CN102854322A - Vascular endothelial growth factor (VEGF) receptor enzyme linked diagnosis kit and preparation method thereof - Google Patents
Vascular endothelial growth factor (VEGF) receptor enzyme linked diagnosis kit and preparation method thereof Download PDFInfo
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- CN102854322A CN102854322A CN2012102713485A CN201210271348A CN102854322A CN 102854322 A CN102854322 A CN 102854322A CN 2012102713485 A CN2012102713485 A CN 2012102713485A CN 201210271348 A CN201210271348 A CN 201210271348A CN 102854322 A CN102854322 A CN 102854322A
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Abstract
The invention discloses a vascular endothelial growth factor (VEGF) receptor enzyme linked diagnosis kit. A VEGF receptor coated enzyme linked reaction plate, an enzyme conjugate prepared through a horse radish peroxidase (HRP)-marked monoclonal antibody and a protecting agent, VEGF protein freeze-dried powder serving as positive comparison, protein freeze-dried powder serving as negative comparison, sample diluting liquid, PBST concentrated washing liquid, a chromogenic substrate prepared through 3,3',5,5'-tetramethyl benzidine, 2MH2S04 serving as stop buffer and shrouding adhesive paper are arranged in a kit body. The kit can be used for diagnosing and prognosis of diseases related to VEGF receptors such as cancer, angiopathy, diabetes retina disease and rheumatoid arthritis, has the advantages of being accurate, special, flexible, stable, convenient and the like, and has wide application prospects.
Description
Technical field
The invention belongs to the biological medicine technology field, relate in particular to elisa kit for detecting of a kind of detection human vascular endothelial growth factor (VEGF) and preparation method thereof.
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 Prognostic significance is rapid 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 of regulating new Angiogenesis.Studies show that vascular endothelial growth factor (VEGF) has irreplaceable effect in the Development And Differentiation of vascular system.
VEGF is a basic protein that molecular weight is the high glycosylation of 45KD, has two identical subunits to form by disulfide-bonded, and isoelectric point is 8.5, and very strong heat-resisting and acid-fast ability is arranged.Human VEGF gene has 5 kinds of different isomeride, is respectively VEGF206, VEGF189, VEGF165, VEGF145, VEGF121.The VEGF high affinity combined sites only is positioned at 3 kinds of vegf receptors of sunlight on the vascular endothelial cell, i.e. VEGFR-1, VEGFR-2, VEGFR-3, and these 3 kinds of acceptors mainly are distributed in Surface of Vascular Endothelial Cells, all are transmembrane receptors.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.Lot of documents report is arranged, and VEGF is obviously different with concentration among the cancer patient the normal person, and the VEGF concentration among the normal person is very low or can't detect, and VEGF is dense in the cancer patient.Therefore, detect the 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 large, 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.Therefore VEGF must detect the index that can be used as 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.
About the biological detecting method of VEGF, there are the problems such as sensitivity is lower, stability is not good in the prior art.
Summary of the invention
For the problems referred to above that prior art exists, the purpose of this invention is to provide the detection kit of a kind of enzyme linked immunosorbent detection human vascular endothelial growth factor (VEGF).Kit provided by the invention as a result accurate stable, high specificity, highly sensitive, easy to use, be convenient to promote.
Another object of the present invention provides manufacture and the using method of above-mentioned elisa kit for detecting.
For achieving the above object, the invention provides a kind of vegf receptor enzyme connection diagnostic kit, comprising: 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 coated enzyme-linked reaction plate of vascular endothelial growth factor (VEGF) acceptor;
Enzyme connection thing is the horseradish peroxidase-labeled vascular endothelial growth factor monoclonal antibody.
As further preferred version, described positive control is the vascular endothelial growth factor protein freeze-dried powder;
Described negative control is the animal blood serum freeze-dried powder;
Described sample diluting liquid is 0.05% Tween-20～80, l% bovine serum albumin(BSA), l0mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4M NaCl, pH6.5;
Described concentrated cleaning solution is 137mM NaCl, 2.7mM KC1,10mMNa2HP04,2mMKH
2P0
4, 0.05% polysorbas20, Ph7.4:
Described developer A liquid is H
2O
2Solution, described developer B liquid is TMB solution;
Described stop buffer is 2MH
2S0
4Solution.
As further preferred version, above-mentioned each amount of substance is take the hole count of elisa plate as standard.
As further preferred version, described elisa plate is 96 holes.
A kind of preparation of above-mentioned vegf receptor elisa kit for detecting and method of operating, key step is:
(a) vascular endothelial growth factor receptor is coated on the solid support enzyme-linked reaction plate;
(b) seal with confining liquid;
(c) special biological sample joins in the pre-coated enzyme-linked reaction plate reacting hole, and the vascular endothelial growth factor in the biological sample is combined with vascular endothelial growth factor receptor specifically;
(d) detect vascular endothelial growth factor with the vascular endothelial growth factor monoclonal antibody;
(e) chromogenic substrate is TMB solution.
As further preferred version, the described vascular endothelial growth factor receptor insect cell expression of step a.
As further preferred version, the described biological sample of step c is serum, blood plasma, cerebrospinal fluid, saliva, tissue extract or the urine of cancer, angiosis, diabetic retinopathy and rheumatoid arthritis bion.
As further preferred version, the described vascular endothelial growth factor monoclonal antibody of steps d is with vascular endothelial growth factor immune mouse gained.
As further preferred version, the described monoclonal anti body and function of steps d horseradish peroxidase-labeled.
The uniqueness of kit of the present invention is, its uses the coated elisa plate of vegf receptor, can guarantee that VEGF can be detected by this kit, can have accurately sample and measure.VEGF in this kit detection of biological sample is preferably from cancer, angiosis patient or other diseases patient's sample.The develop of the method for the invention has great significance for detection and the prognosis of the identical disease of vegf receptor, the method is with a wide range of applications and social demand also for control, early screening, the diagnostic work of vegf receptor disease provide accurate, easy, effective monitoring means simultaneously.In a word, active adaption of the present invention the need of work in the fields such as modern biology, prevention and health care, clinical diagnosis and the needs of human nature service, so its application prospect is very wide.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that following embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Ratio and number percent are based on weight, unless stated otherwise.
The preparation of embodiment 1VEGF acceptor enzyme connection diagnostic kit
Kit of the present invention can be following material for example:
(1) the coated elisa plate of vegf receptor;
(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: the 0.050r6 polysorbas20, the lor6 bovine serum albumin(BSA), the 10mM disodium ethylene diamine tetraacetate, the 0.050r6 thimerosal, 0.4M NaCl, pH6.5,1 bottle,
(5) enzyme connection thing: horseradish peroxidase-labeled vascular endothelial growth factor monoclonal antibody, 1 bottle;
(6) concentrated cleaning solution is: 137mM NaCl, 2.7mM KC1,10mM Na2HP04,2rrrMKH2P04,0.050r6 polysorbas20, Ph7.4,1 bottle;
(7) developer A liquid level H.O。Solution, developer B liquid are 3,37,5,5 tetramethyl biphenyl amine aqueous solutions, each 1 bottle;
(8) stop buffer is 2MH, s04,1 bottle;
(9) the shrouding gummed paper is 2.
Above-mentioned various amount all take elisa plate as 96 holes (12 8 holes or 8 12 holes) calculate as example, if the hole count of elisa plate greater or less than 96 holes, then each amount also should be more or less corresponding.
The preparation method of above-mentioned vegf receptor enzyme connection diagnostic kit:
(1) VEGF gene and vegf receptor gene cloning
Carry out PCR (PCR) take the human placenta cDNA library as template.According to the report primers of VEGF gene, part is held a memorial ceremony in PCR reaction: 95 ℃ of sex change 40 seconds, and 52 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, 30 rear 72 ℃ of insulations of circulation 10 minutes.
Reaction system:
The fragment of pcr amplification is reclaimed in gel electrophoresis, is cloned among the carrier pMT18-T, transforms escherichia coli DH5a, and the extraction recombinant plasmid carries out enzyme and cuts evaluation.The recombinant plasmid called after bVT18-1 of VEGF gene, the recombinant plasmid called after pMT18-2 of vegf receptor gene.Dna sequence analysis is finished by the rich fertile bioengineering company limited in Shanghai.
(2) structure of Vero fibrocyte expression vector
With Sal I, BamH 1 difference digested plasmid pMT18-1 and pMT18-2, reclaim respectively VEGF genetic fragment and vegf receptor genetic fragment.Use simultaneously Sal I, BamHI digested plasmid pFastBac-HT, reclaim the 6.7kb fragment.The VEGF genetic fragment is connected with the 6.7kb fragment with the vegf receptor genetic fragment is connected carrier construction pFastBac-1 and pFastBac-2.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 DHlOBac 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 2mLLB nutrient culture media, 37 ℃, 250rpm shaken cultivation 12 hours.
The plasmid method of extracting in the bacterium liquid of cultivating is as follows:
(a) 1.5mL bacterium liquid is changed in the centrifuge tube, the centrifugal 30s of 10000rpm abandons supernatant, and centrifuge tube is stood upside down on paper handkerchief, makes bacterial precipitation dry as far as possible;
(b) add 300uL solution I (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH8.O), mixing vibrates on the earthquake device;
(c) add the solution II (0.2N NaOH, lor6SDS) that 300uL now joins, mixing, room temperature was placed 5 minutes;
(d) add the solution III (5M KAc, pH5.5) of 300uL precooling, ice bath 5 minutes;
(e) 12000rpm is centrifugal 15 minutes;
(f) shift in the new centrifuge tube of supernatant to, add the absolute ethyl alcohol of 2 times of volumes, mixing;
(g) 10000-12000rpm is centrifugal 10 minutes;
(h) with 70% and absolute ethanol washing precipitation;
(i) dry up, be dissolved among the TE (lOmM Tris-HCl, ImM EDTA, pH8.0) that 50uL adds RNase, placed one hour in 37 ℃, place 20 ℃ for subsequent use.
The plasmid that the extracts performing PCR that spouts is identified, the PCR reaction is according to aforesaid method.
(3) transfection Vero cell
The Vero cell is incubated in the Grace nutrient solution, adds 150r6 hyclone, penicillin 50U/mL, streptomysin 50ug/mL, and transfection method is as follows:
(a) the previous day is assigned to 24 orifice plates with cell in transfection;
(b) transfection is front 2 hours, changes fresh serum-free, unparalleled anti-nutrient solution, washes once, then adds the 500uL nutrient solution:
(c) add respectively 2uL liposome, the aforesaid recombinant plasmid of 5uL 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;
(e) potpourri with previous step is added in the cell, 27 ℃ of cultivations;
(f) transfection sopped up nutrient solution after 4 hours, adds the 1mL fresh bovine serum, two anti-Grace nutrient solutions are arranged, and cultivated 72 hours for 27 ℃;
(g) transfection is after 72 hours, and whether observe has malicious sign;
(h) will there be the baculoviral supernatant to infect new Vero cell.
(4) purifying of Vero expressing protein (Ni column adsorption method)
(a) preparation of Ni post
(I) get ImLNi-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, 500mMKC1, the 20mM imidazoles, the 2mM mercaptoethanol, 10% glycerine, pH8.5) resuspended;
(III) with resuspended liquid to entering in the post, with the wash buffer balance of 5～10 times of volumes.
(b) preparation of cell extract
(1) 500g collected the 50mL cell in centrifugal 5 minutes;
(II) with lysis buffer (50mM Tris-HCl, l00mM KC1,20mM imidazoles, 5mM mercaptoethanol, 1mM phenylmethylsulfonyl fluoride, pH8.5) re-suspended cell;
(III) 10000g is centrifugal 10 minutes, and supernatant is transferred in the new pipe.
(c) protein purification
(I) supernatant with previous step is added in the good Ni post of balance;
(II) with the bufferA of 10 times of volumes (20mM Tris-HCl, 500mM KC1, the 20mM imidazoles, the 5mM mercaptoethanol, 100r6 glycerine pH8.5) is washed post;
(III) with the buffer B of 2 times of volumes (20mM Tris-HCl, 1MKC1, the 20mM imidazoles, the 5mM mercaptoethanol, 10% glycerine pH8.5) is washed post;
(IV) bufferA with 2 times of volumes washes post;
(V) with 5～10 times of buffer C (20mM Tris-HCl, lOOmM KC1, lOOmM imidazoles, 5mM mercaptoethanol, 10% glycerine, pH8.5) wash-out, collection eluent;
(VI) detect concentration and the purity of purified albumen with SDS-PAGE glue.
(5) preparation of VEGF monoclonal antibody, purifying and mark
Take mouse as immune animal, restructuring VEGF is that antigen carries out immunity.
Tire according to mouse immune, select the highest animal of immunizing potency, 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 in the hole～individual clone cell group, central authorities intensive circular to around spread out.Treat all like this and whole test positive in all holes of limiting dilution, can think that the clone finishes.Secreting resulting antibody by the hybridoma in the source of single cell wherein is monoclonal antibody.Caprylic acid ammonium sulfate precipitation method monoclonal antibody purification, purity is more than 95%.
Adopt glutaraldehyde method with horseradish peroxidase (HRP) mark on the VEGF monoclonal antibody.
(6) preparation of kit
The coated employing following steps of elisa plate: antibody dilutes certain multiple with coated damping fluid, and every hole adds 100uL.
In 37 ℃ of water-baths or the incubator coated 2 hours, then be placed on ambient temperature overnight.The PBST cleansing solution is filled it up with in every hole, places about 30 seconds, dries.Every hole adds the 120u1 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 super-clean bench blowing up a few hours, then room temperature is placed and is spent the night.Pack with vacuum packing machine.
The using method of embodiment 2VEFG enzyme connection diagnostic kit
1. from taking out required lath with 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 100uL (or 2).
3. except blank well, add negative control 1 hole, positive control 2 holes, each 100uL 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 350uL, and plate is washed 3 times in injection and sucking-off interval 1530 seconds.(2) wash plate by hand: get rid of liquid in the most hole, every hole adds cleansing solution 350uL, leaves standstill after 30 seconds and gets rid of most liquid, pats dry at thick repeatedly thieving paper, washes 3 times.
5. except blank well, the enzyme-added thing 200uL in every hole (or 4) seals plate hole, 37 ℃ 60 minutes.
6, wash plate 3 times.
7. chromogenic reaction: developer A, each 100ul of B liquid (or 2) 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 50uL (or 1), and mixing is measured the 450nmOD value.
The detection of the embodiment 3 kit ranges of linearity, linearity, specificity, sensitivity, accuracy, accuracy, repeatability
1. specificity
Evaluation on specificity: the vegf receptor detection kit of assembling is for detection of the VEFG acceptor, and the result shows that the specificity of this kit is very high, and is specifically as shown in table 1.
Table 1 detects vegf receptor antigenic content method specific test 450nm absorbance result
The result shows: the specificity of this detection kit is high, does not intersect with multiple acceptors such as TNF-α, IL 22.
2. sensitivity
Detect as shown in table 2 below:
The sensitivity of table 2VEGF receptor antigen detection method
The result shows: content is when 1000-5ng/mL, and OD value 〉=negative control * 2.1, the detection sensitivity of the method are 5ng/mL sensing range and linearity:
Table 3VEGF is subjected to body detecting method linear test absorbance result
The result shows: content when 5-1000ng/mL, continuous 9 point Linears 〉=0.97, the acceptor scope 5ng～1000ng/mL of detection.
3. accuracy
The dilution vegf receptor makes its theoretical content be followed successively by 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1000ng/mL, detects the antigenic content of above-mentioned dilution.
Table 4VEGF is subjected to the body detecting method accuracy testing
The result shows: the 200ng/mL accuracy is: 91%～108%; The 400ng/mL accuracy is: 96%～108%; The 600ng/mL accuracy is: 92%～111%; The 800ng/mL accuracy is: 92%～110%; The 1000ng/mL accuracy is: 92%～109%.Accuracy is between 90%～120%, and exact value is higher.
4. accuracy
According to the content receptor check result, judge the coefficient of variation.
Table 5VEGF is tested by the body detecting method accuracy
The result shows: the variation lines number average is less than 10%, and the coefficient of variation is less, and accuracy is high.
5. repeated
Table 11VEGF is subjected to the body detecting method replica test
The result shows: double revision test shows: cv value＜10% has preferably repeatability.
To sum up state test findings and show, vegf receptor diagnostic kit high specificity provided by the invention, highly sensitive.Detection sensitivity can reach the ng/L level, and stable, the present invention is valid up to 9～12 months in 4 ℃ of preservations, and easy to use, required detecting instrument (microplate reader) generally uses at various big hospital and inspection center.
Be necessary at last in this explanation to be: above embodiment only is used for technical scheme of the present invention is described in more detail; can not be interpreted as limiting the scope of the invention, some nonessential improvement that those skilled in the art's foregoing according to the present invention is made and adjustment all belong to protection scope of the present invention.
Claims (9)
1. vegf receptor elisa kit for detecting, it is characterized in that, described vegf receptor enzyme connection diagnostic kit comprises: 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 coated enzyme-linked reaction plate of vascular endothelial growth factor (VEGF) acceptor;
Enzyme connection thing is the horseradish peroxidase-labeled vascular endothelial growth factor monoclonal antibody.
2. vegf receptor elisa kit for detecting according to claim 1 is characterized in that:
Described positive control is the vascular endothelial growth factor protein freeze-dried powder;
Described negative control is the animal blood serum freeze-dried powder;
Described sample diluting liquid is 0.05% Tween-20～80, l% bovine serum albumin(BSA), l0mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4M NaCl, pH6.5;
Described concentrated cleaning solution is 137mM NaCl, 2.7mM KC1, l0mM Na2HP04,2mMKH
2P0
4, 0.05% polysorbas20, Ph7.4:
Described developer A liquid is H
2O
2Solution, described developer B liquid is TMB solution;
Described stop buffer is 2MH
2S0
4Solution.
3. vegf receptor elisa kit for detecting according to claim 1 is characterized in that: each amount of substance is take the hole count of elisa plate as standard.
4. vegf receptor elisa kit for detecting according to claim 1, it is characterized in that: described elisa plate is 96 holes.
5. the preparation of a vegf receptor elisa kit for detecting claimed in claim 1 and method of operating is characterized in that, key step is:
(a) vascular endothelial growth factor receptor is coated on the solid support enzyme-linked reaction plate;
(b) seal with confining liquid;
(c) special biological sample joins in the pre-coated enzyme-linked reaction plate reacting hole, and the vascular endothelial growth factor in the biological sample is combined with vascular endothelial growth factor receptor specifically;
(d) detect vascular endothelial growth factor with the vascular endothelial growth factor monoclonal antibody;
(e) chromogenic substrate is TMB solution.
6. the preparation of vegf receptor elisa kit for detecting according to claim 5 and method of operating is characterized in that: the described vascular endothelial growth factor receptor insect cell expression of step a.
7. the preparation of vegf receptor elisa kit for detecting according to claim 5 and method of operating, it is characterized in that: the described biological sample of step c is serum, blood plasma, cerebrospinal fluid, saliva, tissue extract or the urine of cancer, angiosis, diabetic retinopathy and rheumatoid arthritis bion.
8. the preparation of vegf receptor elisa kit for detecting according to claim 5 and method of operating is characterized in that: the described vascular endothelial growth factor monoclonal antibody of steps d is with vascular endothelial growth factor immune mouse gained.
9. the preparation of vegf receptor elisa kit for detecting according to claim 5 and method of operating is characterized in that: the described monoclonal anti body and function of steps d horseradish peroxidase-labeled.
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Application publication date: 20130102 |