CN1772766A - Acetylcholinesterase monoclonal antibody related to resisting apoptosis and its use - Google Patents
Acetylcholinesterase monoclonal antibody related to resisting apoptosis and its use Download PDFInfo
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Abstract
On the basis of found apoptosis related acetylcholinesterase (AR-AChE), the present invention immunes mouse with purified AR-AChE as antigen to obtain hybrid tumor through cell fusion and, especially, obtain monoclonal antibody expressing and secreting AR-AChE and its hybrid tumor cell lines ARA-M1, ARA-M5 and ARA-M7, and amplification generating antibodies ARA-M1, ARA-M5 and ARA-M7. These antibodies may be used in screening antitumor medicine, judging chemotherapeutic effect on tumor patient and preparing medicine for diagnosing and/or treating retrogressive nerve diseases, such as AD apoptosis analysis, pre-clinical and clinical research and diagnosis of apoptosis and detection of apoptosis expression level in acute organ damage. The present invention also provides the immunoassay process, kit, etc for the quantitative and/or semi-quantitative analysis on AR-AChE.
Description
Technical field
The present invention relates to biological technical field, the monoclonal antibody and the application thereof of the relevant acetylcholinesterase (AR-AChE) of particularly a kind of anti-apoptotic.
Background technology
(EC 3.1.17, acetylcholinesterase are the lytic enzymes of neurotransmitter acetylcholine AChE) to acetylcholinesterase, belong to glycoprotein.Mainly be present in the electric organ of electric ray, mammiferous cholinergic nerve, neuromuscular junction, erythrocyte membrane (Zakut-H; Et al:J-Clin-Invest.1990,86 (3): (A.Shafferman and B.Velan 900-8) and in the rodentine megalokaryocyte thrombocyte, Multidisplinary approaches to cholinesterase functions.1992 Plenum Press, New York).AChE can be divided into neuromuscular type and erythrocytic form with the position.Can be divided into different subunits poly builds (heteromeric class) and identical subunit's poly build (homomericclass) according to molecular structure.The catalytic subunit of different subunits poly build is connected or is connected with lipid with triple helical collagen subunit.Identical subunit poly build is divided into wetting ability (hydrophilic) and lipotropy hypotypes such as (glycophospholipid-linked) again.The about 260KDa of AChE tetramer molecular weight on the erythrocyte membrane.The function of AChE is the hydrolysis neurotransmitter acetylcholine at cholinergic nerve system, makes it to generate choline and acetate.Also there is report to think that AChE has the hydrolysis of protein activity of serine protease.In recent years, since also detect in the tumor cell line of vitro culture AChE mRNA transcribe and the serum of the ovarian cancer patients after chemotherapy in detect the AChE activity, there is the author to think that AChE with tumour relevant (Lev-Lehman-E takes place, et al:Blood.1997,89 (10): 3644-53).
The contriver finds that at first mammalian cell expresses AChE (Zhang XJ in apoptotic process, Yang L, Zhao Q, CaenJP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF., Induction ofAcetylcholinesterase Expression during Apoptosis in Various Cell Types.
Cell Death And Differentiation9 (8): 790-800,2002.), cell strain (Yang L, Heng-Yi He, the Xue-Jun Zhang Increased expression of intranuclear AChE involved in apoptosis ofSK-N-SH cells. in neural system source
Neuroscience research42 (4): 261-268,2002.) same phenomenon is also arranged.For the further effect of research AChE in apoptosis, the contriver has set up the separation method of this enzyme, and (" obtaining the method for brain type AChE from cells of mamma animals " authorizes.The patent No.: ZL 97 1 25216.5; Qi-Huang JIN, Yu-Fang SHI, Heng-YiHE, Kelvin KW Ng, Hua JIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun Zhang Isolation ofAChE from Apoptotic Human Lung Fibroblast Cells by Antibody Affinity Chromatography.
BioTechniques33 (4): S92-S97,2002.).Since the cell of this expression of enzymes, the time of appearance, the function of execution, the shearing of posttranslational modification even mRNA (" a kind of human acetylcholinesteraseisomer isomer protein white (AR-AChE) and gene coded sequence ", application number: 01105781.5, date of application, 2001/3/23.), be different from the neuromuscular type and the erythrocytic form AChE that have reported fully, the AChE that the contriver expresses apoptotic cell is called AR-AchE (AR-AChE) (Xue-Jun Zhang, Lei Yang, Qi-Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-YiHe, Kelvin NG and Zi-Qing Jiang.Various apoptotic mammalian cells expressapoptosis-related acetylcholinesterase (AR-AChE).
Acta biochimica et biophysica Sinica35 (2): pp213,2003.).Utilize apoptotic cell to express the characteristics of AR-AChE, the contriver can (" screening anti-tumor medicine method ", CN 1186859 have authorized with the method screening antineoplastic drugs that detects this enzymic activity.The patent No.: ZL 97125220.3) and as the apoptotic cell mark.
At neuromuscular type and erythrocytic form and electric ray AChE, people have prepared polyclonal antibody and monoclonal antibody (polyclonal anti-AChE antibody (Institute of Pharmacology and Toxicology, Academyof Military Medical Sciences, Beijing, PR China); Antibody to AChE (C-16) (catalognumber, sc-6430, Santa Cruz Biotechnology) is an affinity-purified goat anti-thecarboxy terminus of human acetylchol inesterase; AChE-antibody (BD Biosciences, SanJose, CA, USA)), the antibody of wherein discerning the neuromuscular type also can be discerned AchE (Zhang XJ, the YangL that apoptotic cell is expressed, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF, Inductionof Acetylchol inesterase Expression during Apoptosis in Various Cell Types.
Cell Death And Differentiation9 (8): 790-800,2002.).Owing to do not have the antibody (AChE its nonrecognition normal nerve synapse on) of specificity at present at AR-AChE, use immunological method, AChE goes to judge that apoptotic cell is also very difficult by identification.
The contriver finds not express cell great expression AChE in apoptosis process of AChE, mainly focuses in the nucleus, and along with nucleus is formed apoptotic body, AChE just is present in the apoptotic body.The contriver has set up with detecting the active method of AChE, the method (method that screening anti-tumor medicine and clinical chemotherapy effect are judged, China Patent No. 97 1 25220.3) of identification apoptotic cell and judgement chemotherapy effect.Because active the detection, can only be to the protein qualitative analysis, and, there is butyrylcholine esterase (BuChE) to have the activity that can disturb AChE in the serum, influence the activity detection of AChE.
If the monoclonal antibody of special anti-AR-AChE is arranged, just can from various types of AChE, distinguish AR-AChE.This specificity has been arranged, just can be used for discerning apoptotic cell, the apoptosis phenomenon is used for fundamental research, the theory and the clinical study of senile dementia (AD), AD auxiliary diagnosis and treatment; Judgement of chemotherapy effect or the like, and can carry out sxemiquantitative and quantitative analysis.Therefore be necessary to develop the monoclonal antibody of special anti-AR-AChE.
Summary of the invention
The purpose of this invention is to provide the monoclonal antibody of the relevant acetylcholinesterase (AR-AChE) of a kind of anti-apoptotic and produce the mouse hybridoma cell system of this antibody.
Another object of the present invention provides the purposes of the monoclonal antibody of anti-AR-AChE, the activity of AR-AChE and expression amount when antibody of the present invention can be used to measure apoptosis.Further, this antibody can be prepared into test kit, screening antineoplastic drugs, measure the effect of antitumor drug treatment, nerve degenerative diseases such as auxiliary diagnosis AD, and detect apoptosis expression level in the acute organ infringement, for the doctor provide patient fall ill with more after reference data.
A further object of the present invention provides a kind of immunoassay of screening the method for antineoplastic chemotherapy medicine and detecting AR-AchE.
A first aspect of the present invention provides a kind of monoclonal antibody, the described antibodies specific recognizing cells apoptosis acetylcholinesterase of being correlated with.
Preferably, said monoclonal antibody is CCTCC NO:-C200413 by preserving number, CCTCC NO:-C200414, the mouse hybridoma cell system secretion of CCTCCNO:-C200415.
Said monoclonal antibody comprises humanized antibody.Preferably, above-mentioned antibody is to comprise inhuman variable region and people's light chain and a kind of chimeric mAb of CH.
A second aspect of the present invention provides a kind of hybridoma cell line, it is characterized in that, described hybridoma cell line produces said monoclonal antibody.Preferable, above-mentioned hybridoma is a mouse hybridoma cell system.Preferably, above-mentioned hybridoma is preserving number CCTCC NO:-C200413, CCTCC NO:-C200414, the mouse hybridoma cell system of CCTCC NO:-C200415.The present invention also provides above-mentioned hybridoma cell line excretory monoclonal antibody.
A third aspect of the present invention provides a kind of immunoassay, and described method is carried out with at least a said monoclonal antibody.Sample in the above-mentioned immunoassay is a blood, tissue or cerebrospinal fluid, preferred blood supernatant.Above-mentioned immunoassay can use elisa technique or immunochromatography technique to carry out.Preferably, said monoclonal antibody is preserving number CCTCC NO:-C200413, and CCTCC NO:-C200414, the mouse hybridoma cell of CCTCC NO:-C200415 are the monoclonal antibody of one or more hypotypes of excretory.
A fourth aspect of the present invention provides a kind of test kit, and described test kit contains at least a said monoclonal antibody.Preferably, said monoclonal antibody is selected from ARA-M1, ARA-M5, among the ARA-M7 (hybridoma cell line is seen the 7th page of explanation) any.Test kit of the present invention can be used for screening antineoplastic drugs, judge the antitumor drug result of treatment, diagnosis of neurodegenerative diseases and detect the AR-AchE expression level, the especially expression level of AR-AChE etc. in the acute organ infringement.
A fifth aspect of the present invention provides a kind of composition, and described composition comprises at least a said monoclonal antibody and pharmaceutically acceptable carrier and/or vehicle.Preferably, above-mentioned composition comprises preserving number CCTCC NO:-C200413, and CCTCCN0:-C200414, the mouse hybridoma cell of CCTCC NO:-C200415 are the monoclonal antibody of one or more hypotypes of excretory.The present composition is used for the treatment of the caused disease of AR-AchE unconventionality expression, comprises nerve degenerative diseases.
A fifth aspect of the present invention provides the application of said monoclonal antibody in screening antineoplastic drugs.
A sixth aspect of the present invention provides a kind of method of screening antineoplastic drugs, and it may further comprise the steps:
(1) cultivator tumor cell line;
(2) add drug candidate in the cell strain nutrient solution in step (1), detect the expression amount of AR-AchE with said monoclonal antibody; Promote that the drug candidate that AR-AchE occurs or expression amount increases is exactly the medicine that promotes tumor death, otherwise described drug candidate is invalid.
Preferably, said monoclonal antibody is selected from ARA-M1, ARA-M5, among the ARA-M7 any.
Preferably, after adding drug candidate, detect AR-AChE in 18-24 hour.
A seventh aspect of the present invention provides the application of said monoclonal antibody in judging the antitumor drug result of treatment.
A eighth aspect of the present invention provides a kind of method of judging the antitumor drug result of treatment, it comprises step: detect the expression amount that tumour patient uses AR-AchE in the 8-168 hour peripheral blood in antitumor drug treatment back, and with treatment before relatively; The expression amount of AR-AchE increases, and shows that antitumor drug is effective, and no change or expression amount do not increase, and then antitumor drug is invalid.
Be 12-120 hour preferred detection time.
The 9th aspect of this aspect provides the application of said monoclonal antibody in the medicine of preparation diagnosis or treatment nerve degenerative diseases.
The tenth aspect of this aspect provides the application of said monoclonal antibody in preparing the test kit that detects AR-AchE expression level in the acute organ infringement.
The contriver once found the isomer (isoform) of acetylcholinesterase (AChE) from apoptotic cells, called after AR-AChE, and applied for patent (a kind of human acetylcholinesteraseisomer isomer protein white (AR-AChE) and gene coded sequence.Application number: 01105781.5, date of application, 2001/3/23.)。The contriver found that a lot of cell strains expressed AChE (Zhang XJ when apoptosis afterwards, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression during Apoptosis in VariousCell Types.
Cell Death and Differentiation9 (8): 790-800,2002.), because distribution in the type of time of this expression of enzymes, cell, the cell, posttranslational modification and functional character are different from the AChE in cynapse type on the neurone and the AChE on the motor end-plate, the AChE on the erythrocyte membrane, the megalokaryocyte, therefore, the AChE that the contriver expresses apoptotic cell be called AR-AchE (apoptosis-related acetylcholinesterase, AR-AChE).The contriver is the antibody of the AR-AChE that expresses at apoptotic cell, is the anti-apoptotic acetylcholinesterase antibody (antiapoptosis-related acetylcholinesterase antibody) of being correlated with.At AR-AChE, the contriver can prepare monoclonal antibody and polyclonal antibody.
Present anti-spinous process type AChE antibody on the market also is different from the antibody of AChE on the anti-erythrocyte film.Antibody of the present invention is only discerned the AChE that apoptotic cell is expressed, the contriver is referred to as (AR-AchE, apoptosis-relatedacetylchol inesterase, abbreviation AR-AChE), though and anti-spinous process type AChE antibody may be discerned AR-AChE on the market, but do not have specificity, they do not discern the specificity of apoptotic cell, therefore can not be as the mark of apoptosis.The contriver has obtained as antigenic AR-AchE (AR-AChE) (Xue-Jun Zhang, Lei Yang, Qi-HuangJin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG and Zi-Qing Jiang.Variousapoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR-AChE) .Acta biochimica et biophysica sinica_35 (2): pp213,2003.).The contriver has prepared the monoclonal antibody of anti-AR-AChE on this basis.The anti-AR-AChE monoclonal antibody of special generation has been arranged, just can from various types of AChE, distinguish AR-AChE.This specificity has been arranged, just can be used for discerning apoptotic cell, the apoptosis phenomenon is used for fundamental research, AD research, AD auxiliary diagnosis and treatment; Judgement of chemotherapy effect or the like.This is the monoclonal antibody of the AR-AChE that expresses of the specific recognition apoptotic cell invented first in the world.
Therefore, (ZL 97 1 25216.5 with Chinese patent for the contriver, the method of acquisition brain type AChE from cells of mamma animals) from apoptotic cell, isolates AR-AChE, (Qi-Huang JIN, Yu-Fang SHI, Heng-Yi HE, Kelvin KW Ng, HuaJIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun Zhang Isolation of AChE from ApoptoticHuman Lung Fibroblast Cells by Antibody Affinity Chromatography.
BioTechniques33 (4): S92-S97,2002.).With its immune BaLb/c mouse, filter out 3 clones, special generation anti-apoptotic acetylcholinesterase (AR-AChE) monoclonal antibody of being correlated with.
Monoclonal antibody of the present invention can produce by hybridoma of the present invention, is to obtain from the nutrient solution of cultivating hybridoma of the present invention therefore.Yet, produce monoclonal antibody method of the present invention and be not particularly limited, if but genetically engineered antibody capable combine with the AR-AChE protein-specific, it is also within the scope of the invention.
Hybridoma of the present invention produces the monoclonal antibody of identification AR-AChE, and can be by merging acquisition through the splenocyte of the animal of AR-AChE protein immunization or lymph-node cell and myeloma cell.
Hybridoma cell line ARA-M1 of the present invention, ARA-M5, ARA-M7 is deposited in Chinese typical culture collection center (Wuhan) according to " microorganism that is used for patented procedure preserves Budapest pact " on September 21st, 2004, preserving number respectively is CCTCC NO:-C200413, CCTCC NO:-C200414, CCTCC NO:-C200415.
Hybridoma of the present invention can produce with cell-fusion techniques known in the art.Therefore, as the animal of immunogen immune except the people, splenocyte or lymph-node cell and myeloma cell's fusion with this animal produce hybridoma with AR-AChE, discern the hybridoma of the monoclonal antibody of AR-AChE from wherein selecting generation, thereby obtained hybridoma of the present invention.
The immunogen of above-mentioned preparation hybridoma is not particularly limited, and can be reorganization AR-AChE, the AR-AChE albumen of purifying or contain the tumour cell of AR-AChE.For example can or contain the engineering strain of the proteic fusion rotein of AR-AChE by culture expression AR-AChE albumen on suitable medium.
Go up when antibody that inhuman subject produced or the antibody that derives from non-human antibody's genetic expression is used for human body when treatment, they may produce immunne response being identified as external antibody in varying degrees in patient's body.Making this immune response minimize or eliminate this immunoreactive a kind of method is to produce the chimeric antibody derivative, promptly unites the antibody molecule of inhuman animal variable region and people's constant region.Therefore, the present invention also comprises humanized antibody, and the application of humanized antibody in the medicine of preparation treatment nerve degenerative diseases (as AD).This area skill industry personnel can utilize Monoclonal Antibody humanized antibody of the present invention.
These antibody remain with the epi-position binding specificity of primary monoclonal antibody, but may less immunogenicity when to people's administration, therefore more may be stood by the patient.
Can prepare chimeric mAb with recombinant DNA technology known in the art.For example, the gene of the gene substitution coding non-human antibody molecule constant region of usefulness coding human constant region (referring to, Robinson etc., PCT patent disclosure text PCT/US86/02269; Akira, etc., european patent application 184,187; Or Taniguchi, M., european patent application 171,496).The variable region part that part replaces not relating to conjugated antigen that is equal to of variable region that can be by personnel selection further makes chimeric antibody " humanization ".
The Application Areas of monoclonal antibody of the present invention is not particularly limited, and if can be used for immunoassay, comprises quantitatively or half-quantitative detection.The existence that monoclonal antibody of the present invention can detect AR-AChE whether, as the foundation of judging the chemotherapy of tumors result.
Antigen-binding fragments of antibodies of the present invention be meant can with the epitope bonded antibody fragment of AR-AchE.
The animal that produces hybridoma of the present invention through immunity is not particularly limited, and for example comprises: goat, sheep, cavy, mouse, rat and rabbit.Mouse preferably therein.
Immunoassay of the present invention is carried out with at least a monoclonal antibody of the present invention, and method can only be carried out with a kind of antibody, also can combine with the AR-AChE polyclonal antibody and carry out, or use two or more monoclonal antibodies (as ARA-M1 and ARA-M5) simultaneously.
As immunoassay of the present invention, comprise technology such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), western blotting, immunochromatography.Above-mentioned various immunoassay can be used for competition law or sandwich assay, with antigen or the TPPA target antigen or the antibody of marker mark.In above-mentioned various immunoassays, ELISA and immunochromatography technique are preferred.
Above-mentioned sandwich assay for example comprises: AR-AChE is clipped between the monoclonal antibody of the present invention and monoclonal antibody of the present invention or anti-AR-AChE polyclonal antibody with another hypotype of marking agent mark of a kind of hypotype of fixed, add substrate at marker (for example enzyme) etc. then, Show Color etc., thus the AR-AChE in the sample detected.Usually consider the superb monoclonal antibody of sensitivity as preferred traget antibody, because the polyclonal antibody that ground unrest is high with specificity is low compares, the specificity height of monoclonal antibody and ground unrest is low makes detection more accurate.
Above-mentioned competitive method is based on for example: the AR-AChE of the mark of AR-AChE and known quantity and monoclonal antibody quantitative competitive association reaction of the present invention in the test specimen.In the above-mentioned competitive method of mentioning especially, what add predetermined amount in containing the sample solution of AR-AChE is fixed on the antibody on the carrier and the AR-AChE with the marking agent mark of predetermined amount.Then, it is active or be not retained in marking agent activity on the carrier to measure the marking agent be retained on the carrier.To this, preferably almost add antibody and labelled antigen simultaneously.
For above-mentioned marking agent, can use radio isotope, for example
125I, enzyme, enzyme substrates, phosphorus, fluorescent substance, vitamin H and coloring material.In with these marking agents and antigen or antibodies, can use maleimide method (J.Biochem. (1976), 79,233), activation vitamin H method (J.Am.Chem.SOC. (1978), 100,3585) or hydrophobic bond method.
For the above-mentioned enzyme of mentioning, comprise for example peroxidase, alkaline phosphatase, beta-galactosidase enzymes and glucose oxidase.For the substrate that is used for this occasion, can select to be fit to the substrate of the used enzyme of this occasion, for example comprise: ABTS, luminol,3-aminophthalic acid cyclic hydrazide-H
2O
2, o-phenylenediamine-H
2O
2(at peroxidase), p-nitrophenyl phosphate, phosphoric acid methyl are paid shape ester, 3-(2 '-volution diamantane)-4-methoxyl group-4-(3 '-phosphorus acyloxy) phenyl-1,, 2-dioxetane (at alkaline phosphatase), p-nitrophenyl-BETA-D-semi-lactosi (at beta-galactosidase enzymes).
Can be 4-40 ℃ of reaction 1 minute-18 hours, color or fluorescence, phosphorescence or the colour developing amount of measurement result demonstration are carried out above-mentioned test then.In addition, can use so-called speed trial, it is incubated in 4-40 ℃ of scope carries out.
Can carry out radio-labeling easily with commercially available Bolton-Hunter reagent to above-mentioned antigen or antibody.For example, can be by Bolton-Hunter reagent being added in the solution (this solvent prepares by antigen or antibody are dissolved in the 0.1M sodium bicarbonate aqueous solution), passed through then 1-2 hour, and removed unreacted Bolton-Hunter reagent part with G-25 desalting column etc.
In addition, can use chloramine T method, the former method of iodine etc., carry out easily
125The I radio-labeling.
Above-mentioned fluorescent substance as fluorescein and Luo Daming, can be convenient to use active ester method or isocyanic ester method (" enzyme immunoassay technique ", Igaku Shoin published in 1987) and carry out mark.For above-mentioned coloring material, can use painted latex particle and Radioactive colloidal gold.
Immunoassay of the present invention is used monoclonal antibody of the present invention, therefore has outstanding characteristic, only identification is present in the AR-AChE in all apoptotic cells (comprising tumour or neurocyte), and can be owing to cross reaction other materials of error-detecting and normal AChE, therefore can carry out the very high mensuration of specificity.
In order to implement immunoassay of the present invention, can use test kit of the present invention.Test kit of the present invention contains at least a monoclonal antibody of the present invention, and can only contain a kind of monoclonal antibody.The monoclonal antibody of the present invention that is used for test kit of the present invention is not particularly limited, but can be the monoclonal antibody of any identification AR-AChE, can also be degradation production or its Fab of monoclonal antibody of the present invention, as F (ab ')
2, Fab ', Fab etc.
In test kit of the present invention, but monoclonal antibody predetermined fixed of the present invention on solid phase, monoclonal antibody of the present invention can also be with above-mentioned marking agent mark.
Test kit of the present invention can detect the sample contain AR-AChE with immunological method, as blood, and tissue or cerebrospinal fluid, whether and the variation of expression amount the existence that therefore can judge AR-AChE.
The solid phase that is used for test kit of the present invention is not particularly limited, but comprises for example polymkeric substance, as polystyrene, granulated glass sphere, magnetic-particle, microtiter plate, immunochromatography filter paper, glass filter or other insoluble carriers.
Test kit of the present invention also can contain other assemblies.Above-mentioned other assemblies are not particularly limited, but comprise enzyme, its substrate, radio isotope, phosphorus, fluorescent substance, coloring material, damping fluid and culture dish that mark is for example used, and the above-mentioned composition of mentioning.
Test kit of the present invention can be the test kit that is used to implement above-mentioned competitive method or above-mentioned sandwich assay.Yet preferably use the test kit of sandwich assay.Above-mentioned sandwich assay has advantage, and is promptly highly sensitive, and the reaction times that needs is short, and the accuracy rate height is easy to operate.Use above-mentioned immunochromatography technique, can prepare a kind of test kit, it makes testing method convenient and simple, and can be simply by the visual inspection judged result with it.When with elisa technique and when using above-mentioned sandwich assay, the formation of enzyme reaction product is determined by the test objective amount of substance, therefore can set up a kind of system, can pass through colour developing etc. easily, determine whether existing of AR-AChE with visual inspection, its O.D value of also available instrument test is carried out quantitative and semiquantitative detection.
The used sample of test kit of the present invention is not particularly limited, but for example comprises: blood and cerebrospinal fluid or tissue, preferred blood.
Monoclonal antibody of the present invention and test kit of the present invention can be used for screening antineoplastic drugs, auxiliary judgment antitumor drug result of treatment, the medicine of preparation diagnosis or treatment nerve degenerative diseases detects expression level and the variation of the AR-AChE that apoptosis produces in the acute organ infringement.
The method of above-mentioned screening antineoplastic drugs may further comprise the steps:
(1) cultivator tumor cell line;
(2) add drug candidate in the cell strain nutrient solution in step (1), detect the expression amount of AR-AchE with said monoclonal antibody or mentioned reagent box; Promote that the drug candidate that AR-AchE occurs or expression amount increases is exactly the medicine that promotes apoptosis of tumor cells, otherwise described drug candidate is invalid.
According to the present invention, discern AR-AChE with monoclonal antibody, can eliminate the influence that is present in other material in the sample, thus the existence of sensitivity that can be very high and specific detection AR-AChE.Since set up the hybridoma that produces at the monoclonal antibody of AR-AChE, now can the same monoclonal antibody of almost nonvolatil generation.
Diagnostic kit of the present invention uses monoclonal antibody, has high-caliber detection sensitivity, does not produce false negative or false positive problem, batch with criticize between without any difference.As the antibody of mark preferably use with solid phase carrier on the monoclonal antibody of monoclonal antibody different subtype, then be more convenient for controlling quality product and formulating unified standard.
The AR-AChE detection kit detects principle, with the anti-AR-AChE monoclonal antibody of the polyclonal antibody of the monoclonal antibody of anti-AR-AChE and anti-AR-AChE or another hypotype as immobilization antibody and enzyme labelled antibody, if there is AR-AChE in the testing sample, then form antibody-antigen-hrp-antibody complex, add TMB (enzyme is exempted from the series product that develop the color) substrate and produce color reaction, otherwise then do not have color reaction.The double-antibody sandwich elisa method is a kind of very sensitive detection technique, and ELISA susceptibility height, easy and simple to handle, and the development of necessary instrument equipment makes schedule of operation standardization and automatization, thereby has further improved stability.
The invention provides the hybridoma cell line of the anti-AR-AChE monoclonal antibody of special generation, can produce anti-people AR-AChE monoclonal antibody, can prepare test kit, be used for fundamental research, AD research, AD auxiliary diagnosis and treatment with this antibody by these clones; Judgement of chemotherapy effect or the like.
Up to the present, utilize serological method to judge the reagent of chemotherapy effect, have only M30 antibody.Two companies are arranged at present, and ALEXIS Biochemicals Corporation company and DiaPhqrma Group Inc. have released test kit-M30-Apoptosense
TMELISA Kit.This test kit main component is exactly a M30 antibody, a kind of mouse authentic monoclonal antibody, and hypotype is IgG2b, what its was discerned is a small segment of Keratin sulfate (Cytokeratin) 18 (CK18) C end.CK18 is epithelial moderate fiber, and during the epithelium apoptosis, activated kinases (Caspase) 3,6,7,9 is with the next small segment of the C end-grain cutting of CK18.Because this small segment only appears in the apoptotic cell, therefore, becomes the specific marker of epithelial cell apoptosis.Use M30 antibody, can discern the apoptosis of histocyte and culturing cell, also can detect the variation of M30 in the serum, judge chemotherapy effect with ELISA.But it is only at the relevant tumour of epithelium, and costs an arm and a leg.
The purposes of monoclonal antibody of the present invention is:
1. experiment in vitro screening antineoplastic drugs:
Can the research anti-cancer agent will screen in numerous drug candidates, inducing apoptosis of tumour cell be an important indicator.The inventive method can be screened anti-cancer agent in order to experiment in vitro.The human tumor cell line of vitro culture, the antitumor drug candidate that adds the various dose that will detect, after 18-20 hour, detect cell or cultivate liquid, occur and the change of amount by AR-AChE, judge screened medicine whether effectively, if AR-AChE increases, illustrate that this medicine induced apoptosis, it is exactly the effective antitumour medicine.
2. clinical tumor patient chemotherapy effect is judged
For different tumour (comprising leukemia), different patients is different to the reaction of various chemotherapeutics, and curative effect is fine on one's body this patient for the antitumor drug that has, and just bad to another patient's curative effect.The inventive method can help the clinician to select tumor chemotherapeutic drug targetedly, shoots the arrow at the target, and finds optimal drug in the shortest time, brings glad tidings to the patient.
Chemotherapeutics mainly is at tumour cell (normal cell is also arranged), makes it to take place apoptosis, thereby reaches the purpose of treatment.The present invention is based upon on the newfound basis of scientific research.Its principle is the cell great expression AR-AChE in apoptosis process that does not express AChE under the normal circumstances, and this enzyme is present in the apoptotic cell more stablely.Normal people or tumour patient are under the situation that does not have treatment, though in the body apoptosis is arranged constantly, quantity is few, and the AR-AChE in the macrophage phagocytic around the very fast quilt of apoptotic cell, apoptotic cell is not discharged in the blood flow.But, if effective chemotherapeutics, make a large amount of apoptosis of tumour cell, scavenger cell can't be engulfed all apoptotic cells very soon, perhaps be subjected to drug influence, the macrophage phagocytic ability reduces, and can not engulf apoptotic cell very soon, and at this moment a large amount of AR-AChE enters in the blood with apoptotic cell and apoptotic body.Get the protein content that the patients serum measures serum AR-AChE this moment, will find that the protein content of the AR-AChE of treatment beginning back 72 hours (the 3rd day) exceeds more than 1 times before treating.Importantly the contriver find cord blood CD 34+the hemopoietic stem cell apoptosis time do not express AR-AChE, measure the more representative meaning of blood AR-AChE.Therefore, measure patient treatment front and back serum AR-AChE protein content, just how know chemotherapy effect, help the clinician in the shortest time, to find the tumor chemotherapeutic drug of targeted the best, shoot the arrow at the target,, strive for valuable time for saving patient's life.If chemotherapy is invalid, can in time change medicine, thereby provide a kind of means that detect chemotherapy effect fast and effectively to the doctor, really realize individualized treatment.Certainly, also can analyze the result of treatment of other antitumor drugs with monoclonal antibody of the present invention.
3. research nerve degenerative diseases, particularly AD have diagnosis of being beneficial to and treatment
By brain tissue slice and cerebrospinal fluid analysis, can obtain the evidence of apoptosis amount.AChE can quicken the precipitation of beta-amyloyd peptide, this effect and irrelevant (the Inestrosa NC of classical enzyme catalysis, Alvarez A, Perez CA, Moreno RD, Vicente M, Linker C, Casanueva OI, Soto C, Garrido J, Acetylcholinesteraseaccelerates assembly of amyloid-beta-peptides into Alzheimer ' s fibrils:possiblerole of the peripheral site of the enzyme, Neuron, 199616 (4); 881-891.), and can form mixture with beta-amyloyd peptide, and mixture toxicity is than bigger (the Alvarez A of single beta-amyloyd peptide toxicity, Alarcon R, Opazo C, Campos EO, Munoz FJ, Calderon FH, Dajas F, Gentry MK, Doctor BP, De MelloFG, Inestrosa NC, Stable complexes involving acetylchol inesterase and amyloid-betapeptide change the biochemical properties of the enzyme and increase theneurotoxicity of Alzheimer ' s fibrils, J Neurosci, 199818 (9); 3213-3223.).The experiment of relevant AChE antisense nucleic acid directly illustrates AChE (the Shohami E that works in neuron loss, Kaufer D, Chen Y, Seidman S, Cohen O, Ginzberg D, Melamed-Book N, Yirmiya R, Soreq H, Antisenseprevention of neuronal damages following head injury in mice, J Mol Med, 200078 (4); 228-236.).This monoclonal antibody, except being used for discerning apoptosis evidence in brain tissue slice and the cerebrospinal fluid, outside diagnostic reagent, also may be used for as medicine (after particularly being prepared into humanized antibody), as be used to prevent that AChE from quickening the precipitation of beta-amyloyd peptide, stop AD to carry out the effect of sexual development thereby reach.Described medicine comprises monoclonal antibody of the present invention and pharmaceutically acceptable carrier and/or vehicle.
4. detect the development of acute organ infringement with more after
Disease such as fulminant hepatitis for example.The infringement of these acute organ has a large amount of apoptosis when taking place, and contains the AR-AChE of several times even tens of times high in the serum, by detecting its variation, can judge the development and change and the prognosis situation of the state of an illness.
Advantage of the present invention: the inventive method is by detecting by the antitumor drug interaction in vitro behind the cell of not expressing AChE, detect the AR-AChE activity of cell expressing, or measure serum AR-AChE content and identify whether apoptosis takes place, apoptotic cell AChE reacting positive, illustrate that this medicine induced apoptosis, it is the effective antitumour medicine, take this screening antineoplastic drugs easily and effectively, also can help the doctor to select responsive especially chemotherapeutics, to improve result of treatment at patient.Detect AR-AChE content with the inventive method and identify that the result of the apoptotic cell of vitro culture is consistent with classical way.And accurate positioning, method is simple, does not need expensive equipment, does not also need too expensive reagent, can do sxemiquantitative and quantitative analysis.
Description of drawings
Fig. 1. five months human fetal brain tissue frozen sections of induced labor (amplifying 800 times, * 800)
A. be immunofluorescence, 1 antibody is mouse anti human AR-AChE monoclonal antibody, and 2 anti-are goat anti-mouse igg antibody, Luo Daming (Rhodamine) mark, and 2 apoptotic cells show positive.
B. be Hoechst No.33258 dyeing, show all nucleus.
C. be the overlapping of A and B.
D. be the visible light photo.It is positive that this photo shows that 2 apoptotic cells show, other does not have the positive.
Fig. 2. the photo of Cord blood smear (* 800)
A. be immunofluorescence, 1 antibody is mouse anti human AR-AChE monoclonal antibody, and 2 anti-are goat anti-mouse igg antibody, the Luo Daming mark, and the karyocyte of apoptosis shows positive, red corpuscle is negative;
B. be Hoechst No.33258 dyeing, the showed cell nuclear substance, the full visual field has only a karyocyte, and all the other all are red corpuscle;
C. be the visible light photo, visible a large amount of red corpuscle, there is 1 karyocyte the centre.This figure illustrates that monoclonal antibody of the present invention discerns the AR-AChE of apoptotic cell specifically, and the AChE on the nonrecognition erythrocyte membrane.
Fig. 3. cracked apoptosis HELA experiment
Among the figure from left to right for apoptosis HELA lysate respectively a. do not add substrate, but replace with the PBS of equivalent; B. do not add antibody, do substrate reactions, as a result the positive; C. with d. for adding excessive antibodies, form mixture, centrifugal, get supernatant (c) and do substrate reactions respectively with precipitation (d), both are negative entirely as a result.
Fig. 4. detect the HELA cell with monoclonal antibody of the present invention
The HELA cell of cultivating, antibody is the mouse anti human AR-AChE monoclonal antibody (ARA-M5) that the contriver prepares, 1 antibody directly connects HRP, DAB reaction, normal HELA cell feminine gender, the apoptotic cell positive.
Fig. 5 .WESTERN BLOT
A. (CA USA) is WESTERN BLOT for BD Biosciences, San Jose, and antigen is from the HELA cell of apoptosis with the antibody of buying;
B. be WESTERN BLOT with ARA-M5, antigen is from the HELA cell of apoptosis;
C. be WESTERN BLOT with ARA-M5, antigen is from torpedo acetylcholinesterase (Sigma).
Fig. 6. the photo (* 400) of Cord blood karyocyte smear
A. be the visible light photo, black arrow points apoptotic cell, white arrow points normal cell;
B. be Hoechst No.33258 dyeing, the showed cell nuclear substance, the nuclear of apoptotic cell has formed apoptotic body, and Normocellular nucleus is complete;
C. be immunofluorescence, 1 antibody is mouse anti human AR-AChE monoclonal antibody, and 2 anti-are goat anti-mouse igg antibody, the Luo Daming mark, and there only have the cell of apoptotic body to show to be positive, and normal cell is negative, and this monoclonal antibody specific recognition apoptotic cell is described.
Fig. 7. rat cerebral tissue's section
A. antibody is the mouse anti human AR-AChE monoclonal antibody (ARA-M1) that the contriver prepares, and 1 antibody directly connects Rhodamine, red positive cell (AR-AChE has cross reaction with rat); Large stretch of nervous tissue does not have the positive, and the there has cholinergic nerve to exist, and the acetylcholinesterase of cynapse does not show the positive.
B.TUNEL dyeing, the positive is an apoptotic cell.
C. be A, B's is overlapping.TUNEL dyeing coincide finely with anti-AR-AChE positive region, illustrates that this Dan Kelong antibody discerns the AR-AChE of apoptotic cell specifically, and the AChE of the nerve synapse type that nonrecognition rat cerebral tissue expresses.
Fig. 8. five months human fetal brain tissue frozen section (* 800) photos of induced labor are the same visual field
A. be Hoechst No.33258 dyeing, showed cell nuclear;
B. be cytochemical Ache staining (Konovsky and Root method), isabelline (being Dark grey among the figure) is the AChE positive, as seen evenly there are not showed cell or nuclear profile, illustrate that resultant does not concentrate in cytoplasm or the nucleus, these are different fully with the distribution in the apoptosis that the contriver finds;
C. be the result that visible light photo and Hoechst No.33258 dyeing is taken pictures simultaneously, prove that further the AChE positive is not in nucleus.
Fig. 9. the photo (* 800) of the people SK-N-SH cell of vitro culture
Photo divides A, B, and C, D, E five parts are the same visual field.
A.TUNEL dyeing, the positive is an apoptotic cell;
B. be the visible light photo, there is a pyknocyte that does not have cell process the lower right of cell mass;
C. be immunofluorescence, 1 antibody is mouse anti human AR-AChE monoclonal antibody, and 2 anti-are goat anti-mouse igg antibody, the Luo Daming mark, and it is positive that the lower right has an apoptotic cells to show;
D. be hoechest dyeing, showed cell nuclear;
E. be A, C, D's is overlapping.The people SK-N-SH cell of vitro culture, normally can express a spot of nerve synapse type AChE, it is positive that this photo shows that 1 apoptotic cells shows, other does not have the positive, TUNEL dyeing coincide finely with anti-AR-AChE is positive, illustrate that this Dan Kelong antibody discerns the AR-AChE of apoptotic cell specifically, and the AChE of the nerve synapse type of nonrecognition SK-N-SH cell expressing.
Figure 10. AR-AChE half-quantitative detection in the serum before and after the tumour patient chemotherapy
Figure 11. the photo of P of Rats C12 cell
A. visible light photo.The PC12 cell of apoptosis, the substrate reactions positive is distributed in cytoplasm and the nuclear.
B. normal PC12 cell, copolymerization Jiao (CONFOCAL) photo.FITC shows that AChE expresses in normal viable cell, be distributed on cytoplasm and the film, does not have the positive in the nucleus, and blue look is HOECHST dyeing, showed cell nuclear.
C. visible light photo.Anti-AR-AChE antibody (ARA-M5), the DAB reaction, brown is positive, the dyeing of phenodin background, showed cell nuclear; The PC12 feminine gender alive of adherent growth, the apoptotic cell positive of pyknosis.
A Figure 12 .30 healthy newborns Cord blood and a routine tumour patient serum AR-AChE.
The OD value of normal newborn Cord blood AR-AChE is all below 0.2, near blank.The OD value of the 3rd day serum AR-AChE of one routine tumour patient chemotherapy is 3.1.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The reagent that does not specify among the present invention, instrument etc. are commercially available conventional products.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, Russell, molecular cloning: laboratory manual III (New York:Cold Spring Harbor Laboratory Press, 2001) condition described in, or the condition of advising according to manufacturer.
The preparation of embodiment 1 acetylcholinesterasemonoclonal monoclonal antibody related to resisting apoptosis and hybridoma
Preparation before I, the cytogamy
(1) immunization protocol
A, antigen: according to the method (patent No.: the ZL 97 1 25216.5 of contriver's patent; Qi-Huang JIN, Yu-Fang SHI, Heng-Yi HE, Kelvin KW Ng, Hua JIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun ZhangIsolation of AChE from Apoptotic Human Lung Fibroblast Cells by Antibody AffinityChromatography.
BioTechniquesI33 (4): S92-S97,2002) AR-AChE of acquisition HELA cell expressing.
B, mouse: the BaLb/c mouse, 6～10 ages in week, male mouse was 3.
People AR-AChE adds Fu Shi Freund's complete adjuvant (limit is used for the first time) or freund 's incomplete adjuvant, and equal-volume mixes, and uses two syringes, through T-valve, injects repeatedly, makes the two form water in oil chyle shape.
Initial immunity antigen (Ag) 1～50 μ g adds the subcutaneous multi-point injection of Fu Shi Freund's complete adjuvant
| (0.8～1ml 0.2ml/ point)
After ↓ 3 weeks
Immunizing dose is the same for the second time, and it is subcutaneous to add freund 's incomplete adjuvant
After ↓ 3 weeks
Immunizing dose is the same for the third time, does not add adjuvant, intraperitoneal injection (ip)
| (blood sampling is surveyed it and is tired the detection immune effect after 5～7 days)
After ↓ 2～3 weeks
Booster immunization, dosage 100 μ g, ip
After ↓ 3 days
Getting spleen merges
(2) feeder cell Turnover of Mouse Peritoneal Macrophages
Prepare Turnover of Mouse Peritoneal Macrophages the day before yesterday merging
Adopt 6～10 ages in week of BaLb/c mouse
↓
Draw neck to put to death and be soaked in 75% alcohol, sterilized 3～5 minutes
↓
Cut off skin with sterile scissors, expose peritonaeum
↓
Inject 6～8ml nutrient solution with asepsis injector
↓
Flushing repeatedly, the sucking-off washing fluid
↓
Put into the 10ml centrifuge tube, 1200 rev/mins centrifugal 5～6 minutes
↓
With the nutrient solution suspendible of 20% calf serum (NCS) or foetal calf serum (FCS), adjust cell count 1 * 10
5/ ml
↓
Add 96 orifice plates, 100 μ l/ holes
↓
Put into 37 ℃ of CO
2Incubator is cultivated
(3) myeloma cell
Two weeks before preparing fusion, the recovery myeloma cell.The contriver adopts myelomatosis SP2/0 clone, the RPMI1640 substratum, and the concentration 20% of calf serum, the maximum density of cell is no more than 10
6/ ml, enlarged culturing went down to posterity once with 1: 10 diluted passage in per 3～5 days.
(4) immune spleen cell
The preparation of splenocyte suspension: take out spleen under aseptic condition, the full nutrient solution that toos many or too much for use is washed once, and stainless steel sift is online in the horizontalization ware, counts after grinding to form cell suspension with the syringe nook closing member.
II, cytogamy are selected hybridoma
(1) cytogamy flow process
(1) the myeloma cell SP2/0 that takes the logarithm and grow, centrifugal 5 minutes of 1000rpm abandons supernatant, counts behind the full nutrient solution suspendible cell that toos many or too much for use, and gets required cell count, the full nutrient solution washing 2 times of toing many or too much for use.
(2) prepare the immune spleen cell suspension simultaneously, the full nutrient solution washing 2 times of toing many or too much for use.
(3) with myeloma cell and splenocyte by the mixed of 1: 10 or 1: 5 together, the full nutrient solution that toos many or too much for use in the 50ml plastic centrifuge tube is washed 1 time, 1200rpm, 8 minutes.
(4) abandon supernatant, with the dropper residual liquid that exhausts, in order to avoid influence the concentration of PEG.
(5) gently at the bottom of the attack centrifuge tube, make cell precipitation loosening slightly.
(6) at room temperature merge:
1. the 1ml45%PEG (Merek, molecular weight 4000) that adds preheating in 30 seconds contains 5%DMSO, and the limit edged stirs.
2. acted on for 100 seconds.
3. the incomplete nutrient solution that adds preheating stopped the PEG effect, added 1ml, 2ml, 3ml, 4ml, 5ml and 10ml respectively every 2 minutes.
(7) centrifugal, 800rpm, 6 minutes.
(8) abandon supernatant, earlier with the suspendible gently of 20% calf serum RPMI1640 about 6ml.
(9) according to the quantity of used 96 well culture plates, add complete culture solution, one 96 orifice plate of 10ml.
(10) the back cell suspension be will merge and 96 orifice plates that contain feeder cell, 100 μ l/ holes, 37 ℃, 5%CO added
2Incubator is cultivated.
One 96 orifice plate contains 1 * 10
7Splenocyte.
(2) HAT selects hybridoma
After merging 24 hours, add HAT and select nutrient solution.With 50 * HT and the HAT reagent of storing, add in the 50ml20% calf serum complete culture solution with 1ml.
50×HAT
H：5×10
-3M
A：2×10
-5M
T：8×10
-4M
HAT uses the HT nutrient solution instead after selecting nutrient solution to keep two weeks of cultivation, keeps and cultivates for two weeks, uses general nutrient solution instead.
III, detection of antibodies
The contriver adopts the ELISA screening method.
The cloning of IV, hybridoma and frozen
(1) cloning scheme
With the limiting dilution assay clone, program is as follows:
1. prepare feeder cell suspension (preparing) with before merging
2. the counting of positive porocyte, and accent cell count is 1～5 * 10
3/ ml
3. get 130 cells and put into 6.5ml and contain the feeder cell complete culture solution, i.e. 20 cell/ml, 100 μ l/ holes add A, B, C three rows are 2 cells in every hole.Remaining 2.9ml cell suspension is added the complete culture solution that 2.9ml contains feeder cell, and cell count is 10/ml, and 100 μ l/ holes add D, E, F three rows, are 1 cell in every hole.Remaining 2.2ml cell suspension is added the complete culture solution that 2.2ml contains feeder cell, 5/ml of cell count, and 100 μ l/ holes add G, H two rows, are 0.5 cell in every hole.
4. after cultivating 4～5 days, on inverted microscope, can see little cell clone, add complete culture solution 200 μ l/ holes.
5. the 8th～9 day the time, naked eyes visible cell clone in time carries out antibody test.
(2) hybridoma is frozen
Hybridoma contains 1 * 10 at every ampoule
6More than.
Cells frozen storing liquid: 50% calf serum; 40% incomplete nutrient solution; 10%DMSO (methyl-sulphoxide)
The mass production of V, monoclonal antibody
1. inoculation hybridoma in the body prepares ascites.
The preparation abdominal injection 0.5ml Pristane (pristane) of elder generation of ascites or white oil are in the BaLb/c mouse, and 1～2 all pneumoretroperitoneums inject 1 * 10
6Individual hybridoma, inoculating cell were put to death mouse after 7～10 days, collected ascites with dropper.
The evaluation of embodiment 2 AR-AChE antibody
1. the evaluation of antibodies specific: use the ELISA method, except that carrying out the detection of antibodies with immunogen (antigen), also use and human albumin, Actin muscle, electric ray AChE carries out cross matching, result and people AR-AChE strong reaction, with electric ray AChE the weak positive is arranged, with human albumin, the Actin muscle reaction (see figure 5) that is negative, immuning tissue and cytochemistry detect, nonrecognition human brain AChE (see figure 1) and HRBC AChE (see figure 2).
2.McAb the Ig class and the evaluation of subclass: screen with enzyme target second antibody, determined the Ig type of antibody basically.Test kit (Mouse Typer sub-isotyping Kit catalog number 172-2051) with (BioRed) company is analyzed, and ARA-M1 number (preserving number CCTCC-C200413) is IgG; ARA-M5 number (preserving number CCTCC-C200414) is IgG2 α, and ARA-M7 number (preserving number CCTCC-C200415) is IgG1.
3.McAb in and active evaluation: join among the AR-AChE with antibody, can block this enzymatic activity.For the monoclonal antibody AR-AChE whether the specific recognition apoptotic cell is expressed that differentiates the contriver, and behind antibody and the antigen formation mixture, whether esterase activity changes, the contriver has done the immunosuppression experiment, the contriver uses Karnovsky, MJRoots, and the L.A method detects acetylcholine esterase active (Karnovsky, MJRoots, L.A " Direct-Coloring " Thiocholine Methodfor Cholinesterases.J Histochem Cytochem 1964; 12:219-21.), in 96 holes, under 405nm, detect enzymic activity then with microplate reader.
With cracked apoptosis HELA, divide 3 to organize greatly
A. apoptosis HELA lysate does not add substrate, but replaces with the PBS of equivalent
B. do not add antibody, do substrate reactions, as a result the positive
C. add excessive antibodies, form mixture, centrifugal, supernatant is done substrate reactions respectively with precipitation, as a result both complete negative (Fig. 3).Among the contriver C supernatant and precipitation are WESTERN BLOT, showing in the supernatant does not have AChE, and in the sedimentary immune complex, the AChE These positive bands is arranged.
The comparison of embodiment 3 AR-AChE antibody and commercially available antibody performance
General character: 1. identification AChE (general character being arranged) with commercial AChE antibody
AR-AChE antibody: normal HELA cell feminine gender, the apoptotic cell positive (commodity the results are shown in paper that hypomere draws, monoclonal antibody is seen Fig. 4).The same band (see figure 5) of AR-AChE antibody and commodity AChE antibody recognition
The contriver reports (Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, AlemanyM, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression during Apoptosisin Various Cell Types.
Cell Death and Differentiation9 (8): 790-800,2002.), the HELA cell of normally living is not expressed AChE, and the HELA cell expressing AChE of apoptosis, rather than butyrylcholine esterase (BuChE).The contriver is with two affinity columns (concanavalin A-Sepharose and edrophonium-Sepharose) (Ji Young Son, Sook Shin, Kwang Ho Choi, and In Kook Park.Purification of solubleacetylcholinesterase from Japanese quail brain by affinity chromatography.Theinternational journal of biochemistry ﹠amp; Cell biology.34 (2): 204-210,2002) from the HELA cell of apoptosis, isolates AChE.The contriver is WESTERN BLOT with separating the protein that obtains, and the result shows that antibody of buying and contriver's monoclonal antibody are discerned is same band (66kDa).(Fig. 5)
2. inhibitory enzyme activity, the AChE activity of apoptosis HELA can (among the McAb and active evaluation, Fig. 3), the contriver will go up cleer and peaceful precipitation and be WESTERN BLOT, and showing in the supernatant does not have AChE, and in the precipitation, the AChE These positive bands is arranged by the blocking-up of contriver's monoclonal antibody.With existing antibody difference: specificity.
Antibody of the present invention is only discerned the AR-AChE that apoptotic cell is expressed, and is different from now commercial antibody, the cellular immunization analysis, and this monoclonal antibody is only discerned the AR-AChE that apoptotic cell is expressed, physical evidence (Fig. 1,6).Fig. 6 will be placed room temperature following 18 hours from the isolating karyocyte of Cord blood, part cell generation apoptosis.Photo is the same visual field.
1. fail to see others and rat cynapse type (brain) AChE (Fig. 1, Fig. 7).Rat cerebral tissue's section among Fig. 7, AR-AChE immunohistochemistry and TUNEL double staining.This rat is handled (Longa EZ through the arteria cerebri media ischemic, Weinstein PR, CarlsonS.Cummins R.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke.1989 Jan; 20 (1): 84-91.), take from damage perienchyma.Picture A, B, C is the same visual field.
2. nonrecognition human erythrocyte membrane AChE (Fig. 2 C).Cord blood was placed room temperature following 18 hours, part cell generation apoptosis.A, B, C photo are the same visual field among Fig. 2.
Present anti-spinous process type AChE antibody on the market also is different from the antibody of AChE on the anti-erythrocyte film.Antibody of the present invention is only discerned the AChE that apoptotic cell is expressed, the contriver is referred to as (AR-AchE, apoptosis-relatedacetylcholinesterase, abbreviation AR-AChE), and anti-spinous process type AChE antibody capable identification AR-AChE on the market still, does not have specificity, they do not discern the specificity of apoptotic cell, therefore can not be as the mark of apoptosis.The contriver has obtained as antigenic AR-AchE (AR-AChE) (Xue-Jun Zhang, Lei Yang, Qi-Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG and Zi-Qing Jiang.Various apoptoticmammalian cells express apoptosis-related acetylcholinesterase (AR-AChE) .Acta
Biochimica et biophysica sinica35 (2): pp213,2003.).The contriver has prepared the monoclonal antibody of anti-AR-AChE on this basis.The anti-AR-AChE monoclonal antibody of specificity has been arranged, just can from various types of AChE, distinguish AR-AChE.This specificity has been arranged, just can be used for discerning apoptotic cell, the apoptosis phenomenon is used for fundamental research, AD research, AD auxiliary diagnosis and treatment; Judgement of chemotherapy effect or the like.This is the monoclonal antibody of the AR-AChE that expresses of the specific recognition apoptotic cell invented first in the world.
Distribution and enzyme and nuclear relation that embodiment 4 observes normal brain activity type AChE
Five months human fetal brain tissues of induced labor (red house hospital, and agree through family members) frozen section, 4% Paraformaldehyde 96 is fixed, and remakes acetylcholine esterase active dyeing.Preparation acetylcholinesterase substrate reactions liquid (0.1M phosphoric acid buffer Ph 6.0150ml, acetyl thio choline 100mg/200ml, 0.1M Trisodium Citrate 11ml, 30mM copper sulfate 20ml, 5mM Tripotassium iron hexacyanide 20ml) carries out enzyme reaction.When detecting the cell acetylcholine esterase active, cell room temperature in reaction solution was hatched 3-4 hour, be the positive reaction of tawny (figure Oxford gray) (seeing Fig. 8 B).As seen evenly do not have showed cell or nuclear profile, illustrate that resultant does not concentrate in cytoplasm or the nucleus, these are different fully with the distribution in the apoptosis of contriver's discovery.Fig. 8 C proves that further the AChE positive is not in nucleus.
Embodiment 5 observes a small amount of nerve cell apoptosis phenomenon in the growth course
Five months human fetal brain tissue frozen sections of induced labor, 4% Paraformaldehyde 96 is fixed, do immunohistochemical reaction and Hoechst dyeing, showed cell is examined and AR-AChE positive cell (see figure 1) respectively, the large stretch of acetylcholinesterase that exists of antibody nonrecognition of the present invention is described, and the relevant AChE of recognizing cells apoptosis only.
Embodiment 6 utilizes the people SK-N-SH cell proof monoclonal antibody specificity of the present invention of vitro culture
Utilize the people SK-N-SH cell of vitro culture, illustrate that antibody of the present invention only discerns apoptotic cells, and the normal acetylcholinesterase of nonrecognition because the SK-N-SH cell can be expressed acetylcholinesterase, is distributed on the cytolemma.See Fig. 9, photo divides A, B, and C, D, E five parts are the same visual field.1 antibody is mouse anti human AR-AChE monoclonal antibody (ARA-M7) among the C, 2 anti-are goat anti-mouse igg antibody (1: 100 dilution, Rhodamine conjugated anti-mouse IgG-R, Santa Cruz), it is positive that the lower right has an apoptotic cells to show.The people SK-N-SH cell of vitro culture, normally can express a spot of nerve synapse type AChE, 1 apoptotic cells of this photo shows positive, other does not have the positive, TUNEL dyeing coincide finely with anti-AR-AChE is positive, illustrate that this monoclonal antibody discerns the AR-AChE of apoptotic cell specifically, and the AChE of the nerve synapse type of nonrecognition SK-N-SH cell expressing.
Embodiment 7 uses monoclonal antibody of the present invention to judge the chemotherapy of tumors effect
Do the semi-quantitative analysis of acetylcholinesterase in the human serum with antibody of the present invention, can be used for judging the quality of chemotherapy of tumors (comprising leukemia) effect.This is with the monoclonal antibody of the anti-people AR-AChE of the present invention (ARA-M5 and ARA-M1 pairing), is ELISA with the filled loaf method, detects AR-AChE content in the serum of tumour patient PAD, TZH and GB chemotherapy front and back respectively.As seen patient PAD and TZH after chemotherapy in 72 hours serum the content of AR-AChE exceed more than 1 times before than treatment, illustrate that curative effect is better.The content of AR-ACHE does not change in the serum of patient GB treatment front and back, then the unsatisfactory curative effect (see figure 10).
The specificity experiment of embodiment 8 monoclonal antibody identification apoptotic cells of the present invention
Utilize the P of Rats C12 cell of vitro culture, illustrate that monoclonal antibody of the present invention only discerns apoptotic cells, and the normal acetylcholinesterase of nonrecognition because P of Rats C12 cell can be expressed acetylcholinesterase, is distributed on the cytolemma and (sees Figure 11 B).Normal PC12 cell, CONFOCAL photo FITC show that AChE expresses in normal viable cell, be distributed on cytoplasm and the film, do not have the positive in the nucleus; The PC12 cell of apoptosis, the substrate reactions positive is distributed in cytoplasm and the nuclear (seeing Figure 11).
Embodiment 9 uses monoclonal antibody half-quantitative detection 30 healthy newborns Cord bloods of the present invention and a routine tumour patient serum AR-AChE
Do the semi-quantitative analysis of acetylcholinesterase in the 30 healthy newborns Cord blood serum with antibody of the present invention, visible OD value is all below 0.2, near blank.The OD value of the 3rd day serum AR-AChE of one routine tumour patient chemotherapy is more than 3.1.This is with the monoclonal antibody of the anti-people AR-AChE of the present invention (pairing ARA-M5 and ARA-M1), is ELISA with the filled loaf method, detects AR-AChE content in tumour patient and the normal newborn Cord blood serum respectively.As seen the patient after chemotherapy in 72 hours serum the content of AR-AChE exceed the normal newborn Cord blood more than 20 times.The content of AR-AChE is very low in the normal newborn Cord blood serum, and the OD value is below 0.2, near blank (seeing Figure 12).
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (19)
1. a monoclonal antibody is characterized in that, the described antibodies specific recognizing cells apoptosis acetylcholinesterase of being correlated with.
2. the described antibody of claim 1 is characterized in that, described antibody is CCTCC NO:-C200413 by preserving number, CCTCC NO:-C200414, the mouse hybridoma cell system secretion of CCTCC NO:-C200415.
3. the described monoclonal antibody of claim 2 is characterized in that, described antibody is humanized antibody.
4. the described monoclonal antibody of claim 2 is characterized in that, described antibody is to comprise inhuman variable region and people's light chain and a kind of chimeric mAb of CH.
5. a hybridoma cell line is characterized in that, described hybridoma cell line produces the described monoclonal antibody of claim 1.
6. the described hybridoma cell line of claim 5 is characterized in that, the preserving number of described hybridoma cell line is CCTCC NO:-C200413, CCTCC NO:-C200414, CCTCC NO:-C200415.
7. an immunoassay is characterized in that, this method is carried out with the described monoclonal antibody of at least a claim 1.
8. the described immunoassay of claim 7 is characterized in that, sample is a blood, tissue or cerebrospinal fluid.
9. the described immunoassay of claim 7 is characterized in that, described assay method is carried out with elisa technique.
10. the described immunoassay of claim 7 is characterized in that, described assay method is carried out with immunochromatography technique.
11. a test kit is characterized in that, this test kit contains the described monoclonal antibody of at least a claim 1.
12. the described test kit of claim 11 is characterized in that, described test kit contains the described any monoclonal antibody of claim 2.
13. a composition is characterized in that, it comprises the described monoclonal antibody of at least a claim 1 and pharmaceutically acceptable carrier and/or vehicle.
14. the application of the described monoclonal antibody of claim 1 in screening antineoplastic drugs.
15. the method for a screening antineoplastic drugs is characterized in that, it may further comprise the steps:
(1) cultivator tumor cell line;
(2) add drug candidate in the cell strain nutrient solution in step (1), detect the expression amount of AR-AchE with the described monoclonal antibody of claim 1; Promote that the drug candidate that AR-AchE occurs or expression amount increases is exactly the medicine that promotes apoptosis of tumor cells, otherwise described drug candidate is invalid.
16. the application of the described monoclonal antibody of claim 1 in judging the antitumor drug result of treatment.
17. a method of judging the antitumor drug result of treatment is characterized in that it comprises step: detect behind the tumour patient antineoplaston expression amount of AR-AchE in 8-168 hour peripheral blood, and with treatment before relatively; The expression amount of AR-AchE increases, and shows that antitumor drug is effective, and no change or expression amount do not increase, and then antitumor drug is invalid.
18. the application of the described monoclonal antibody of claim 1 in the medicine of preparation diagnosis and/or treatment nerve degenerative diseases.
19. the application in the test kit of the described monoclonal antibody of claim 1 apoptosis expression level in the infringement of preparation detection acute organ.
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| CNA2004100681470A CN1772766A (en) | 2004-11-12 | 2004-11-12 | Acetylcholinesterase monoclonal antibody related to resisting apoptosis and its use |
| PCT/CN2005/001888 WO2006050667A1 (en) | 2004-11-12 | 2005-11-10 | MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF |
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| CN102921000A (en) * | 2011-08-08 | 2013-02-13 | 中国科学院上海生命科学研究院 | Application of acetylcholinesterase as nuclease |
| CN103087154A (en) * | 2012-12-05 | 2013-05-08 | 江苏澳格姆生物科技有限公司 | Polypeptide for preparing monoclonal antibody for detecting acetylcholin esterase and application of polypeptide |
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| DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
| CA2631195C (en) | 2005-11-30 | 2016-04-05 | Abbott Laboratories | Monoclonal antibodies against amyloid beta protein and uses thereof |
| KR20080090408A (en) | 2005-11-30 | 2008-10-08 | 아보트 러보러터리즈 | Anti-abeta; globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies |
| US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
| WO2008104386A2 (en) | 2007-02-27 | 2008-09-04 | Abbott Gmbh & Co. Kg | Method for the treatment of amyloidoses |
| MX360403B (en) | 2010-04-15 | 2018-10-31 | Abbvie Inc | Amyloid-beta binding proteins. |
| EP2603524A1 (en) | 2010-08-14 | 2013-06-19 | AbbVie Inc. | Amyloid-beta binding proteins |
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| GB8927389D0 (en) * | 1989-12-04 | 1990-01-31 | Jehanli Ahmed M T | Monoclonal antibodies |
| GB9606040D0 (en) * | 1996-03-22 | 1996-05-22 | Isis Innovation | Active peptide |
| CN1072722C (en) * | 1997-12-30 | 2001-10-10 | 中国科学院上海细胞生物学研究所 | Method of screening anti-tumour medicine and determining clinical chemotherapy effect |
| CN1059702C (en) * | 1997-12-30 | 2000-12-20 | 中国科学院上海细胞生物学研究所 | Method for cerebral acetylcholinesterase obtained from cells of mamma animals |
| IL140071A0 (en) * | 2000-12-04 | 2002-02-10 | Yissum Res Dev Co | Human antibodies to specific acetylcholinesterase variants and their use as diagnostic agents for cholinergic neurodeterioration processes |
| CN1244700C (en) * | 2001-03-23 | 2006-03-08 | 中国科学院上海细胞生物学研究所 | Human acetylcholinesterase isomer protein (AR-ACHE) and its gene coding sequence |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102921000A (en) * | 2011-08-08 | 2013-02-13 | 中国科学院上海生命科学研究院 | Application of acetylcholinesterase as nuclease |
| CN102921000B (en) * | 2011-08-08 | 2015-02-18 | 中国科学院上海生命科学研究院 | Application of acetylcholinesterase as nuclease |
| CN103087154A (en) * | 2012-12-05 | 2013-05-08 | 江苏澳格姆生物科技有限公司 | Polypeptide for preparing monoclonal antibody for detecting acetylcholin esterase and application of polypeptide |
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