CN1355882A - Method for quantifying transforming growth factor-beta 1 and method for detecting cancer by using same - Google Patents
Method for quantifying transforming growth factor-beta 1 and method for detecting cancer by using same Download PDFInfo
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- CN1355882A CN1355882A CN 00806081 CN00806081A CN1355882A CN 1355882 A CN1355882 A CN 1355882A CN 00806081 CN00806081 CN 00806081 CN 00806081 A CN00806081 A CN 00806081A CN 1355882 A CN1355882 A CN 1355882A
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Abstract
The amount of TGF- beta 1 in a sample is quantified by treating the sample with a TGF- beta 1 specific receptor to from a complex between TGF- beta 1 and the receptor and measuring the amount of the complex.
Description
The field of the invention
The present invention relates to the method for the concentration of transforming growth factor-beta 1 (TGF-β 1) in a kind of quantitative a kind of body fluid, a kind of by using identical method to detect method for cancer, a kind of composition that detects cancer, and the special monoclonal antibody of a kind of TGF-β 1.
Background of the present invention
Transforming growth factor (TGF-β) is regulated the growth and the differentiation of several cells, its binding mode depend on the existing of the chromosome configuration of cell and other growth factor (Sporn etc. science (Science) 233,532-534 (1986); And Roberts and Sporn. cancer research progress (Adv.Cancer Res.) 51,107-145 (1998)).
The TGF-β factor that in mammal, has three kinds of forms, TGF-β 1, TGF-β 2, TGF-β 3, and, in the middle of these, it is believed that TGF-β 1 brings into play crucial effect in physiological mechanism and progression of disease.It is unusual to have reported that it acts in a kind of pathogenic process, and for example, cancer takes place.This hint TGF-β 1 is useful as a kind of tumor-marker in the cancer diagnosis, and the high-precision method of the TGF-β 1 in a kind of quantitative a kind of body fluid may be conclusive in cancer diagnosis.
European patent discloses the 0 722 773 A1 number (EP Publication No.0 722 773 A1) and discloses a kind of by using a kind of adsorbent, the hydroxyapatite of OH-carbonating, contact a kind of blood sample that contains TGF-β 1, thereby absorption TGF-β 1 wherein, the TGF-β 1 that is adsorbed with a kind of buffer solution elution, and determine the detection method for cancer of the amount of eluted TGF-β 1 with ultraviolet spectrophotometry.Yet this method is being stood limited susceptibility and is being coarse problem that great fluctuation process proved of measured value.
Therefore, the needs that had the method for the amount of TGF-β 1 in a kind of improved quantitative blood plasma of exploitation.
General introduction of the present invention
Therefore, an object of the present invention is to provide the method with high precision and susceptibility of the amount of TGF-β 1 in a kind of quantitative a kind of sample.
Another object of the present invention provides a kind of by using said method to detect method for cancer.
Further purpose of the present invention provides a kind of composition that detects cancer.
Another purpose of the present invention provides a kind of TGF-β 1 special monoclonal antibody and a kind of hybridoma cell line of producing this monoclonal antibody.
According to an aspect of the present invention, the method of the amount of TGF-β 1 in a kind of quantitative a kind of sample is provided, comprise: form a kind of compound thereby handle this sample therewith between the acceptor at TGF-β 1, and measure the amount of this compound with a kind of TGF-β 1 special acceptor.
The accompanying drawing summary
Above-mentioned purpose of the present invention and feature will become apparent together with accompanying drawing from the description of following embodiment preferred, wherein:
Fig. 1 illustrates the TGF-β 1III type of acquisition in embodiment 1 and the optical density-TGF-β 1 concentration correlation curve of II receptor respectively;
Fig. 2 describes from healthy people, patients with gastric cancer, separately TGF-β 1 CONCENTRATION DISTRIBUTION in the plasma sample that liver cancer patient and patient with breast cancer obtain; And
Fig. 3 describes from healthy people, patients with lung cancer, carcinoma of the colon and rectum patient, separately TGF-β 1 CONCENTRATION DISTRIBUTION in the plasma sample that patients with prostate cancer and cervix cancer patient obtain.
Detailed description of the present invention
The TGF-β 1 special acceptor that can be used in the present invention comprises TGF-β I type, II type, III receptor (R I, RII and RIII), and preferred TGF-β 1 III receptor, RIII. These TGF-β1receptors can be according to a kind of method (Burand of routine, J.P. etc. virology (Virology) 101,286-290 (1980)) obtain by in a kind of mammal or insect cell line, expressing a kind of TGF-β1receptor gene. For example, this TGF-β1receptor can followingly obtain: with a kind of a kind of insect cell line of recombinate shape virus infection that contains a kind of TGF-β1receptor gene, for example, Sf21 (Invitrogen, Netherlands); Be extracted in the water-fast receptor protein of this expressed in insect cells; Dissolve this water-fast receptor protein with guanidine hydrochloride or urea; Again fold dissolved receptor protein by removing guanidine or urea, thereby recover the affinity to TGF-β 1.
Operable TGF-β 1 special antibody can be by preparing with TGF-β 1 or a kind of mammal of one of them partial immunity in the present invention. This TGF-β 1 special antibody can be only TGF-β 1 to be had specific a kind of monoclonal antibody or a kind of polyclonal antibody.
A kind of quantitative a kind of body fluid, for example, the method for optimizing of the amount of the TGF-β 1 in blood plasma or the urine, according to the present invention includes:
(a) receptors bind that a kind of TGF-β 1 is special is to a kind of solid support;
(b) thus a kind of humoral sample added form a kind of TGF-β1-receptor compound in this acceptor that is supported;
(c) the special antibodies of the TGF-β that a kind of and a kind of label combined 1 is to this compound; With
(d) use this kind to measure the amount of TGF-β 1 as a kind of label of certification mark.
The representative label that can be used in the present invention comprises horseradish peroxidase, biotin and fluorescence.
First preferred embodiment of the present invention comprises: the TGF-β1Shou Ti is attached on a kind of solid support, for example, the hole of a microtiter plate; A kind of sample that contains TGF-β 1 of suitable dilution is joined in this TGF-β1Shou Ti, thereby make TGF-β 1 can form a kind of compound between the TGF-β1Shou Ti therewith; (PBS) washs this holder with a kind of phosphate buffer; Join anti--TGF-beta 1 antibodies and form the enzyme that adds lustre to wherein adding a kind of enzyme that adds lustre to; Thereby and the quantitative content of TGF-β 1 in this sample of the optical density of measuring the solution produced.
In second preferred embodiment of the present invention, a kind of liquid that contains the special acceptor of a kind of TGF-β 1 can be used to replace the TGF-β1Shou Ti that is supported.In this method, the amount of the TGF-β 1 in a kind of sample can be following quantitatively: this sample is added in this liquid that contains the special acceptor of TGF-β 1; Add the special antibody that combines with a kind of label wherein of a kind of TGF-β 1; Precipitate a kind of antibody-TGF-β1Shou Ti compound; And measure its optical density.
Method of the present invention can detect at 30 pg/ml or the lower unusual TGF-β 1 of low strength range.
Above method is particularly useful in cancer diagnosis, because TGF-β 1 concentration in cancer patient's body fluid obviously is different from healthy people that.Therefore, thereby cancer can be by repeating quantitatively patient's humoral sample of above method, TGF-β 1 level in blood plasma or the urine for example, and with that comparison of this TGF-β 1 concentration and healthy people and detected.
A preferred embodiment of a kind of method for cancer of detection of the present invention comprises:
(a) a kind of TGF-β 1 is special receptors bind is to a kind of solid support;
(b) thus a kind of humoral sample added form this TGF-β1-Shou Ti compound in this acceptor that is supported;
(c) a kind of TGF-β 1 is the special antibodies that combines with a kind of label is to this compound;
(d) use this kind to measure the amount of TGF-β 1 as a kind of label of diagnostic marker; With
(e) with that comparison of this TGF-β 1 amount with healthy people.
Above method is detecting cancer of the stomach, liver cancer, and breast cancer, lung cancer, carcinoma of the colon and rectum, effective especially in prostate cancer and the cervix cancer.
Detect a kind of composition that can be used in a kind of method for cancer at this and comprise a kind of TGF-β1Shou Ti, the antibody that preferred RIII and a kind of TGF-β 1 are special.
In order to improve susceptibility, this monoclonal antibody can followingly obtain: according to a kind of cell fusion method of routine, use TGF-β 1 or a kind of antigenic determinant part wherein as a kind of immunogene, prepare a kind of hybridoma cell line of producing the special monoclonal antibody of TGF-β 1; And from then on hybridoma cell line separates this monoclonal antibody.For example, a kind of like this hybridoma cell line can be prepared as follows: personnel selection TGF-β 1 immune a kind of mouse; According to Kohler and the described cell fusion method of Milstein (European Journal of Immunology (Eur.J.Immunol.), 6,511-519 (1976)), mouse boosting cell and myeloma cell are merged; The ELISA selection is a kind of only to have specific hybridoma cell line to people TGF-β 1 by using; Use a kind of method of immunodiffusion, determine the subclass of the monoclonal antibody that this hybridoma cell line produces; And, select a kind of hybridoma cell line of high antibody titer that has of IgG secretion 1 subclass.This hybridoma cell line of Huo Deing is named and is hTGF-46 like this, and according to being deposited in (address: #52, Korea S typical case culture collection center (Korean Collection for TypeCulture) on April 20th, 1998 about clause (The Budapest Treaty on the International Recognition ofthe Deposit of Microorganism for the Purpose of Patent Procedure) because of the budapest treaty of the world identification of the microbial preservation thing of proprietary program, Oun-dong, Yusong-ku, Taejon 305-600, Republic ofKorea), its preserving number is KCTC 0460BP.
Hybridoma cell line hTGF-46 originates from β-lymthoma, and divides continuously in the antibody that produces the special IgG1 subclass of TGF-β 1.This hybridoma cell line can RPMI 1640 nutrient culture media that contain 10% hyclone (Gibco-BRL, USA) at 37 ℃ at 5%CO
2Air in and 100% humidity under cultivate.Cell number doubled in 12 to 14 hours.This hybridoma cell line is suspended in this nutrient culture media, self is not joined to the bottom of culture flask, and is rounded, and diameter is 15 to 20 μ m.
For with the special monoclonal antibody of a large amount of TGF-β of this hybridoma cell line production 1, this hybridoma cell line is expelled in a kind of mouse, thereby and the ascites of when expand in its abdominal cavity, obtaining the hybridoma that contains high concentration separate this monoclonal antibody from wherein.
As TGF-β 1 ,-β 2 and-β 3 carries out electrophoresis when carrying out the Western trace subsequently, this monoclonal antibody of the present invention is only discerned TGF-β 1, but nonrecognition TGF-β 2 or-β 3.This hints that this monoclonal antibody has unique specificity to TGF-β 1.This monoclonal antibody of the present invention also shows high-affinity to people TGF-β 1, and is attached to the epitope regions corresponding to the 5th to 80 amino acid residue of TGF-β 1.
Plan to further specify the present invention, and do not limit its scope with following embodiment.
Further, the percentage of the potpourri of the solid that below provides in liquid, liquid potpourri and the potpourri of solid in liquid in liquid is respectively based on wt/wt, and vol/vol and wt/vol are unless offer some clarification in addition.(the preparation of step 1) TGF-β 1 III receptor of 1 pair of receptor sensitivity of embodiment 1:TGF-β
Use primer RIII and RIII2 (SEQ ID Nos:1 and 2), plasmid pCEP4 (Invitrogen with the full-length cDNA that contains people TGF-β 1 an III receptor, Netherlands) carry out PCR (PCR), thereby obtain the dna fragmentation of an extracellular domain that coding comprises this acceptor of 400 amino acid residues (1 to 400).The dna fragmentation of Huo Deing is inserted into baculovirus vector pCRBac (Invitrogen Netherlands), thereby obtains recombinant plasmid pCRBac-TGFR like this.PCRBac-TGFR has transformed the E.coli cell with this recombinant plasmid, and at a kind of selection nutrient culture media, contains the E.coli cell of having selected quilt to be transformed on the LB nutrient culture media of ampicillin.
Use liposome transfection method (Burand, J.P., virology (Virology), 101,286-290 (1980)), with carrier pCRBac-TGFR and Bac-N-Blue DNA (Invitrogen, Netherlands) import insect cell line Sf21 (Invitrogen jointly, Netherlands) in, and cultivate 3 days, thereby obtain a kind of viral product.After 3 days, as a kind of selected marker, the virus of such acquisition has been carried out the plaque analysis, thereby selected this recombinant virus with the lacZ gene.Use forward primer (SEQ ID NO:3) and reverse primer (SEQ ID NO:4), the recombinant virus of such acquisition has been carried out PCR, thereby confirmed the existence of this TGF-β1Shou Ti gene.Wild baculoviral has shown 839 bp PCR products, and this recombinant virus has provided one 1.5 kbp PCR product.
With this recombinant virus infection insect cell line SF21, and after this cultivating 5 days.Centrifugal this culture, thus remove cell fragment and collect the supernatant that contains virus.
With this supernatant inoculation insect cell line Sf21, and after this containing 10% hyclone (FBS) at 27 ℃, the Grace insect nutrient culture media of 7.3% TC yeastolate and 73% lactalbumin hydrolysate (Invitrogen, Netherlands) the middle cultivation 72 days.Centrifugal this culture, thereby collecting cell, and wash this cell with PBS.To wherein adding protein cleavage solution (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM beta-mercaptoethanols, 1%Triton X-100 and 2 mMBMSF), and the solution that is after this produced 100 ℃ of heating 10 minutes, thereby prepare a kind of sample.
In 12.5% SDS-polyacrylamide gel, carried out SDS-PAGE with this sample, and gel-colored with Coomassie brilliant blue to what produced.Following this gel has been carried out the Western trace: will be on albumen electrotransfer to a film separated on the gel, from R﹠amp; D Systems Inc., albumen on this film of antibodies of the TGF-β1Shou Ti that USA (American Studies and development system incorporated company) obtains, and after this using the anti-IgG second antibody that combines horseradish peroxidase (HRP) to analyze TGF-β 1 (Chemicon, USA), thus confirm the expression of TGF-β 1 III receptor.
Because this TGF-β1Shou Ti is not dissolved in water, thus in this sample, add the 8M guanidine hydrochloride, and stirred the solution that produced 1 hour.The solution that is produced is 7, centrifugal 40 minutes of 000rpm, and after this supernatant is adjusted to the 2mg/ml protein concentration.In order to recover the activity of this TGF-β1Shou Ti in conjunction with TGF-β 1, the solution that is produced is added (100mMTris, 0.5M arginine, 0.2M EDTA in a kind of folding damping fluid, pH8.0), to protein concentration be 150 μ g/ml, and preserved 40 hours at 10 ℃, the solution that is produced is with 20mM Tris solution (pH8.0), adjoining land is according to per 4 hours twice, spend the night afterwards once, and twice dialysis in after this per 2 hours, thereby folding again effectively this TGF-β1Shou Ti.(step 2) TGF-β 1III receptor is to the susceptibility of TGF-β 1
The TGF-β 1III receptor of each part 2 μ g that will obtain in step 1 is positioned in the hole of a microtiter plate, and institute's generation plank was preserved 24 hours in environment temperature, thus with this receptors bind on this plate.TGF-β 1 (R﹠amp with the purifying of 2ng; D systems Inc. USA) is dissolved among the PBS, and serial dilution.The amount of each dilute solution by 100 μ l added in the hand-hole, and after this preserving 3 hours, thereby make this TGF-β 1 can be in conjunction with this acceptor in environment temperature.Wash each hole with the PBS that contains 0.05%Tween 20 (PBST), and after this to the anti--TGF-beta 1 antibodies (R﹠amp that wherein adds in conjunction with HRP; D systems Inc., USA).With the plank that produces room temperature preservation 1.5 hours.Wash each hole with PBST.To the TMB-ELISA that wherein adds 100 μ l (Gibco-BRL, USA), a kind of HRP substrate, thus and the plank that produced placed colour developing in 20 minutes in room temperature.Stopped this chromogenic reaction by adding 25 μ l 2N sulfuric acid.Measure the optical density of this reaction mixture at the tuning wavelength of the measurement wavelength of 450nm and 570nm, and with drafting pattern as a result, thereby obtained the optical density-concentration correlation curve of a standard.
Fig. 1 illustrates that the correlation curve of such acquisition is that a related coefficient is 0.999, and slope is 0.28 straight line.This slope representative employed receptor sensitivity in these are measured, and this TGF-β 1III receptor is considered to TGF-β 1 in conjunction with having good sensitivity.This method that also illustrates correlation curve in Fig. 1 can detect the TGF-β 1 of extremely low concentration, is low to moderate 10pg/ml.
Use TGF-β 1II receptor (R﹠amp; D systems Inc. USA) has repeated above process, thereby determines the susceptibility of TGF-β 1II receptor.These results also are plotted among Fig. 1, illustrate that the correlation curve that uses TGF-β 1II receptor to obtain also is that a related coefficient is 0.999, and slope is 0.57 straight line.Therefore, the II receptor also can be used to the concentration of quantitative TGF-β 1, but its susceptibility is lower than that of III receptor significantly.Embodiment 2:TGF-β 1III receptor is to the specificity of TGF-β 1
Except use contains 2,000pg/ml TGF-β 1,2,000pg/ml TGF-β 2 and 2,000pg/ml TGF-β 3 (R﹠amp; D Systems Inc., potpourri USA) has been replaced outside the TGF-β 1, has repeated the process of the step 2 of embodiment 1.Use 2,000pg/ml TGF-β 1 has repeated the process of the step 2 of embodiment 1 as a contrast.The result is displayed in the Table I.
Table I
| ?TGF-β1 | ?TGF-β1+TGF-β2+TGF- ??????????β3 | |
| TGF-β 1III receptor | ??100％ | ?????????91.5％ |
As can seeing from Table I, TGF-β 1III receptor only combines with TGF-β 1, with TGF-β 2 or TGF-β 3 cross reaction does not take place.Embodiment 3: plasma TG F-β 1 concentration of using monoclonal anti bulk measurement cancer patient
From 101 healthy people, 111 patients with gastric cancer, 100 liver cancer patients and 151 patient with breast cancers obtain blood sample.Collect blood sample with containing 0.081ml as a kind of vacuum tank of 15% ethylenediamine tetraacetic acid (EDTA) of anti-coagulants, and after this with the potpourri that obtained 3, centrifugal 20 minutes of 000rpm, thus obtain a kind of plasma sample.This plasma sample of 0.1ml is added in 2.5N acetate/10M urea liquid of 0.1ml.With the potpourri that produced room temperature preservation 10 minutes, and with the 2.7N NaOH neutralization that contains 1M ethyl piperazidine ethyl sulfonic acid (HEPES) of 0.1ml.Doubly dilute the blood plasma that is activated of such acquisition with PBST4, thereby obtain a kind of plasma sample that carries out the process of its TGF-β 1 concentration of following measurement.
0.1ml the plasma sample solution of such acquisition, and TGF-β 1 standard solution (0 of 0.1ml portion, 100,1,000 and 2,000pg/ml) joined respectively in the hole of a 96-orifice plate that contains TGF-β 1III receptor, room temperature preservation 3 hours, and, washed 3 times with PBST after this.The TGF-β 1 monoclonal antibody-HRP compound (Sigma) of purifying is added each hole, and after this with this plate room temperature preservation 1.5 hours, after this wash these holes 3 times with PBST.With the TMB-ELISA of 100 μ l (Gibco-BRL, USA), a kind of HRP substrate adds each hole, thereby and with this plate in room temperature preservation colour developing in 20 minutes.Stopped this chromogenic reaction by the 2N sulfuric acid that adds 25 μ l.Measured the optical density of this reaction mixture at the tuning wavelength of the measurement wavelength of 450nm and 570nm.Based on the calibration curve that uses standard solution to obtain, determined TGF-β 1 concentration of this plasma sample, and will the results are shown in Table II and Fig. 2.
Table II
| The plasma sample group | Mean+/-standard error (ng/ml) | Scope | |
| Cancer of the stomach (n=111) | ????6.53±0.31 | ????1.5-16.35 | |
| Liver cancer (n=100) | ????5.89±0.3 | ????1.77-14.76 | |
| Breast cancer | Before the operation (n=117) | ????5.49±0.32 | ????0.87-13.44 |
| Operation back (n=34) | ????2.15±0.42 | ????0.46-9 | |
| Healthy people (n=101) | ????1.03±0.08 | ????0.27-8 | |
As can seeing in Fig. 2 of the plasma TG F-β 1 CONCENTRATION DISTRIBUTION pattern of describing each patient's group, cancer patient's plasma sample presents that the obvious different TGF-β 1 concentration pattern with healthy people's group.This hints that above-mentioned cancer can be detected by measuring plasma TG F-β 1 concentration according to above process.Embodiment 4: use monoclonal to measure cancer patient's plasma TG F-β 1 concentration
Use is from 288 healthy people, 29 patients with lung cancer, 48 rectum-colon cancer patients, 50 patients with prostate cancer and 88 blood samples that the cervix cancer patient is obtained, repeated the process of embodiment 3, thereby measured each plasma TG F-β 1 concentration, and the result has been presented among Table III and Fig. 3.
Table III
| Plasma sample | Mean+/-standard error (ng/ml) | Standard deviation |
| Lung cancer (n=20) | ????8.48±1.27 | ????4.16 |
| Carcinoma of the colon and rectum (n=48) | ????5.19±0.87 | ????3.69 |
| Prostate cancer (n=50) | ????4.12±0.53 | ????2.30 |
| Cervix cancer (n=88) | ????8.55±0.92 | ????5.25 |
| Healthy people (n=288) | ????1.77±0.05 | ????0.05 |
P＜0.01
Can seeing in Fig. 3 of the plasma TG F-β 1 CONCENTRATION DISTRIBUTION pattern of organizing each patient of explanation, cancer patient's plasma sample presents that the obvious different TGF-β 1 concentration pattern with healthy people's group.This hints that above-mentioned cancer can be detected by measuring plasma TG F-β 1 concentration according to above process.Embodiment 5: use polyclonal antibody to measure cancer patient's plasma TG F-β 1 concentration
Except having used a kind of polyclonal antibody to replace this monoclonal antibody, use from 50 healthy people 50 liver cancer patients, with 50 blood samples that the patient with breast cancer obtained, repeat the process of embodiment 3, thereby measured each plasma TG F-β 1 concentration, and will the results are shown in Table IV.
Table IV
| Blood plasma | Mean+/-standard error (ng/ml) | Standard deviation | Scope (ng/ml) |
| Liver cancer (n=50) | ??5.14±0.57 | ????2.92 | ???1.44-16.96 |
| Breast cancer (n=50) | ??5.31±0.46 | ????1.67 | ???2.07-10.27 |
| Healthy people (n=50) | ??1.19±0.08 | ????0.29 | ???0.70-1.9 |
P＜0.05
As can seeing in Table IV, cancer patient's plasma sample presents that the obvious different TGF-β 1 concentration pattern with healthy people's group.This hints that above-mentioned cancer can be detected by measuring plasma TG F-β 1 concentration according to above process.Embodiment 6: preparation (the step 1) immune mouse of producing a kind of hybridoma cell line of TGF-β 1 monoclonal antibody
TGF-β 1 is mixed with isopyknic Freund's complete adjuvant, become liquid, and the potpourri that is produced is expelled to the tail vein of the Balb/c mouse in one 7 age in week by the amount of 100 μ l/ mouse until this potpourri.After 2 weeks, will mix with incomplete Freund with injection for the first time in the TGF-β 1 of same amount, be expelled to the tail vein of this mouse.After 4 to 5 days, obtain blood in a small amount from tail, and confirmed the existence of TGF-beta 1 antibodies by ELISA.After this, cell fusion process below 3 to 4 days before is with people TGF-β 1 intravenous injection that is dissolved in 30 μ g among the 0.85%PBS.(step 2) Fusion of Cells
In this cell fusion process, use myeloma cell SP2/0Ag14 (ATCC CRL 1581) as a germ mother cell.This mother cell is cultivated in RPMI 1640 nutrient culture media that contain 10%FBS, keeps 5 * 10 simultaneously
5The maximum cell density of/ml.
Use that etherization obtained in step 1 by mice immunized, and extract its spleen, thereby use Potter-Elvehjem Tissue Grinders homogenate.With the homogenate that is produced be suspended in HBSS (Gibco-BRL, USA) in, and be positioned in the 15ml centrifuge tube suspending liquid that is produced centrifugal.Repeat this process twice, thereby wash splenocyte up hill and dale.With this mother cell, the SP2/0Ag14 cell, be suspended among the HBSS and centrifugal it.Repeat twice of this process.Thereby splenocyte and SP2/0Ag14 are suspended in the cell quantity of counting among the HBSS of 10ml in each suspending liquid respectively.Will be from 10 of each suspending liquid acquisition
8Individual splenocyte and 10
7Individual SP2/0Ag14 cell mixes in a centrifuge tube, and is after this centrifugal, thereby makes cell precipitation.Disperse precipitated cell thereby rap dozen this centrifuge tube, and, preserved 1 minute at 37 ℃ after this with light finger.Last 1 fen clockwise and wherein add the HBSS that contains 45%PEG (w/v) and 5%DMSO of 1ml, after this shook this pipe 1 minute.Last 3 fens clockwise and wherein add the RPMI nutrient culture media of 9ml, and, shake this pipe simultaneously after this becoming 50ml to wherein adding the cumulative volume of RPMI nutrient culture media until cell suspending liquid.The suspending liquid that is produced is centrifugal, and the cell precipitation that will obtain like this is by 1-2 * 10
5The concentration of/ml be suspended in again the HAT nutrient culture media (Gibco-BRL, USA) in.The suspending liquid that is produced of 0.2ml portion is placed the hole of a 96-hole microtiter plate, and after this cultivating a couple of days in incubator, condition maintains 37 ℃, 5%CO
2With 100% humidity.(the selection of the hybridoma cell line of step 3) manufacture order clonal antibody
As following described, end user TGF-beta 1-6 antigen has carried out ELISA to the hybridoma cell line that obtains in step 2, thereby obtains specifically the cell with TGF-β 1 reaction.
People TGF-beta 1-6 antigen is added in the hole of a microtiter plate by the amount in 50 μ l (2 μ g/ml)/hole, and room temperature preservation 12 hours, thereby antigen is attached on the surface in hole.Wash these holes with PBST, thereby removed the antigen that does not have combination.
This Hybridoma Cell Culture thing that will obtain in step 2 adds in each hole by the amount of 50 μ l/ cells, and preserves 1 hour at 37 ℃.Wash these holes with PBST, thereby remove this culture.(Sigma USA), room temperature preservation 1 hour, and washs with PBST to wherein adding sheep anti-mouse igg-HRP.(OPD Sigma), room temperature preservation 20 minutes, and has measured the optical density of the reaction mixture that is produced at 492nm to the substrate solution that wherein adds 100 μ l.
Selected the hybridoma cell line of secretion at first at the antibody with high specific of people TGF-beta 1-6 antigen, and end user TGF-β 1,-β 2 and-β 3 has carried out ELISA in these hybridoma cell lines each, thereby screening only has specific hybridoma to people TGF-beta 1-6 antigen.In the hybridoma of such acquisition each is carried out limiting dilution, thereby obtained 7 hybridoma cell lines clones that produce a kind of monoclonal antibodies, hTGF-7 ,-8 ,-31 ,-46 ,-70 ,-119 and-207.Each clone is frozen drying.
Centrifugal Hybridoma Cell Culture thing, and supernatant carried out ELISA, thus determine this antibody titer, and (Sigma USA) analyzes, thereby determines the subclass of this antibody after this using immunophenotyping kit (immunotype kit).To the results are shown in Table V.
Table V
| Clone number | Optical density (492nm) | Subclass |
| ????HTGF-46 | ????2.125 | ????IgG1 |
| ????HTGF-7 | ????1.644 | ????IgG1 |
| ????HTGF-70 | ????2.590 | ????IgG1 |
| ????HTGF-8 | ????2.395 | ????IgG1 |
| ????hTGF-207 | ????1.735 | ????IgG1 |
| ????hTGF-119 | ????2.462 | ????IgG1 |
| ????HTGF-31 | ????2.282 | ????IgG1 |
Can seeing in Table V, whole 7 clones are IgG1.
In the middle of these 7 clones, selected to have the clone of high titre, hTGF-46, and intraperitoneal injection is given a mouse.After this, collect its ascites, and carried out the Western trace.This hybridoma cell line of presentation of results clone hTGF-46 secretion is a kind of to have the monoclonal antibody of high specific to people TGF-β 1.According to about clause because of the budapest treaty of the world identification of the microbial preservation thing of proprietary program, on April 20th, 1998 this hybridoma cell line hTGF-46 is deposited in (address: #52, Korea S typical case culture collection center, Oun-dong, Yusong-ku, Taejon305-600, Republic of Korea), its preserving number is KCTC 0460BP.The production of the monoclonal antibody of embodiment 7:TGF-β 1
For the monoclonal antibody of using in embodiment 6 hybridoma cell line that obtains to produce TGF-β 1, give the norphytane of having injected 0.5ml in the Balb/c mouse peritoneum, and after 1 week, given each injected in mice 5 * 10
6Individual hybridoma.Obtain the ascites of the hybridoma that contains high concentration from the mouse in abdominal cavity, and 10,000rpm is centrifugal, thereby removes this hybridoma with expansion.Supernatant is stored in-20 ℃.
Pillar is filled with the Protein G pearl, and is after this using 1 * PBS to wash 4 times.With the supernatant of 2ml by 5 droplets/minute speed drop by drop on the ground sample to this pillar, thereby and 0.1M glycocoll-hydrochloric acid solution introduced this pillar wash-out IgG by 1/10 minutes speed.
HRP is activated in containing 1.25% glutaraldehyde 0.1M sodium phosphate buffer (pH6.5), and the HRP that is activated is dialysed to carbonate buffer solution (pH9.2).The HRP that will be dialysed IgG therewith reacts, thereby obtains the IgG of a kind of HRP of combining.After this reaction is finished, determined RZ value (A by the optical density of measuring this reaction mixture at 280nm and 403nm
403/ A
280).In order to determine the activity of enzyme len antibody, by each hole of a microtiter plate, and combine the IgG reaction of HRP therewith after this with TGF-β 1 bag of 1 μ g, thereby determine activity.Further, the antibody that this is combined HRP has carried out the Western trace, thereby determines its activity.
Embodiment 8:Western trace
For check in embodiment 7 the special monoclonal antibody of the TGF-β that obtains 1 whether with TGF-β 2 and TGF-β 3 reactions, following this monoclonal antibody has been carried out SDS-PAGE and Western trace.
On the 10%SDS-polyacrylamide gel to people TGF-β 1 ,-β 2 and-β 3 albumen have carried out SDS-PAGE, and on after this with its electrotransfer to one nitrocellulose filter.With this film monoclonal antibody reactive a few hours therewith.The film that is produced was handled 12 to 14 hours with bovine serum albumin(BSA) in room temperature, thereby sealed the non-specific responding of these albumen.Wash this film 3 times with the PBS that contains 0.5%Tween 20, and in that after this (Sigma is USA) at room temperature reaction with the anti-mouse IgG that combines HRP.Wash this film 3 times with the PBS that contains 0.5%Tween 20, after this, add a kind of substrate buffer solution (TMB, Gibco-BRL, thereby USA) colour developing to this film.
Presentation of results this monoclonal antibody of the present invention only with people TGF-β 1 reaction, and not with people TGF-β 2 and-β 3 reactions.Therefore, this monoclonal only has specificity to people's TGF-β 1.
Although about above specific embodiment, the present invention has been described, should admit that the skilled person in this area can carry out various modifications and changes and still in by the scope of additional claims defined the present invention.
The international preservation of microorganism proves (translation)
| The preservation people | Choe，Yong-Kyung |
| The address | Taejon, Korea |
| Preserving number | KCTC?0480BP |
| The microorganism classification name | HTGF-46 |
| International depositary institution title | Korea S's bio-science and Bioteknologisk Institut Korea S typical case's culture collection center |
| Agency affixes one's seal |
＜110〉＜120〉-β1＜130〉PCA00418/HMY＜150〉KR 1999-12568＜151〉1999-04-09＜150〉KR 1999-43935＜151〉1999-10-12＜160〉4＜170〉PatentIn version 3.1＜210〉1＜211〉18＜212〉DNA＜213〉＜220〉＜223〉Primer RIII1＜400〉1atggcagtga catcccac 18＜210〉2＜211〉12＜212〉DNA＜213〉＜220〉＜223〉Primer RIII2＜400〉2atttgggctt cc 12＜210〉3＜211〉24＜212〉DNA＜213〉＜220〉＜223〉＜400〉3tttactgttt tcgtaacagt tttg 24＜210〉4＜211〉21＜212〉DNA＜213〉＜220〉＜223〉＜400〉4caacaacgca cagaatctag c 21
Claims (22)
1. the method for the amount of TGF-β 1 in the quantitative sample, thus comprise that handling sample with a kind of TGF-β 1 special acceptor forms a kind of compound therewith at TGF-β 1 between the acceptor, and measure the amount of this compound.
2. the method for claim 1 comprises
(a) TGF-β 1 is special receptors bind is to a kind of solid support;
(b) thus this sample is added this acceptor that is supported forms TGF-β1-Shou Ti compound;
(c) a kind of TGF-β that combines a kind of label 1 is special antibodies is to this compound; With
(d) use this label to measure the amount of TGF-β 1 as certification mark.
3. claim 1 or 2 method, wherein TGF-β 1 special acceptor is TGF-β 1 an III receptor.
4. the method for claim 2, wherein TGF-β 1 special antibody is a kind of by with TGF-β 1 or the prepared antibody of a kind of mammal of one of them partial immunity.
5. the method for claim 4, wherein this antibody is a kind of monoclonal antibody or polyclonal antibody antibody.
6. the method for claim 5, wherein this monoclonal antibody is produced with a kind of hybridoma cell line hTGF-46 (KCTC 0460BP).
7. claim 1 or 2 method, wherein the concentration of TGF-β 1 is 30pg/ml or lower in this sample.
8. the method for claim 2, wherein this label is a horseradish peroxidase, biotin or fluorescence.
9. one kind is detected method for cancer in the patient, thereby comprises with the special acceptor of TGF-β 1 and handle the humoral sample of taking from the patient at this acceptor and be present between the TGF-β 1 in this sample and form compound; Thereby the amount of measuring this compound is the concentration of the TGF-β 1 in this sample quantitatively; And with comparing of this TGF-β 1 concentration and healthy people.
10. the method for claim 9 comprises
(a) TGF-β 1 is special receptors bind is to a kind of solid support;
(b) thus this sample is added this acceptor that is supported forms TGF-β1-Shou Ti compound;
(c) a kind of TGF-β that combines a kind of label 1 is special antibodies is to this compound;
(d) use this label to measure the amount of TGF-β 1, thereby determine the concentration of TGF-β 1 in this sample as certification mark; With
(e) with this TGF-β 1 concentration and healthy people's comparison.
11. the method for claim 9 or 10, wherein this TGF-β 1 special acceptor is TGF-β 1 an III receptor.
12. the method for claim 10, wherein this TGF-β 1 special antibody is a kind of by using TGF-β 1 or the prepared antibody of a kind of mammal of one of them partial immunity.
13. the method for claim 13, wherein this antibody is a kind of monoclonal antibody or a kind of polyclonal antibody.
14. the method for claim 13, wherein this monoclonal antibody is produced with a kind of hybridoma cell line hTGF-46 (KCTC 0460BP).
15. the method for claim 9 or 10, wherein the concentration of the TGF-β 1 in this sample is 30pg/ml or lower.
16. the method for claim 10, wherein this label is a horseradish peroxidase, biotin or fluorescence.
17. the method for claim 9 or 10, wherein this cancer is selected from cancer of the stomach, liver cancer, breast cancer, lung cancer, carcinoma of the colon and rectum, prostate cancer and cervix cancer.
18. the method for claim 9 or 10, wherein this body fluid is blood plasma or urine.
19. a composition that detects cancer comprises antibody that acceptor that a kind of TGF-β 1 is special and a kind of TGF-β 1 are special.
20. the composition of claim 19, wherein this TGF-β 1 special acceptor is a TGF-β 1III receptor.
21. a hybridoma cell line, it is the hTGF-46 (KCTC 0460) that produces the special monoclonal antibody of people TGF-β 1.
22. the monoclonal antibody that a TGF-β who is produced by hybridoma cell line hTGF-46 (KCTC 0460BP) 1 is special.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1999/12568 | 1999-04-09 | ||
| KR19990012568 | 1999-04-09 | ||
| KR19990043935 | 1999-10-12 | ||
| KR1999/43935 | 1999-10-12 |
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| Publication Number | Publication Date |
|---|---|
| CN1355882A true CN1355882A (en) | 2002-06-26 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00806081 Pending CN1355882A (en) | 1999-04-09 | 2000-04-10 | Method for quantifying transforming growth factor-beta 1 and method for detecting cancer by using same |
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| Country | Link |
|---|---|
| EP (1) | EP1166112A4 (en) |
| JP (1) | JP2002541479A (en) |
| CN (1) | CN1355882A (en) |
| AU (1) | AU768029B2 (en) |
| BR (1) | BR0009607A (en) |
| CA (1) | CA2369892A1 (en) |
| ID (1) | ID30316A (en) |
| MX (1) | MXPA01009942A (en) |
| NZ (1) | NZ514596A (en) |
| TR (1) | TR200102917T2 (en) |
| WO (1) | WO2000062062A1 (en) |
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| MXPA03004675A (en) * | 2000-11-28 | 2003-09-05 | Amgen Inc | Transforming growth factor-beta-related molecules and uses thereof. |
| JP4834835B2 (en) * | 2006-07-27 | 2011-12-14 | 国立大学法人浜松医科大学 | Diagnostic agent for autism |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59195162A (en) * | 1983-04-21 | 1984-11-06 | Toyo Jozo Co Ltd | Reagent for assay of transforming gross factor |
| JPS59216058A (en) * | 1983-05-23 | 1984-12-06 | Toyo Jozo Co Ltd | Determining method of transforming gross factor |
| JPS59226864A (en) * | 1983-06-07 | 1984-12-20 | Toyo Jozo Co Ltd | Method for measuring enzyme immune of transforming gross factor |
| JPS6018764A (en) * | 1983-07-12 | 1985-01-30 | Toyo Jozo Co Ltd | Immunological measurement of transforming growth factor |
| JPH02126157A (en) * | 1988-11-04 | 1990-05-15 | Tosoh Corp | Method for immunologically measuring human transforming growth factor-beta |
| WO1993010215A1 (en) * | 1991-11-15 | 1993-05-27 | Memorial Sloan-Kettering Cancer Center | Purified proteoglycan betaglycan, compositions, and methods |
| GB9601081D0 (en) * | 1995-10-06 | 1996-03-20 | Cambridge Antibody Tech | Specific binding members for human transforming growth factor beta;materials and methods |
-
2000
- 2000-04-10 MX MXPA01009942A patent/MXPA01009942A/en unknown
- 2000-04-10 EP EP00921123A patent/EP1166112A4/en not_active Withdrawn
- 2000-04-10 CN CN 00806081 patent/CN1355882A/en active Pending
- 2000-04-10 JP JP2000611074A patent/JP2002541479A/en active Pending
- 2000-04-10 BR BR0009607-5A patent/BR0009607A/en not_active IP Right Cessation
- 2000-04-10 ID IDW00200102145A patent/ID30316A/en unknown
- 2000-04-10 NZ NZ514596A patent/NZ514596A/en unknown
- 2000-04-10 AU AU41471/00A patent/AU768029B2/en not_active Ceased
- 2000-04-10 CA CA002369892A patent/CA2369892A1/en not_active Abandoned
- 2000-04-10 TR TR2001/02917T patent/TR200102917T2/en unknown
- 2000-04-10 WO PCT/KR2000/000329 patent/WO2000062062A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU4147100A (en) | 2000-11-14 |
| BR0009607A (en) | 2002-01-08 |
| MXPA01009942A (en) | 2003-07-14 |
| EP1166112A4 (en) | 2004-11-10 |
| NZ514596A (en) | 2003-03-28 |
| TR200102917T2 (en) | 2002-03-21 |
| CA2369892A1 (en) | 2000-10-19 |
| JP2002541479A (en) | 2002-12-03 |
| ID30316A (en) | 2001-11-22 |
| EP1166112A1 (en) | 2002-01-02 |
| AU768029B2 (en) | 2003-11-27 |
| WO2000062062A1 (en) | 2000-10-19 |
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