.0), on earthquake device, vibration mixes;
(c) add the solution II (0.2N NaOH, 1%SDS) that 300 μ l now join, mix, room temperature is placed 5 minutes;
(d) add the solution III (5M KAc, pH5.5) of 300 μ l precoolings, ice bath 5 minutes;
(e) 12, centrifugal 15 minutes of 000rpm;
(f) shift in the new centrifuge tube of supernatant to, add the absolute ethyl alcohol of 2 times of volumes, mix;
(g) 10,000-12, centrifugal 10 minutes of 000rpm;
(h) with 70% and absolute ethanol washing precipitation;
(i) dry up, be dissolved in the TE(10mM Tris-HCl that 50 μ l add RNase; 1mM EDTA, pH8.0) in, in 37 DEG C place one hour, be placed in-20 DEG C for subsequent use.
The plasmid extracting is carried out to PCR qualification, and PCR reaction is according to aforesaid method.
(3) transfection insect cell (taking sf9 cell as example, but be not limited only to sf9 cell, adopt sf21, sf158, c127 cell line can obtain identical effect, in this not narration one by one).
Sf9 cell is incubated in Grace nutrient solution, adds 15% hyclone, penicillin 50U/ml, streptomysin 50ug/ml, and transfection method is as follows:
(a) the previous day is assigned to 24 orifice plates by cell in transfection;
(b) first 2 hours of transfection, changes fresh serum-free, without dual anti-nutrient solution, washes once, then adds 500 μ l nutrient solutions;
(c) at 50 μ l serum-frees, add respectively 2 μ l liposomes, the aforesaid recombinant plasmid of 5 μ l in without dual anti-nutrient solution, room temperature is placed 5 minutes, and plasmid is joined in liposome, and room temperature is placed 20 minutes;
(e) potpourri of previous step is added in cell to 27 DEG C of cultivations;
(f) transfection, after 4 hours, sops up nutrient solution, adds 1ml newly to rarely have serum, has dual anti-Grace nutrient solution, cultivates 72 hours for 27 DEG C;
(g) transfection, after 72 hours, is observed and whether is had malicious sign;
(h) will there is baculoviral supernatant to infect new sf9 cell.
(4) purifying of insect expressing protein (Ni column adsorption method)
(a) preparation of Ni post
(I) gets 1ml Ni-NTA in a centrifuge tube, centrifugal 5 minutes of 1500g;
(II) removes supernatant, and Ni-NTA is resuspended with the lavation buffer solution (20mM Tris-HCl, 500mM KCl, 20mM imidazoles, 2mM mercaptoethanol, 10% glycerine, pH8.5) of 1 times of volume;
(III) by resuspended liquid to entering in post, by the wash buffer balance of 5-10 times of volume.
(b) preparation of cell extract
(I) 500g collects 50ml cell for centrifugal 5 minutes;
(II) uses lysis buffer (50mM Tris-HCl, 100mM KCl, 20mM imidazoles, 5mM mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, pH8.5) re-suspended cell;
(III) 10, centrifugal 10 minutes of 000g, transfers to supernatant in one new pipe.
(c) protein purification
(I) is added to the supernatant of previous step in the Ni post that balance is good;
(II) washes post with the buffer A (20mM Tris-HCl, 500mM KCl, 20mM imidazoles, 5mM mercaptoethanol, 10% glycerine, pH8.5) of 10 times of volumes;
(III) washes post with the buffer B (20mM Tris-HCl, 1M KCl, 20mM imidazoles, 5mM mercaptoethanol, 10% glycerine, pH8.5) of 2 times of volumes;
(IV) washes post with the buffer A of 2 times of volumes;
(V), with 5-10 times of buffer C (20mM Tris-HCl, 100mM KCl, 100mM imidazoles, 5mM mercaptoethanol, 10% glycerine, pH8.5) wash-out, collects eluent;
(VI) detects concentration and the purity of purified albumen with SDS-PAGE glue.
(5) preparation of VEGF monoclonal antibody, purifying and mark
Taking mouse as immune animal, restructuring VEGF is that antigen carries out immunity.Tire according to mouse immune, select the animal that immunizing potency is the highest, get spleen and carry out Fusion of Cells, adopt indirect ELISA method to carry out the screening of positive hybridoma cell strain.Select the positive hole of a cell mass and carry out limiting dilution, only have this clone cell group in a hole, central authorities are intensive circularly to be spread out to surrounding.Treat all like this and whole test positive in all holes of limiting dilution, can think and clone.Secreting by the hybridoma that wherein single cell is originated the antibody obtaining is monoclonal antibody.Caprylic acid-ammonium sulfate precipitation method monoclonal antibody purification, purity is more than 95%.
Adopt glutaraldehyde method by horseradish peroxidase (HRP) mark on VEGF monoclonal antibody.
(6) preparation of kit
The coated employing following steps of elisa plate: antigen dilutes certain multiple with coated damping fluid, and every hole adds 100 μ l.In 37 DEG C of water-baths or incubator coated 2 hours, be then placed on ambient temperature overnight.PBST cleansing solution is filled it up with in every hole, places about 30 seconds, dries.Every hole adds 120 μ l confining liquids, and room temperature is placed 2 hours.Confining liquid formula mainly contains BSA, some salts etc.Get rid of confining liquid, pat and remove remaining confining liquid.On super-clean bench, dry up.Or super-clean bench blowing up a few hours, then room temperature is placed and is spent the night.Pack with vacuum packing machine.
Embodiment 2: kit using method
1. from taking out required lath with balance to the sealing bag of room temperature, other lath please seal puts back to 4 DEG C.
2. staying a hole is blank well, and other each holes add sample diluting liquid 100 μ l(or 2).
3. except blank well, add negative control 1 hole, positive control 2 holes, the each 100 μ l of serum specimen to respective aperture, flick and mix.Seal reacting hole with shrouding gummed paper, 37 DEG C 60 minutes.
4. wash plate 3 times: (1) automatic washer: get rid of liquid in most hole, requiring the cleansing solution injecting is 350 μ l, inject the second with sucking-off interval 15-30, wash plate 3 times.(2) wash plate by hand: get rid of liquid in most hole, every hole adds cleansing solution 350 μ l, leave standstill and get rid of most liquid after 30 seconds, repeatedly on thieving paper, pat dry thick, wash 3 times.
5. except blank well, the enzyme-added thing 200 μ l(in every hole or 4), seal plate hole, 37 DEG C 60 minutes.
6. wash plate 3 times.
7. chromogenic reaction: by developer A, the each 100 μ l(of B liquid or 2) be added in 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), mix, measure 450nmOD value.
The present invention has advantages of:
1. result high specificity.It is the coated elisa plate of associating encrusting substance that the present invention adopts vegf receptor and monoclonal antibody, and therefore, the specificity that detects VEGF is high.
2. highly sensitive.Detection sensitivity can be got to ng/L level.
4. stable.The present invention is in 4 DEG C of preservations, and the term of validity reaches 9-12 month.
5. convenience.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 (3)
1. a preparation method for elisa kit for detecting, is characterized in that,
Consisting of of described elisa kit for detecting: elisa plate, positive control, negative control, sample diluting liquid, enzyme connection thing, concentrated cleaning solution, developer A & B, stop buffer and shrouding gummed paper;
Wherein:
Elisa plate is the enzyme-linked reaction plate of vascular endothelial growth factor receptor and monoclonal antibody combined packet quilt;
Positive control is vascular endothelial growth factor protein freeze-dried powder;
Negative control is animal blood serum freeze-dried powder;
Sample diluting liquid is 0.05% Tween-20,1% bovine serum albumin(BSA), 10mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4MNaCl, pH6.5;
Concentrated cleaning solution is 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, 0.05% Tween-20, pH7.4;
Developer A, B, wherein A liquid is H
2o
2solution, B liquid is 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution;
Stop buffer is 2M H
2sO
4;
Enzyme connection thing is horseradish peroxidase-labeled vascular endothelial growth factor monoclonal antibody, this monoclonal antibody is prepared as follows, purifying and mark: taking mouse as immune animal, restructuring VEGF is that antigen carries out immunity, tire according to mouse immune, select the animal that immunizing potency is the highest, get spleen and carry out Fusion of Cells, adopt indirect ELISA method to carry out the screening of positive hybridoma cell strain, select the positive hole of a cell mass and carry out limiting dilution, in a hole, only has this clone cell group, central authorities are intensive circularly to be spread out to surrounding, treat all like this and whole test positive in all holes of limiting dilution, can think and clone, secreting by the hybridoma that wherein single cell is originated the antibody obtaining is monoclonal antibody, caprylic acid-ammonium sulfate precipitation method monoclonal antibody purification, purity is more than 95%, adopt glutaraldehyde method by horseradish peroxidase (HRP) mark on VEGF monoclonal antibody,
Wherein, the preparation method of this elisa kit for detecting comprises following key step:
(a) vascular endothelial growth factor receptor of gene engineering expression and monoclonal antibody are coated on solid support enzyme-linked reaction plate;
(b) seal with confining liquid;
Wherein, the described monoclonal antibody of step (a) is with vascular endothelial growth factor immune mouse gained; The vascular endothelial growth factor receptor insect cell expression of the gene engineering expression described in step (a); Described insect cell is sf9, sf21, sf158 or c127 cell line.
2. preparation method according to claim 1, is characterized in that, the vascular endothelial growth factor receptor of described insect cell expression is purified with Ni post.
3. preparation method according to claim 2, is characterized in that, described elisa plate is 96 holes.
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| CN102778452A (en) * | 2012-08-08 | 2012-11-14 | 北京健平九星生物医药科技有限公司 | Chemiluminescence detection kit and preparation method thereof |
| CN102830235B (en) * | 2012-08-28 | 2015-08-12 | 邹检平 | A kind of luminescence detection kit and preparation method thereof |
| CN108931638A (en) * | 2017-05-23 | 2018-12-04 | 北京健平金星生物科技有限公司 | A kind of colloid gold chromatographic test paper strip and preparation method thereof |
| WO2019074886A1 (en) | 2017-10-09 | 2019-04-18 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| CN110031616B (en) * | 2019-04-03 | 2020-07-28 | 浙江众意生物科技有限公司 | Detection kit for auxiliary diagnosis of diseases |
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