CN1260492A - Tumor prognosis evaluation kit and preparation process thereof - Google Patents
Tumor prognosis evaluation kit and preparation process thereof Download PDFInfo
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- CN1260492A CN1260492A CN 99115902 CN99115902A CN1260492A CN 1260492 A CN1260492 A CN 1260492A CN 99115902 CN99115902 CN 99115902 CN 99115902 A CN99115902 A CN 99115902A CN 1260492 A CN1260492 A CN 1260492A
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- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 22
- 238000011156 evaluation Methods 0.000 title claims abstract description 9
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- 238000004393 prognosis Methods 0.000 title claims abstract description 9
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims abstract description 24
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 239000013641 positive control Substances 0.000 claims abstract description 5
- 238000007789 sealing Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 12
- 238000002965 ELISA Methods 0.000 claims description 8
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- 230000004927 fusion Effects 0.000 claims description 4
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
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- 206010003445 Ascites Diseases 0.000 claims description 2
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- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
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- 238000002649 immunization Methods 0.000 claims description 2
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- 239000007924 injection Substances 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
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- 238000012216 screening Methods 0.000 claims description 2
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- 238000005406 washing Methods 0.000 abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 3
- 239000007853 buffer solution Substances 0.000 abstract 2
- 239000003292 glue Substances 0.000 abstract 1
- 239000003550 marker Substances 0.000 abstract 1
- 239000012089 stop solution Substances 0.000 abstract 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 5
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- 210000002700 urine Anatomy 0.000 description 5
- 101100372758 Danio rerio vegfaa gene Proteins 0.000 description 4
- 101150030763 Vegfa gene Proteins 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 4
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- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 description 2
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
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Abstract
The invention provides a tumor prognosis evaluation kit, which comprises a positive control (VEGF), normal saline, a secondary antibody marker, a sealing glue, an enzyme label plate buffer solution, a VEGF monoclonal antibody or polyclonal antibody, an enzyme substrate solution, a stop solution and a washing solution, and also provides a preparation process of the VEGF monoclonal antibody, other buffer solutions and reagents, and the kit can be quickly and simply used for tumor prognosis evaluation.
Description
The present invention relates to a kind of tumor prognosis evaluation reagent kit and preparation technology thereof.
The growth of normal structure medium vessels remains static, and only occurs adjustable, of short duration propagation in the organ of injured or cyclic regeneration; But in the tumor tissues, angiogenic growth is uncontrolled, continues to carry out.Tumour cell can produce a series of cell factors that promote the vascularization function that have under certain condition, as VEGF, and bFGF, ANG etc.In tumor vascular growth, play an important role.VEGF is by combine different its biologically active that is situated between with the vascular endothelial cell surface receptor, by plasminogen activation activator and fibrinolytic system, and degraded basilar memebrane composition, thus the stimulating endothelial cell migration, immerse, form tubular structure.
The generation of tumour, development, transfer must depend on the generation of new vessels, in tumor vascular forming process, the growth factors such as VEGF relevant with vascularization begins to express, after tumour is subjected to the chemotherapy and radiation effect, tumour cell is suppressed or damages together with the tumor vascular endothelial cell growth, and the vegf expression amount descends or do not express.The height of vegf expression amount is relevant with tumor size and growth conditions, and the knurl body is big, the tumor tissues vegf expression amount height fast of growing; And the knurl body is little, and the slow tumor tissues vegf expression amount of silk ribbon attached to an official seal or a medal of growing is low.And the knurl body is constant relatively, and it is relative constant to breed the tumor tissues VEGF suitable with apoptosis speed.Gross tumor volume, the speed of growth are subjected to the influence of factors such as patient's state of an illness, methods of treatment, result of treatment, and the active situation and the fluctuation of these genes of monitoring cancer patient change the variation tendency that can indicate the state of an illness, and be significant.This kit provides the dynamic observing of vascularization gene molecule level of tumour patient for the clinician, for result of treatment, the PD of tumour patient, prognosis changes that basis for estimation is provided.
The object of the present invention is to provide the preparation technology of VEGF monoclonal antibody and other buffering agent in a kind of tumor prognosis evaluation ELISA kit and the kit thereof, reagent, can be used for the prognosis evaluation of tumour fast simply.
The every box of this kit is made up of following material:
Bag is cushioned 1 bottle of liquid
1 bottle of positive control (VEGF)
1 bottle of negative control (physiological saline)
1 bottle of the monoclonal antibody of VEGF or polyclonal antibody
1 bottle of two anti-label
1 bottle of enzyme substrate solution
1 bottle of stop buffer
1 bottle of cleansing solution
1 of sealing compound
1 of ELISA Plate
Wherein, positive control (VEGF), negative control (physiological saline), two anti-labels, sealing compound, ELISA Plate are existing material, and bag is cushioned the Monclone antibody of liquid, VEGF or restrains gallery antibody, enzyme substrate solution, stop buffer, cleansing solution for being prepared from by technology of the present invention more.
Preparation technology of the present invention realizes by following steps:
1. MONOCLONAL ANTIBODIES SPECIFIC FOR
1.1 in mouse immune BAL B/C mouse 6-8 week, add Fu Shi with 50-100ug VEGF
The Freund's complete adjuvant lumbar injection is behind the At intervals of two to three weeks, with same dosage VEGF abdomen
Chamber booster immunization 2-3 time, 3-4 days extracting spleen cells merge after the last immunity.
1.2 antigen VEGF, research institute provides by the auspicious gloomy genetically engineered drug in Shaanxi.
1.3 Fusion of Cells, immune mouse spleen cell and Sp2/0 cell mix in 10: 1 ratio
Close, under the effect of 50%PEG (MW=1500), carry out Fusion of Cells, use ELISA
The indirect method preliminary screening goes out positive colony, again with in and inhibition test turn out to be sun
After the sex clone, clone 2-3 time, filter out the secretory antibody sun through limiting dilution assay
The property single clone, stable going down to posterity 3 months, preparation ascites and liquid nitrogen cryopreservation.
1.4 the evaluation of monoclonal antibody
1.4.1 hybridoma cell line chromosome analysis: the hybridoma in the growth period of taking the logarithm
Handle with colchicine, through Giemsa dyeing, the microscopy counting.
1.4.2Ig class and IgG subclass are measured: with the anti-mouse Ig Asia of U.S. Sigma company
Class antiserum and the Hybridoma Cell Culture supernatant that is concentrated into former volume 1/20
Liquid is made the two-way immunodiffusion of agar.
1.4.3 the specific mensuration of monoclonal antibody: VEGF is carried out electrotransfer, then
Carry out ELISA dyeing respectively with each strain monoclonal antibody.
1.4.4 the monoclonal antibody specific activity is measured: the albumen of each strain monoclonal antibody is carried out quantitatively,
Measure the titre of each strain then by different dilutabilitys.
2. polyclone and clone ELISA measure the VEGF in the urine sample
2.1 the compound method of various damping fluids and reagent
A. bag is cushioned liquid: the Na of 0.05M PH9.6
2CO
3-NaHCO
3
Na
2CO
3(MW 105.99) 1.59 grams
NaHCO
3(MW 84.01) 2.93 grams
Distill water-soluble to 1000ml
B. the PBS-Tween 20 of cleansing solution: PH 7.4
NaCl (MW 58.44) 8.0 grams
KH
2PO
4(MW 136.09) 0.2 gram
Na
2HPO
412 H
2O (MW 358.14) 2.9 grams
(perhaps NaHPO
41.14 gram)
KCl (MW 74.56) 0.2 gram
Distill water-soluble to 1000ml
Tween?20?0.5ml
C. enzyme labeling thing dilution: add sheep blood serum in above-mentioned cleansing solution, concentration is 2%.
D. enzyme substrate solution: o-phenylenediamine (O.P.D)-H
2O
2
(1) phosphoric acid-citrate buffer solution PH5.0
①0.2M?Na
2HPO
4
Get Na
2HPO
412 H
2O 71.63 grams are water-soluble to 1000ml with distillation.
2. 0.1M citric acid solution
Get citric acid 19.2 grams, water-soluble with distillation to 1000ml, get 1. liquid
25.7ml, 2. liquid 24.3ml, adding distil water 50ml again.
(2)O.P.D-H
2O
2
O-phenylenediamine (O.P.D) 10mg
Phosphoric acid-citrate buffer solution 25ml
3%H
2O
2(analyzing pure) 0.4ml
E. stop buffer 2MH
2SO
4
The concentrated sulphuric acid (95-98%) 22.2ml
Distilled water 177.3ml
(timing slowly splashes into the concentrated sulphuric acid in the distilled water, and the limit edged shakes up.)
2.2 operation steps
Get monoclonal or polyclonal antibody, the parallel bag of normal mice IgG (all being diluted to 10mg/ml) is by 40 orifice plates (by monoclonal or polyclonal antibody, the B package is by normal mice IgG as the A package), 0.1ml/ wet 4 ℃ of refrigerator overnight of box are put in the hole, use cleansing solution wash plate 3 times, each 3 minutes, dry.Add tested antigen 0.1ml/ hole in the hole of parallel bag quilt, hatched 1 hour for 37 ℃, the same washing 3 times dries.Add monoclonal or polyclonal antibody (being diluted to 10mg/ml) again in each hole, hatched 1 hour for 37 ℃ in the 0.1ml/ hole, and the same washing 3 times dries, and adds enzyme target two anti-(being diluted to 1: 4000) in each hole, 0.1
Add enzyme substrate solution 0.1ml/ hole, 37 ℃ of lucifuges were hatched 20 minutes.At each
Add 2M H in the hole
2SO
40.025ml/ the hole, cessation reaction is surveyed with the enzyme joint inspection
Instrument is measured the absorbance of the 490nm in each hole.
2.3 the result judges
The OD ratio calculation:
Embodiment: one, kit is formed:
96 person-portions, 48 person-portions, 20 person-portions
Bag is cushioned liquid bottle 10ml 5ml 2.5ml
Positive control (VEGF) bottle 1ml 0.5ml 0.25ml
Negative control (physiological saline) bottle 1ml 0.5ml 0.25ml
The monoclonal antibody of VEGF
Or polyclonal antibody bottle 10ml 5ml 5ml
Two anti-label bottle 10ml 5ml 5ml
Enzyme substrate solution bottle 6ml 3ml 3ml
Stop buffer bottle 6ml 3ml 3ml
Washing liquid bottle 25ml 12.5ml 6ml
Seal 10 10 1 of blob of viscose
ELISA Plate piece 96 orifice plates 48 orifice plates 24 orifice plates two, specimen collection
1. urina sanguinis, dehydration and a large amount of transfusion patient should not be adopted urine.
2. best fresh urine sample can be preserved 48 hours for 4 ℃, can preserve for 4 weeks for-20 ℃.
3. urine can not multigelation.
Three, reagent is stored
1.4 ℃ preservation, the term of validity are 4-6 month.
2. coating buffer must cover tightly, 4 ℃ of preservations, for a long time need not, should more renew coating buffer.Four, detect step
1. taking-up ELISA Plate, every hole add 2 of coating buffers (wherein 3 holes do not add, directly add sun,
Each 2 of negative controls, a hole gives over to blank).
2 add patient's urine 10ul, with the coating buffer mixing.Add a cover and seal film, put wet box, 4 ℃
Spend the night or 37 ℃ more than 3.5-4 hour.
3 then take out, and inhale and remove coating buffer, and every hole adds cleansing solution 200ul, washes 3 times, and is each
Add leave standstill 1 minute after the washing lotion after, get rid of only, pat dry surplus liquid.
4 add one anti-2,37 ℃ of wet boxes 60 minutes.
5 with step 3, washes 3 times.
6 add two anti-2,37 ℃ of wet boxes 60 minutes.
7 with step 3, washes 3 times.
8 every holes drip substrate A and B each 1 (blank is not dripped), and mixing is put wet box 37
℃ * 30 minutes (or room temperature 45 minutes) lucifuges.Then, every hole adds stop buffer 1
Drip, survey the OD value at microplate reader 490mm place.Five, judged result
The OD ratio calculation:
Six, clinical assessment reference
1 sample OD ratio to be checked 〉=2.1 show vascularization peptide gene table
Active.
2 sample OD ratios to be checked are suspicious between 1.5-2.0.
3 sample OD ratios to be checked are below 1.5, and the expression of expression vascularization peptide gene is in quiet
The phase of ending.
Claims (3)
1, tumor prognosis evaluation ELISA kit comprises positive control (VEGF), physiological saline, two anti-labels, sealing compound, ELISA Plate, it is characterized in that, wherein also comprise the monoclonal antibody or polyclonal antibody, enzyme substrate solution, stop buffer and the cleansing solution that are cushioned liquid, VEGF.
2, kit according to claim 1 is characterized in that, said bag is cushioned the Na that liquid is 0.05M, PH9.6
2CO
3-NaHCO
3, said enzyme substrate solution is o-phenylenediamine (O.P.D)-H
2O
2, said stop buffer 2MH
2SO
4, said cleansing solution is the PBS-Tween 20 of PH 7.4.
3, kit according to claim 1 is characterized in that, the monoclonal antibody of said VEGF adopts following method preparation:
(1) in mouse immune BAL B/C mouse 6-8 week, adds Fu Shi with 50-100ug VEGF
The Freund's complete adjuvant lumbar injection is behind the At intervals of two to three weeks, with same dosage VEGF abdomen
Chamber booster immunization 2-3 time, 3-4 days extracting spleen cells merge after the last immunity;
(2) Fusion of Cells, immune mouse spleen cell and Sp2/0 cell mix in 10: 1 ratio
This mixing under the effect of 50%PEG (MW=1500), is carried out Fusion of Cells,
Go out positive colony with ELISA indirect method preliminary screening, in using again and inhibition test
After turning out to be positive colony, clone 2-3 time, filter out branch through limiting dilution assay
Secrete the single clone of antibody positive, stable going down to posterity 3 months, preparation ascites and liquid
Nitrogen is frozen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99115902 CN1260492A (en) | 1999-11-09 | 1999-11-09 | Tumor prognosis evaluation kit and preparation process thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99115902 CN1260492A (en) | 1999-11-09 | 1999-11-09 | Tumor prognosis evaluation kit and preparation process thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1260492A true CN1260492A (en) | 2000-07-19 |
Family
ID=5278791
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99115902 Pending CN1260492A (en) | 1999-11-09 | 1999-11-09 | Tumor prognosis evaluation kit and preparation process thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1260492A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316249C (en) * | 2004-08-25 | 2007-05-16 | 北京健平九星生物医药科技有限公司 | Enzyme-linked assay kit and producing process thereof |
| WO2011088740A1 (en) * | 2010-01-21 | 2011-07-28 | 北京大学 | Enzyme linked immunosorbent assay (elisa) method and kit for detecting soluble programmed cell death protein 5 (pdcd5) |
| CN102203606A (en) * | 2008-10-30 | 2011-09-28 | 公共健康研究中心 | Biomarkers |
| 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 |
| CN102426240A (en) * | 2011-09-19 | 2012-04-25 | 北京华创远航科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| CN102435743A (en) * | 2011-09-15 | 2012-05-02 | 北京华创远航科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| CN1973205B (en) * | 2004-06-24 | 2012-07-18 | 贝林格尔·英格海姆维特梅迪卡有限公司 | Kits for the diagnosis of Lawsonia intracellularis |
-
1999
- 1999-11-09 CN CN 99115902 patent/CN1260492A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1973205B (en) * | 2004-06-24 | 2012-07-18 | 贝林格尔·英格海姆维特梅迪卡有限公司 | Kits for the diagnosis of Lawsonia intracellularis |
| CN1316249C (en) * | 2004-08-25 | 2007-05-16 | 北京健平九星生物医药科技有限公司 | Enzyme-linked assay kit and producing process thereof |
| CN102203606A (en) * | 2008-10-30 | 2011-09-28 | 公共健康研究中心 | Biomarkers |
| WO2011088740A1 (en) * | 2010-01-21 | 2011-07-28 | 北京大学 | Enzyme linked immunosorbent assay (elisa) method and kit for detecting soluble programmed cell death protein 5 (pdcd5) |
| 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 |
| CN102435743A (en) * | 2011-09-15 | 2012-05-02 | 北京华创远航科技有限公司 | Enzyme-linked 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 |
| CN102426240A (en) * | 2011-09-19 | 2012-04-25 | 北京华创远航科技有限公司 | Enzyme-linked detection kit and preparation method thereof |
| 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 |
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