CN101074264A - Recombinant anti-CTLA4 monoclonal antibody, its production and use - Google Patents
Recombinant anti-CTLA4 monoclonal antibody, its production and use Download PDFInfo
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Abstract
An anti-CTLA4 mono-clone antibody, its DNA molecule for encoding antibody, its carrier for expressing antibody and eukaryotic host cell converted by the expression carrier are disclosed. Light-chain variable zone has amino-acid sequence with SEQ ID NO:1, and heavy-chain variable zone has amino-acid sequence with SEQ ID NO:2. It can inspect CTLA4-Fc concentration in blood serum for examinee and patients with malignant diseases in hematopoietic system fast.
Description
Technical field
The present invention relates to the antibody drug technical field.Specifically, the present invention relates to the anti-cytolytic T lymphocyte antigen 4 of a kind of new reorganization monoclonal antibody, and its production and use.
Background technology
The lymphocytic activation of T needs at least 2 signals, first signal by T cell antigen receptor (TCR) and the surperficial MHC/ antigenic peptide complexes of antigen presenting cell (APC) in conjunction with being produced, the combination of these two kinds of parts needs specific recognition, with immunoreactive specificity substantial connection is arranged.Second signal is that other accessory molecule of T cell surface combines generation with the respective ligand on antigen presenting cell surface, and this signal is called costimulatory signal again, is antigen non-specific.
T cell, antigen presenting cell surface participate in producing altogether, and the molecule of hormesis is referred to as costimulatory molecules.Generally acknowledge that at present the most important path that stimulates altogether is the B7-CD28 path.B7 is positioned at antigen presenting cell, combines with the CD28 molecule of T cell surface and can produce common stimulation.Behind t cell activation, its cell surface can occur another kind can with B7 bonded molecule---cytotoxic t lymphocyte-associated antigen 4 (Cytotoxic TLymphocyte associate Antigen-4, CTLA4), it is a negative regulatory factor of keeping T Cell Homeostasis in the body.The expression of CTLA4 is subjected to strict regulation and control in the cell.Detect less than this albumen at static T cell surface, only transcribe and increase Shi Caike and measure its mRNA at the T cell activation.Even activated T cell, also this albumen of part T cell surface expression only.The avidity of CTLA4 and B7 is 500-2500 times of CD28.The CTLA4 molecule combines with B7, can the inducing T cell apoptosis, and it is the important molecule of performance immunoregulation effect behind t cell activation.
Utilized engineered method with surface of cell membrane CTLA4 molecule and immunoglobulin Fc fragment combination by Linsley etc. in 1991, form a kind of artificial constructed fusion protein molecule CTLA4-Fc, the avidity of it and B7 is much higher than CD28, can seal the B7 on the APC, blocking-up stimulates path, the activation of suppressor T cell altogether.
The N-end of CTLA4-Fc contains the extracellular section of human CTLA 4, i.e. the 1-125 amino-acid residue of N-end is followed heavy chain hinge area, CH2 and CH3 district by the human IgG1.The said gene sequence is packed into after the suitable expression, changes zooblast (as the CHO engineering cell) over to, can give expression to the protein molecular of dna sequence dna correspondence.Owing to kept 2 cysteine residues of hinge area, CTLA4-Fc with etc. dimeric form secretion.In addition, 2 N-glycosylation sites are arranged on every peptide chain, CTLA4 and Ig part respectively have 1.Its molecular weight is approximately 97KDa.
The immunosuppression mechanism of CTLA4-Fc: because CTLA4-Fc kept and part B7 bonded CTLA4 film outskirt, it can combine with B7, thereby the CD28 molecule of T cell surface can't be combined with B7 with after expression has the antigen presenting cell of B7 molecule to contact.This moment, TCR only accepted first signal from antigen presenting cell MHC, owing to do not have the common hormesis of subsidiary signal, and cause t cell proliferation.Can cause this clone's T cell to be eliminated in vivo, cause clonal anergy and immunological tolerance.But it does not have influence for the T lymphocyte that is not subjected to first token stimulus.By contrast, other immunosuppressor tends to selectively not kill and wound all T cell clones, causes tangible systemic reaction and toxic side effect.
Carry out in the process of preclinical study and clinical study at CTLA4-Fc, and become in the future in the process that the patient uses behind the medicine, all need to detect experimenter (animal or human) or patient's change of serum C TLA4-Fc concentration, in the malignant disease of some hemopoietic system, CTLA4 is high expression level unusually, therefore, in this class patient's diagnosis and treatment process, also need to detect CTLA4 concentration in patient's serum.So press for a kind of can be effectively, the product of rapid detection CTLA4-Fc class medicine and CTLA4.
Summary of the invention
One of technical issues that need to address of the present invention provide the anti-cytolytic T lymphocyte antigen 4 of a kind of reorganization monoclonal antibody, and this antibody is used for the concentration of vitro detection CTLA4.
Second technical problem that the present invention need solve provides a kind of dna molecular of the above-mentioned anti-cytolytic T lymphocyte antigen 4 monoclonal antibody of encoding.
The 3rd technical problem that the present invention need solve provides a kind of expression vector.
The 4th technical problem that the present invention need solve provides a kind of eukaryotic host cell.
The 5th technical problem that the present invention need solve provides a kind of purposes of said monoclonal antibody.
The 6th technical problem that the present invention need solve provides a kind of this MONOCLONAL ANTIBODIES SPECIFIC FOR method.
For solving the problems of the technologies described above, one aspect of the present invention provides a kind of anti-cytolytic T lymphocyte antigen 4 antibody of reorganization, this antibody contains variable region of heavy chain and variable region of light chain, it is characterized in that, variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:1, and variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:2.
The present invention provides the dna molecular of coding said monoclonal antibody on the other hand.This dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:3, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of the coding shown in the SEQ IDNO:4.
Third aspect present invention provides a kind of expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations.
Fourth aspect present invention provides a kind of host cell, it is characterized in that, it is transformed by above-mentioned expression vector.In a preferable example, this host cell is a Chinese hamster ovary celI.
The present invention the 5th side and provide a kind of anti-cytolytic T lymphocyte antigen 4 antibody test CTLA4 concentration method, it is characterized in that described method is the ELISA method.
Sixth aspect present invention provides a kind of method for preparing said monoclonal antibody, it is characterized in that, this method comprises:
A) provide an expression vector, the expression regulation sequence that this expression vector contains above-mentioned dna sequence dna and links to each other with this series of operations;
B) with the described expression vector transformed host cell of step a);
C) host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With
D) separation and purification obtains described monoclonal antibody.
The invention has the advantages that monoclonal anti physical efficiency of the present invention carries out in the process of preclinical study and clinical study at CTLA4-Fc, and become in the future in the process that the patient uses behind the medicine rapid detection experimenter (animal or human) or patient's change of serum C TLA4-Fc concentration; Simultaneously in the malignant disease of some hemopoietic system, unusual high expression level CTLA4, monoclonal antibody provided by the invention in this class patient's diagnosis and treatment process, also can the rapid detection patients serum in CTLA4 concentration.Antibody of the present invention detects CTLA4-Fc fusion rotein high specificity, highly sensitive with the ELISA method, significantly is better than commercially available antibody.
Description of drawings
Fig. 1: carrier pMG18, and indicated element and restriction enzyme site wherein, wherein, hCMV pro is the main early promoter of human cytomegalic inclusion disease virus; Ck is a people κ constant region of light chain gene; IgG1 constant region behaviour γ 1 weight chain constant area gene; PA is the poly-adenosine signal; DHFR is a dihydrofolate reductase gene; AmpR is an ampicillin resistance gene.
Fig. 2: the pM carrier that the present invention is constructed, wherein, Ck still is people κ chain (light chain) constant region gene, IgG1 constant is mouse γ 1 weight chain constant area gene.
Fig. 3: pM (H+L) carrier that the present invention is constructed, contain recombinant anti-CTLA 4 heavy chain of antibody total length and light chain full-length gene.
Fig. 4: anti-CTLA 4 monoclonal antibody of the present invention and commercially available antibody are respectively applied for the sensitivity comparative result (■ is a monoclonal antibody of the present invention, and zero is commercially available antibody) that detects the CTLA4-Fc fusion rotein.
Embodiment
The present invention relates to a kind of anti-CTLA 4 monoclonal antibody of reorganization, this antibody comprises variable region of heavy chain and variable region of light chain, it is characterized in that variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:1, variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO:2.
Monoclonal antibody can make with the whole bag of tricks well known to those skilled in the art.For example, monoclonal antibody can use hybridoma method (by Kohler etc., Nature, 256:495 (1975) at first proposes) to make, or makes with recombinant DNA method (U.S. Patent No. 4,816,567).The also available for example Clackson of monoclonal antibody etc., Nature, 3 52:624-628 (1991) and Marks etc., J.Mol.Biol., the described technology of 222:581-597 (1991) is separated acquisition from phage antibody library.
The present invention also provides the dna molecular of code book invention recombinant anti-CTLA 4 monoclonal antibody.In a preferable example, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:3, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the SEQ ID NO:4.
Behind the nuclear former times acid sequence that obtains code book invention recombinant anti-CTLA 4 monoclonal antibody variable region of heavy chain and variable region of light chain, can prepare monoclonal antibody of the present invention by the following method usually.
At first, provide nucleotide sequence that contains code book invention monoclonal antibody and the expression vector of the expression regulation sequence that links to each other with this series of operations.
The dna sequence dna of code book invention monoclonal antibody can make with conventional means well known to those skilled in the art.For example, can be according to sequence synthetic disclosed by the invention or with PCR method amplification obtain the encoding nucleotide sequence of this monoclonal antibody variable region of heavy chain and variable region of light chain.Then, these nucleotide sequences are inserted in the suitable expression vector by selecting proper restriction site with the whole bag of tricks well known in the art, make them respectively before expression vector entrained CH encoding sequence and constant region of light chain encoding sequence, and make them in same frame.
Used expression vector is a various commercially available expression vector well known by persons skilled in the art among the present invention, for example available from the expression vector of Qiagen and Promega company, and the expression vector pMG 18 that can buy (" carrying out the exploitation of the instrument of environmental monitoring according to the INCP-9 plasmid sequence " (DEVELOPMENT OFTOOLS FORENVIRONMENTAL MONITORING BASED ON INCP-9PLASMIDS SEQUENCES), A.Created, R.Krasowiak, M.Titok, C.M.ThomasSchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State UniversityScoring Av.4, Minsk 220080 Belarus).
Subsequently, the expression vector with above-mentioned acquisition transforms proper host cell." host cell " generally comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.In the present invention, preferable host cell is a Chinese hamster ovary celI.Method with the expression vector transformed host cell has a variety of, used Transformation Program to depend on host to be transformed.Power one method that the former times acid of allos multinuclear is imported in the mammalian cell is known in the art, it comprises transfection, calcium phosphate precipitation, the Polybrene (1 of dextran mediation, 5-dimethyl-1,5-phenodiazine 11 methylene radical gather Methobromide) mediation transfection, protoplastis fusion, electroporation, liposome-mediated transfection and with the dna direct microinjection in karyon.In the present invention, preferred methods is electroporation or liposome mediated-method etc.For example can adopt the liposome method test kit of Invitrogen company to come transfection CHO cell.
Then, under the condition that is fit to monoclonal antibody expression of the present invention, cultivate the host cell that transforms gained.Use conventional immunoglobulin purification step then, as albumin A-Sepharose, conventional separation and purification means purifying well known to those skilled in the art such as light basic phosphatic rock chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, sieve chromatography or affinity chromatography obtain recombinant anti-CTLA 4 monoclonal antibody of the present invention.
The gained monoclonal antibody can be identified with conventional means.The binding specificity of monoclonal antibody can be measured with immunoprecipitation or external combination test (as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).The binding affinity of monoclonal antibody is available Munson etc. for example, Anal.Biochem., and the Scatchard of 107:220 (1980) analyzes and measures.
Below in conjunction with embodiment the present invention is described in further detail.Yet should be appreciated that and enumerate these embodiment, and be not to be used for limiting the present invention just for an illustration.
Do not indicate among the embodiment that being of source is commercially available.
1) structure of mouse source antibody library
CTLA4-Fc fusion rotein (R﹠amp; D, 325-CT/CF) with freund's adjuvant immunity Balb/C mouse, after the immunity 4 times, 1: 500 dilution back of mice serum shows strong positive reaction with CTLA4, gets immunity back mouse spleen, according to people J.Mol.Biol. such as Marks, 222,581-597.Hoogenboom and Winter, J.Mol.Biol., 227, people J Immunol Methods.2001 Nov1 such as 381-388.Haidaris CG; 257 (1-2): 185-202, Griffiths, people EMBO J. such as A.D., 13,3245-3260 (1994) .Nissim, people EMBO J. such as A., 13, the method for describing among the 692-698 (1994) makes up mouse source antibody library.
2) screening
The antibody library bacterial strain of recovery is added 14 milliliters of fresh LB substratum for 1 milliliter, in 50 milliliters of triangular flasks, cultivated 16 hours for 37 ℃.
12000rpm high speed centrifugation 10 minutes is transferred to supernatant liquor in 50 milliliters of aseptic centrifuge tubes, preserves standby.Guarantee that its titre should be 2 * 10
11More than.At first use the human IgG1 as antigen, the ordinary method bag is by 25 ml cells culturing bottles.Add in the cell bottle behind the bag quilt and be no less than 3 * 10
10Phage, 37 ℃ of incubations 1 hour.Collect the supernatant liquor in the culturing bottle, standby.The CTLA4-Fc fusion rotein is as antigen, with the ordinary method bag by 25 ml cells culturing bottles.Bag by after the cell bottle in add to go up the supernatant liquor that the step collects, 37 ℃ of incubations 1 hour.Outwell the liquid in the bottle, wash culturing bottle 10 times with 10 milliliters of PBS that added 1%Tween-20.The TG1 cell that adds 1 milliliter of logarithmic phase in culturing bottle, 37 ℃ of incubation concussions were cultivated 16 hours.
Repeat the described step 4 of epimere time.
Cell dilution to 10 with above-mentioned acquisition
5Cultivate being added with on 1.5% agar plate of 0.1 penbritin after the cells/ml, obtain mono-clonal.
Get being cloned on the 96 hole depth orifice plates on the above-mentioned flat board and cultivate, the clone in every hole does 960 clones (10 96 orifice plates) altogether.
Above-mentioned deep-well plates after centrifugal 20 minutes, is transferred to new aseptic deep-well plates with supernatant at 5000RPM on the 96 orifice plate whizzers, be preserved in after sealing 4 the degree standby.
Get 10 of 96 orifice plates, add in every hole the conventional bag of CTLA4 (10 mcg/ml) 10 microlitres by after, add supernatant 10 microlitres of above-mentioned preservation respectively, 37 ℃ of incubations after 1 hour with the PBS washing that contains 1%Tween-20 20 times.The goat-anti M13 monoclonal antibody (available from Pharmacia company) that adds 1 microlitre HRP mark, 37 ℃ of incubations use the PBS of 1%Tween-20 to wash 10 times after 30 minutes.
Add and contain PBS 200 microlitres of 0.025%DAB developer and the H of 1 microlitre 1%
2O
2The 37 ℃ of incubation colour developing was read 595 nanometers after 20 minutes on plate reading machine photoabsorption.
Determine the hole that color reaction is strong according to the photoabsorption reading, the corresponding clone in these holes is the stronger antibody variable region clone of avidity.This test-results filters out 357 positive colonies altogether, determines wherein 5 the strongest clones of avidity according to its reading, is used for next step research.
3) the antibody variable region encoding sequence is to the clone of expression vector
Above-mentioned 5 clones' bacterial strain is increased, then according to the plasmid DNA extracting and purifying test kit (Wizard of manufacturer's specification sheets in 100 milliliters of LB substratum with Promega company
Plus SV Minipreps DNAPurification System) plasmid DNA purification.
With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after NheI Restriction Enzyme enzyme is cut above-mentioned plasmid DNA, get band about 363bp and carry out glue and reclaim, the gained fragment is variable region of heavy chain.Behind the pcr amplification, carry out sequencing.With XbaI with on 1.5% agarose gel electrophoresis, separate endonuclease bamhi after BsiWI Restriction Enzyme enzyme is cut above-mentioned plasmid DNA, get band about 336bp and carry out glue and reclaim, the gained fragment is variable region of light chain.Behind the pcr amplification, carry out sequencing.After determining correct sequence, chemical synthesis is synthesized the light chain full length sequence, 5 ' end design XbaI enzyme cutting site, and 3 ' end design BamHI restriction enzyme site, constant region is a mouse κ constant region of light chain sequence.
The PCR method is from mouse boosting cell amplification IgG1 CH sequence, and the CH1 initial amino acid sported Ala-Ser, contained the NheI restriction enzyme site simultaneously, 3 ' end design BamHI restriction enzyme site, after amplification back sequence verification was errorless, NheI and BamHI double digestion IgG1 constant region fragment and pMG18 were connected into pMG18 with the IgG1 constant region, the novel vector called after pM that makes up, as shown in Figure 2.
Cut expression vector pM with XbaI and NheI Restriction Enzyme enzyme.The restriction enzyme mapping of this expression vector as shown in Figure 2.Above-mentioned variable region of heavy chain is inserted in the XbaI/NheI site of this expression vector then.Equally, utilize XbaI and BamHI Restriction Enzyme the light chain of antibody full-length cDNA to be inserted in the XbaI/BamHI site of pM carrier, thereby make up the expression vector pM (H+L) that is contained recombinant anti-CTLA 4 heavy chain of antibody total length and light chain full-length gene respectively, as shown in Figure 3.
4) screening of the transfection of Chinese hamster ovary celI and recombinant clone
The expression vector that has antibody gene of above-mentioned structure is inoculated in 100 milliliters of LB substratum in the transformed into escherichia coli DH5 α bacterial strain respectively and increases, with ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA Purification Kit) the extracting and purifying plasmid DNA of Qiagen company.The liposome method test kit of employing Invitrogen company (lipofectamine 2000 transfection reagent, 11668-027), with the plasmid DNA cotransfection Chinese hamster ovary celI of above-mentioned purifying, carry out with reference to the specification sheets of producer by operation.
The Chinese hamster ovary celI that transforms carries out the selection in continuous 9 weeks on MTX selection substratum, cultivate in the enterprising limit by row dilution of 96 orifice plates at last, carries out continuously 3 times, carry out mono-clonalization.
The monoclonal cell of choosing ties up on RPMI 1640 substratum and cultivates, and supernatant is carried out the test of Western trace, judges expression intensity according to staining reaction, picks out 12 and expresses strong clone as the candidate cell strain.
5) Purification of Monoclonal Antibodies
Go out said monoclonal antibody with the direct separation and purification from cells and supernatant of Protein A affinity column, prove that through the SDA-PAGE electrophoresis products therefrom purity is greater than 90%.The product of affinity chromatography passes through sieve chromatography once more, has obtained the sample of purity＞98%.These samples are used for following further analysis and research.
The expression intensity of embodiment 2 antibody genes in Chinese hamster ovary celI
12 high expression level candidate clones that above-mentioned screening is obtained are incubated in the tissue culture ware of 10cm, the expression amount of measuring antibody with the ELISA method as described below.Sheep anti-mouse igg (Fc) is wrapped quilt in elisa plate, 4 ℃ are spent the night, sealed 2 hours in 37 ℃ through 2%BSA, add culture supernatant to be measured and standard substance (mouse IgG1), hatched 2 hours for 37 ℃, add HRP-sheep anti-mouse igg (κ)) carry out association reaction, hatched 1 hour for 37 ℃, add TMB in 37 ℃ of effects 10 minutes, use H at last
2SO
4Termination reaction is surveyed A
450Value.The expression amount that has shown 12 candidate clones that above-mentioned screening obtains in the following table 1.
The expression amount of table 1 candidate clone in Chinese hamster ovary celI
| The cell strain numbering | 1A2 | 2B7 | 3C4 | 4D7 | 5E3 | 6F7 | 7G2 | 8H6 | 9A1 | 8F4 | 9H7 | 6D2 | 5F3 |
| Expression amount (mcg/ml) | 289.4 | 142.6 | 302.4 | 112.9 | 178.9 | 146.8 | 201.5 | 295.2 | 165.7 | 132.9 | 167.2 | 156.2 | 129.3 |
As can be seen from Table 1,3C4,1A2 and 8H6 have very high expression level.
The dna sequencing of embodiment 3 anti-CTLA 4 monoclonal antibody genes
According to pedigree, the anti-CTLA 4 antibody gene of the 8H6 cell strain of above-mentioned acquisition is carried out dna sequencing.The result is as follows: SEQ ID NO:3 has shown that the aminoacid sequence of 8H6 chain variable region gene sequence (5 ' to 3 ') its supposition of 336bp is presented among the SEQ ID NO:1; SEQ ID NO:4 has shown 8H6 heavy chain variable region gene sequence ((5 ' to 3 ') 363bp), and the aminoacid sequence of its supposition is presented among the SEQ ID NO:2.
Embodiment 4 monoclonal antibody avidity researchs
Cell culture fluid to each clone carries out purifying by the following method.Centrifugal cell and the cell debris removed of 10000rpm, filter membrane ultrafiltration and concentration to 1/10 volume of 100Kd molecular weight cut-off, the ultrafiltration damping fluid is 100mMTris-HCl, pH7.5.Cross the SPA-sepharose affinity column, sample solution is 100mM Tris-HCl, and pH7.5, elutriant are the 20mM citric acid, pH3.0,100mM NaCl.Molecular sieve (Sephadex G200) chromatography.Elutriant is 100mM Tris-HCl, and pH7.5 gets pure product.
Avidity measure to adopt the Scatchard analytical method (people such as Munson, 1980, Anal.BioChem. 107:220) carries out.The result shows that the avidity of 8H6,3C4 and three kinds of monoclonal antibodies of 1A2 reaches 5.5 * 10 respectively
-10, 8.5 * 10
-9With 8 * 10
-8
Embodiment 5 anti-CTLA 4 antibody test CTLA4:Fc fusion roteins and with the comparison of commercially available antibody
1. reagent and material
1.1 anti-CTLA 4 antibody: the engineering cell of structure (8H6) is through the serum-free fermentation, and the proteinA affinitive layer purification obtains.
1.2 commercial anti CTLA4 monoclonal antibody, R﹠amp; D, MAB325.
1.3 recombinant human CTLA 4-Fc fusion rotein, R﹠amp; D, 325-CT/CF.
1.4 how anti-goat anti-human igg (H+L) is, HRP mark, extent of dilution 1: 1000-4000, Southern Biotech.
1.5HRP chromogenic substrate: TMB, Shanghai Xiamen Kehua divides A liquid and B liquid, face with before, both equal-volumes mix.
1.6 stop buffer: 0.5mol/L sulfuric acid.
1.7pH=7.2 PBS: take by weighing KH
2PO
40.21g, NaCl 9.0g, Na
2HPO
4.12H
2O 0.97g adds the injection water and is dissolved to 1000ml.
1.8 bag is cushioned liquid: the sodium carbonate buffer of 20mmol/l pH=9.6.
1.9 sealing damping fluid: take by weighing the 3g bovine serum albumin, the PBS that adds pH7.2 is dissolved to 100ml.
1.10 lavation buffer solution: get 1ml Tween20, add PBS to 1 liter.
2. method: sandwich ELISA method
2.1 recombinant anti-CTLA 4 antibody and commercially available similar antibody dilution are cushioned in the liquid in bag, concentration 1 μ g/ml wraps by 96 hole enzyme plates 0.1ml/well, 37 ℃ of 2h.
2.2 washing: lavation buffer solution washing 2 times all pats dry on thieving paper at every turn.
2.3 sealing: the sealing damping fluid is filled it up with 37 ℃ of 2h behind the hole.
2.4 washing: lavation buffer solution washing 3 times all pats dry on thieving paper at every turn.
2.5 add antigen: adding is diluted to 25,12.5,6.25,3.1,1.55 with the sealing damping fluid, CTLA4-Fc (ng/mL), and 0.1ml/well establishes 2 multiple holes.
2.6 37℃ 2h。
2.7 washing: lavation buffer solution washing 5 times all pats dry on thieving paper at every turn.
2.8 the goat anti-human igg two of adding HRP mark is anti-: add with two of PBS dilution in 1: 2000 and resist 37 ℃ of 1h.
2.9 washing: lavation buffer solution washing 5 times all pats dry on thieving paper at every turn.
2.10 colour developing: add chromogenic substrate 100 μ L/ holes, lucifuge colour developing 5～20min (developing time is decided on the colour developing situation) adds stop buffer 50 μ L/ holes.
2.11 microplate reader reading: measuring the 450nm photoabsorption, is reference wavelength with 630nm.
2.12 do linear regression equation with standard substance concentration (logarithm) and A450/630nm value.
It is highly sensitive that antibody of the present invention detects CTLA4-Fc with this method, can reach 2ng/mL, high specificity, and commercially available similar antibody test CTLA4-Fc sensitivity has only 20ng/ml.The sensitivity of antibody of the present invention is 10 times of commercially available antibody, as shown in Figure 4.
Antibody of the present invention has high specificity, avidity advantages of higher, and is highly sensitive in commercially available antibody as detecting antibody, relatively has obvious progress with commercially available antibody, and therefore, antibody of the present invention can substitute the commercial anti body and be used to prepare the reagent that detects CTLA4 concentration.
In addition, commercially available detection kit is normally utilized ELISA double antibody sandwich method principle, in advance with antibody sandwich in test kit, be used to detect the corresponding antigen material, this method is easier, quick.Therefore, antibody of the present invention also can substitute the coated antibody in the existing detection kit, is used to prepare the test kit that detects CTLA4 concentration.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Sequence table
＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd
＜120〉a kind of recombinant anti-CTLA 4 monoclonal antibody and its production and use
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<151>2006-05-17
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gaggttcagc tccagcagtc tgggactgtg ctggcaaggc ctggggcttc agtgaagatg 60
tcctgcaagg cttctggcta cacctttacc agctactggg tgccctgggt aaaacagagg 120
cctggacagg gtctggaatg gattggcact atttctcctg gaaatggtgc tactcgctac 180
aaccagaagt tcaagggcaa ggccaaactg actgcagtca catctgccag cactgcctac 240
atggagctca gcagcctgac aaatgaggac tctacggtct attactgttc aagaggcaac 300
gactatcgtt accacagatc ggctgacagg ggccaaggga ctctggtcac tgtctctgca 360
gcc 363
Claims (10)
1. recombinant anti-CTLA 4 monoclonal antibody, it comprises variable region of heavy chain and variable region of light chain, it is characterized in that, and variable region of light chain has the aminoacid sequence shown in the SEQ ID NO:1, and variable region of heavy chain has the aminoacid sequence shown in the SEQ IDNO:2.
2. a dna molecular is characterized in that, the described monoclonal antibody of its coding claim 1.
3. the described dna molecular of claim 2, it is characterized in that, this dna molecular contains the nucleotide sequence of the described monoclonal antibody variable region of light chain of coding shown in the SEQ ID NO:3, and the nucleotide sequence of the described monoclonal antibody variable region of heavy chain of coding shown in the SEQ ID NO:4.
4. an expression vector is characterized in that, the expression regulation sequence that it contains the described dna sequence dna of claim 2 and links to each other with this series of operations.
5. an eukaryotic host cell is characterized in that, it is transformed by the described expression vector of claim 4.
6. the described eukaryotic host cell of claim 5 is characterized in that the eukaryotic host cell of being addressed is a Chinese hamster ovary celI.
7. a method of utilizing the described antibody test CTLA4 of claim 1 concentration is characterized in that, described method is the ELISA method.
8. one kind prepares the described monoclonal antibody method of claim 1, it is characterized in that this method comprises:
A) provide an expression vector, the expression regulation sequence that this expression vector contains the described dna sequence dna of claim 2 and links to each other with this series of operations;
B) transform eukaryotic host cell with the described expression vector of step a);
C) eukaryotic host cell of gained culturing step b under the condition that is fit to described monoclonal antibody expression); With
D) separation and purification obtains described monoclonal antibody.
9. the application of the described monoclonal antibody of claim 1 in the reagent of preparation detection CTLA4 concentration.
10. the application of the described monoclonal antibody of claim 1 in the test kit of preparation detection CTLA4 concentration.
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| CN2007101014904A CN101074264B (en) | 2006-05-17 | 2007-04-23 | Recombinant anti-CTLA4 monoclonal antibody, its production and use |
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| CN101628940B (en) * | 2008-07-15 | 2011-11-23 | 中国科学院生物物理研究所 | Monoclonal antibody and application thereof |
| CN105296433A (en) * | 2014-08-01 | 2016-02-03 | 中山康方生物医药有限公司 | CTLA4 antibody, pharmaceutical composition thereof and application of pharmaceutical composition |
| WO2017198212A1 (en) * | 2016-05-19 | 2017-11-23 | 苏州康宁杰瑞生物科技有限公司 | Single domain antibody for ctla4 and derived protein thereof |
| CN108218987A (en) * | 2016-12-21 | 2018-06-29 | 南京金斯瑞生物科技有限公司 | High-affinity, high specific, more antigen recognizing epitopes the anti-human CTLA4 antibody with higher function |
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| CN108218987A (en) * | 2016-12-21 | 2018-06-29 | 南京金斯瑞生物科技有限公司 | High-affinity, high specific, more antigen recognizing epitopes the anti-human CTLA4 antibody with higher function |
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| CN101074264B (en) | 2011-08-17 |
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