CN102207504A - Enzyme-linked immunosorbent assay kit, and preparation method thereof - Google Patents
Enzyme-linked immunosorbent assay kit, and preparation method thereof Download PDFInfo
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- CN102207504A CN102207504A CN201110069600XA CN201110069600A CN102207504A CN 102207504 A CN102207504 A CN 102207504A CN 201110069600X A CN201110069600X A CN 201110069600XA CN 201110069600 A CN201110069600 A CN 201110069600A CN 102207504 A CN102207504 A CN 102207504A
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Abstract
The invention discloses an enzyme-linked immunosorbent assay (ELISA) kit for detecting vessel endothelial growth factors (VEGF). The kit comprises: an enzyme-linked reaction plate coated with the combination of VEGF acceptors and monoclonal antibodies, enzyme conjugates prepared with horseradish peroxidase (HRP) labeled antibodies and a protective agent, VEGF protein lyophilized powder as a positive control, protein lyophilized powder as a negative control, a sample dilution, PBST concentrated washing liquor, a chromogenic substrate prepared with 3,3<,>,5,5<,>-tetramethylbiphenyl, 2MH2SO4 as a stop buffer, and plate sealing gum paper. The kit can be used in the diagnosis and prognosis of cancer, angiopathy, diabetic retinopathy, and rheumatoid arthritis, with advantages of accurate, specific, sensitive, stable, and convenient.
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 is the necessary condition of tumour generation, transfer and metastasis growth, vascular endothelial growth factor (VEGF) is to regulate the most important cell factor that neovascularity generates.Studies show that vascular endothelial growth factor (VEGF) 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, has two identical subunits to form by disulfide-bonded, and isoelectric point is 8.5, and very strong heat-resisting and acidproof 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 on the vascular endothelial cell, i.e. VEGFR-1, VEGFR-2, VEGFR-3, and these 3 kinds of acceptors mainly are distributed in the vascular endothelial cell surface, 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, 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 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 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 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.
(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) has reported with colorimetric and send out ELISA method into the detection VEGF on basis, 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 the normal person is 8-36ng/l.
Summary of the invention
The detection kit that the purpose of this invention is to provide a kind of enzyme linked immunosorbent detection 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 preparation method of above-mentioned elisa kit for detecting.
For achieving the above object, the invention provides a kind of kit, it consists of:
1. elisa plate; 2. positive control (freeze-drying); 3. negative control (freeze-drying); 4. sample diluting liquid; 5. enzyme joins thing; 6. concentrated cleaning solution; 7. developer A﹠amp; B; 8. stop buffer; 9. shrouding gummed paper.
The invention provides a kind of preparation method of kit, comprise the steps:
(a) with the enzyme-linked reaction plate be solid support, with the vegf receptor of gene engineering expression and monoclonal antibody bag by on solid support;
(b) seal with confining liquid;
(c contacts biological sample and be incubated with vegf receptor and monoclonal antibody on being fixed in elisa plate, and the VEGF in the biological sample combines with vegf receptor and monoclonal antibody specifically;
(d) separating bio sample on vegf receptor and the monoclonal antibody;
(e) the VEGF polyclonal antibody with 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 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 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.
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 be 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-20,1% 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 polyclonal antibody, 2 bottles;
(6) concentrated cleaning solution is: 137mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 2mMKH
2PO
4, 0.05% Tween-20, Ph7.4,1 bottle;
(7) developer A, B, wherein A liquid level H
2O
2Solution, B liquid is 3,3 ', 5,5 '-the tetramethyl biphenyl amine aqueous solution, each 1 bottle;
(8) stop buffer is 2MH
2SO
4, 1 bottle;
(9) 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 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 (PCR).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.
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 Sal I, BamHI difference digested plasmid pMT18-1 and pMT18-2, reclaim VEGF genetic fragment and vegf receptor genetic fragment respectively.Use Sal I, BamH I 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 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, 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;
(b) add 300 μ l solution I (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH8.0), mixing vibrates on the earthquake device;
(c) add the solution II that 300 μ l now join (0.2N NaOH, 1%SDS), mixing, room temperature was placed 5 minutes;
(d) add 300 μ l precoolings solution III (5M KAc, pH5.5), 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, mixing;
(g) 10,000-12, centrifugal 10 minutes of 000rpm;
(h) with 70% and absolute ethanol washing precipitation;
(i) dry up, be dissolved in TE (the 10mM Tris-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, 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:
(a) the previous day is assigned to 24 orifice plates with cell in transfection;
(b) transfection is preceding 2 hours, changes fresh serum-free, unparalleled anti-nutrient solution, washes once, adds 500 μ l nutrient solutions then;
(c) add 2 μ l liposomes, the aforesaid recombinant plasmid of 5 μ l respectively in 50 μ l serum-frees, 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) previous step is rapid potpourri is added in the cell, 27 ℃ of cultivations;
(f) 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 ℃;
(g) transfection is after 72 hours, and whether observe has malicious sign;
(h) will there be the baculoviral supernatant to infect new sf9 cell.
(4) purifying of insect expressing protein (Ni column adsorption method)
(a) 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, the 20mM imidazoles, 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.
(b) preparation of cell extract
(I) 500g collected the 50ml cell in centrifugal 5 minutes;
(II) with lysis buffer (50mM Tris-HCl, 100mM KCl, 20mM imidazoles, 5mM mercaptoethanol, 1mM phenylmethylsulfonyl fluoride, pH8.5) re-suspended cell;
(III) 10, centrifugal 10 minutes of 000g transfers to supernatant in the one new pipe.
(c) protein purification
(I) previous step is rapid supernatant is added in the good Ni post of balance;
(II) with the buffer A of 10 times of volumes (20mM Tris-HCl, 500mM KCl, the 20mM imidazoles, the 5mM mercaptoethanol, 10% glycerine pH8.5) is washed post;
(III) with the buffer B of 2 times of volumes (20mM Tris-HCl, 1M KCl, the 20mM imidazoles, 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 imidazoles, 5mM mercaptoethanol, 10% glycerine, pH8.5) wash-out, collection eluent;
(VI) detect the concentration and the purity of purified albumen with SDS-PAGE glue.
(5) VEGF MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
With the mouse is immune animal, and reorganization VEGF165 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, have only this clone cell group in the hole, 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%.
(6) VEGF Polyclonal Antibody Preparation, purifying and mark
Stimulate animal with the injection of 5-10mg Bacille Calmette-Guerin with rabbit earlier as immune animal.After one week vegf protein is made 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.
(7) preparation of kit
The bag of elisa plate is used following steps: antigen is cushioned the certain multiple of liquid dilution with bag, and every hole adds 100 μ l.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 120 μ l confining liquids, 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. 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 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 require to inject is 350 μ l, injects and sucking-off 15-30 second 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. high specificity as a result.The present invention adopt vegf receptor and monoclonal antibody for associating encrusting substance bag by elisa plate, therefore, the specificity height of detection VEGF.
2. highly sensitive.Detection sensitivity can be got to 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.
Clinical practice of the present invention
Adopt the present invention to detect 33 routine patients with lung cancer and 46 routine normal persons' serum sample.The result shows: sensitivity is 67%; Specificity is 80%; Crude agreement is 75%; Positive predictive value is 71%.As shown in table 1:
Table 1. vascular endothelial growth factor detection kit clinical practice result
Sensitivity (%)=A/ (A+C) * 100%=67%
Specificity (%)=D/ (D+B) * 100%=80%
Crude agreement (%)=(A+D)/(A+B+C+D) * 100%=75%
Positive predictive value (%)=A/ (A+B) * 100%=71%
Claims (13)
1. elisa kit for detecting is characterized in that this kit comprises:
Elisa plate, positive control, negative control, sample diluting liquid, enzyme connection thing, concentrated cleaning solution, developer, 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;
Enzyme connection thing is a horseradish peroxidase-labeled vascular endothelial growth factor polyclonal antibody.
2. according to the kit of claim 1, it is characterized in that:
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 is 0.05% Tween-20,1% bovine serum albumin(BSA), 10mM disodium ethylene diamine tetraacetate, 0.05% thimerosal, 0.4M NaCl, pH6.5;
Concentrated cleaning solution is 137mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 2mMKH
2PO
4, 0.05% Tween-20, Ph7.4;
Developer is developer A﹠amp; B, wherein A liquid is H
2O
2Solution, B liquid is 3,3 ', 5,5 '-the tetramethyl biphenyl amine aqueous solution;
Stop buffer is 2MH
2SO
4
3. 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.
4. according to the kit of claim 1, it is characterized in that described elisa plate is 96 holes.
5. the preparation method of an elisa kit for detecting is characterized in that may further comprise the steps:
(a) with the vascular endothelial growth factor receptor of gene engineering expression and monoclonal antibody bag 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 and monoclonal antibody specifically;
(d) detect vascular endothelial growth factor with the vascular endothelial growth factor polyclonal antibody;
(e) chromogenic substrate be 3,3 ', 5,5 '-the tetramethyl biphenyl amine aqueous solution.
6. according to the method for claim 5, it is characterized in that the described monoclonal antibody of step (a) is with vascular endothelial growth factor immune mouse gained.
7. according to the method for claim 5, it is characterized in that the vascular endothelial growth factor receptor insect cell expression of the described gene engineering expression of step (a).
8. according to the method for claim 7, it is characterized in that described insect cell is sf9, sf12, sf158 or c127 cell line.
9. according to the method for claim 7, it is characterized in that the vascular endothelial growth factor receptor of described insect cell expression Ni post purifying.
10. according to the method for claim 5, 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 patients.
11. the method according to claim 5 is characterized in that, the described vascular endothelial growth factor polyclonal antibody of step (d) prepares the vascular endothelial growth factor of usefulness and also uses Ni post purifying with insect cell expression.
12. the method according to claim 5 is characterized in that, the described polyclonal antibody of step (d) is with vascular endothelial growth factor immunizing rabbit gained.
13. the method according to claim 5 is characterized in that, the described polyclonal antibody horseradish peroxidase-labeled of step (d).
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