WO2006050667A1 - MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF - Google Patents

Abstract

On the basis of discovery of apoptosis-related acetylcholinesterase (AR-AChE), the invention uses purified human AR-AChE as antigen to immunize mouse, fuse cells, get hybridomas, especially expressing and secreting monoclonal antibodies against apoptosis-related acetylcholinesterase and hybridoma cell lines ARA-M1, ARA-M5 and ARA-M7, and produce antibodies. The antibodies could be useful for screening anti-tumor medicines and determining clinical chemotherapy, preparation of medicines for diagnosis and treatment of neuroretrogressive diseases, such as the assay of AD and basal and clinical research and diagnosis of apoptotic cells, measurement of apoptosis expression levels in sharp organ damages. The invention also offers the methods of immunoassay and kits for use in half-quantitative and/or quantitative assays for AR-AChE and otherwise.

Description

 FIELD OF THE INVENTION The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody against apoptosis-associated acetylcholinesterase (AR-AChE) and uses thereof.

 Background Art Acetylcholinesterase (EC 3. 1. 17, acetylcholinesterase, AChE) is a hydrolase of the neurotransmitter acetylcholine and belongs to glycoproteins. Mainly found in electrical organs of electric sputum, mammalian cholinergic nerves, neuromuscular junctions, erythrocyte membranes (Zakut-H; et al: J-Clin-Invest. 1990, 86 (3): 900-8) Rodent megakaryocyte platelets (Α Shafferman and B. Velan, Multidisplinary approaches to cholinesterase functions. 1992 Plenum Press, New York). AChE can be divided into neuromuscular and red blood cell types. According to the molecular structure, it can be divided into different subunits (heteromeric class) and the same subunit multimer type (homomeric class;). The catalytic subunits of the different subunit multimeric types are linked to or linked to the triple helix collagen subunit. The same subunit multimer type is subdivided into subtypes such as hydrophilic and lipophilic-linked. The AChE tetramer on the erythrocyte membrane has a molecular weight of about 260 KDa. The function of AChE, in the cholinergic nervous system, is to hydrolyze the neurotransmitter acetylcholine to produce choline and acetic acid. It has also been reported that AChE has a hydrolyzed protein activity of a serine protease. In recent years, AChE m NA transcription has been detected in in vitro cultured tumor cell lines and AChE activity has been detected in the serum of ovarian cancer patients after chemotherapy. Some authors believe that AChE is associated with tumorigenesis (Lev-Lehman-E, Et al rBlood. 1997, 89 (10) : 3644-53).

The inventors first discovered that mammalian cells express AChE during apoptosis (Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression Cell Apoptosis in Various Cell Types. Cell Death and Differentiation 9 (8) : 790-800, 2002. ), Neural-derived Cell Lines (Yang L, Heng-Yi He, Xue-Jun Zhang Increased expression of intranuclear AChE involved in Apoptosis of SK-N-SH cells. Neuroscience research 42 (4) : 261-268, 2002. ) The same phenomenon occurs. In order to further investigate the role of AChE in apoptosis, the inventors established an isolation method for the enzyme ("Method for obtaining brain-type AChE from mammalian cells", which has been authorized. Patent No.: ZL 97 1 25216. 5; Qi- Huang JIN, Yu-Fang SHI, Heng-Yi HE, Kelvin KW Ng, Hua JIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun Zhang Isolation of AChE from Apoptotic Human Lung Fibroblast Cells by Antibody Affinity Chromatography. BioTechnigues 33 (4 ) :S92-S97, 2002. ). Due to the expression of cells by the enzyme, the time of onset, the function performed, post-translational modification or even raRNA cleavage ("a human acetylcholinesterase isoform protein (AR-AChE) and gene coding sequence", application number: 01150718 . 5 , Application date, 2001/3/23. ), completely different from the already reported neuromuscular and erythrocyte type AChE, the inventors referred to AChE expressed by apoptotic cells as apoptosis-associated acetylcholinesterase (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 apoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR- AChE) . Acta biochimica et biophysica sinica 35 (2): Pp213 ₍ 2003.). Using the characteristics of apoptotic cells expressing AR-AChE, the inventors can screen anti-tumor drugs by detecting the activity of the enzyme ("Anti-tumor drug screening method", CN 1186859 has been authorized. Patent No.: ZL 97 1 25220. 3) and used as a marker for apoptotic cells.

 Polyclonal ant i -AChE antibody (Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, Beijing, PR China); Antibody has been prepared for neuromuscular and erythrocyte types and electroporation AChE. To AChE (C-16) (catalog number, sc-6430, Santa Cruz Biotechnology) is an affinity-purified goat anti-the carboxy terminus of human acetylcholinesterase; AChE- antibody (BD Biosciences, San Jose, CA, USA) ), Antibodies that recognize neuromuscular types also recognize AchE expressed by apoptotic cells (Zhang XJ, 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 Various Cell Types. Cell Death and Differentiation 9 (8): 790-800, 2002. Since there is currently no specific antibody against AR-AChE (which does not recognize AChE on normal neurites), it is still difficult to determine apoptotic cells by identifying AChE using immunological methods.

 The inventors found that cells that do not express AChE express AChE in a large amount during apoptosis, mainly in the nucleus, and AChE is present in apoptotic bodies as the nucleus is formed into apoptotic bodies. The inventors established a method for detecting apoptotic cells and determining the effect of chemotherapy using a method for detecting AChE activity (a method for screening anti-tumor drugs and judging clinical efficacy, Chinese Patent No. 97 1 25220. 3). Due to the activity assay, only qualitative analysis of the protein can be performed, and the presence of butyrylcholinesterase (BuChE) in the serum interferes with the activity of AChE and affects the activity assay of AChE.

 If there is a specific monoclonal antibody against AR-AChE, AR-AChE can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptosis, basic research, theoretical and clinical studies of Alzheimer's disease (AD), AD-assisted diagnosis and treatment, judgment of chemotherapy effects, etc. Semi-quantitative and quantitative analysis is available. Therefore, it is necessary to develop a specific monoclonal antibody against AR-AChE.

 Disclosure of the Invention An object of the present invention is to provide a monoclonal antibody against apoptosis-associated acetylcholinesterase (AR-AChE) and a mouse hybridoma cell line producing the same.

Another object of the present invention is to provide the use of a monoclonal antibody against AR-AChE, which can be used to determine the activity and expression level of AR-AChE upon apoptosis. Further, the antibody can be prepared into a kit, screening anti-tumor drugs, measuring the effect of anti-tumor drugs, assisting in the diagnosis of neurodegenerative diseases such as AD, and detecting urgency. The expression level of apoptosis in sexual organ damage provides doctors with reference data on the onset and recovery of patients.

 A further object of the present invention is to provide a method for screening an antitumor chemotherapeutic drug and an immunoassay for detecting apoptosis-associated acetylcholinesterase.

 A first aspect of the invention provides a monoclonal antibody which specifically recognizes apoptosis-associated acetylcholinesterase.

 Preferably, the above monoclonal antibody is secreted by a mouse hybridoma cell line having the accession number CCTCC NO: - C200413, CCTCC NO: - C200414 or CCTCC NO: - C200415.

 The above monoclonal antibodies include humanized antibodies. Preferably, the above antibody is a chimeric monoclonal antibody comprising a non-human variable region and a human light chain and heavy chain constant region.

 A second aspect of the invention provides a hybridoma cell line, characterized in that said hybridoma cell line produces said monoclonal antibody. Preferably, the hybridoma cell line is a mouse hybridoma cell line. Preferably, the above hybridoma cell line is a mouse hybridoma cell line deposited under CCTCC NO: - C200413, CCTCC NO: - C200414, CCTCC NO: -C200415. The present invention also provides a monoclonal antibody secreted by the above hybridoma cell line.

 A third aspect of the invention provides an immunoassay which is carried out using at least one of the above monoclonal antibodies. The specimen in the above immunoassay is blood, tissue or cerebrospinal fluid, preferably blood supernatant. The above immunoassay can be carried out using an ELISA technique or an immunochromatography technique. Preferably, the above monoclonal antibody is a monoclonal antibody of one or more subtypes secreted by a mouse hybridoma cell line of accession numbers CCTCC NO: -C200413, CCTCC NO: - C200414, CCTCC N0: - C200415.

 A fourth aspect of the invention provides a kit comprising at least one of the above monoclonal antibodies. Preferably, the monoclonal antibody is selected from the group consisting of ARA-Ml, ARA-M5, ARA-M7 (hybridoma cell line, see description on page 7). The kit of the invention can be used for screening anti-tumor drugs, judging the therapeutic effect of anti-tumor drugs, diagnosing neurodegenerative diseases and detecting the expression level of apoptosis-associated acetylcholinesterase, especially the expression level of AR-AChE in acute organ damage.

 A fifth aspect of the invention provides a composition comprising at least one of the above monoclonal antibodies and a pharmaceutically acceptable carrier and/or excipient. Preferably, the above composition comprises a monoclonal antibody of one or more subtypes secreted by a mouse hybridoma cell line of accession number CCTCC NO: - C200413, CCTCC NO: ~C200414, CCTCC NO: - C200415. The composition of the present invention is useful for treating diseases caused by abnormal expression of apoptosis-associated acetylcholinesterase, including neurodegenerative diseases.

 A fifth aspect of the invention provides the use of the above monoclonal antibody for screening an antitumor drug.

 A sixth aspect of the invention provides a method of screening an antitumor drug, which comprises the following steps -

(1) cultivating a human tumor cell line;

 (2) adding a candidate drug to the cell culture medium in the step (1), detecting the expression level of apoptosis-associated acetylcholinesterase by the above monoclonal antibody, and promoting the occurrence or expression of apoptosis-associated acetylcholinesterase. A drug candidate is a drug that promotes tumor apoptosis, and conversely, the drug candidate is ineffective.

Preferably, the monoclonal antibody is selected from any one of ARA-M1, ARA-M5, and ARA-M7. Preferably, AR-AChE is detected within 18-24 hours after the drug candidate is added.

 A seventh aspect of the present invention provides the use of the above monoclonal antibody for determining the therapeutic effect of an antitumor drug. An eighth aspect of the present invention provides a method for determining the therapeutic effect of an antitumor drug, comprising the steps of: detecting the expression level of apoptosis-associated acetylcholinesterase in peripheral blood of a tumor patient after treatment with an antitumor drug, 8 to 168 hours later, And compared with before treatment; the expression of apoptosis-associated acetylcholinesterase increased, indicating that the anti-tumor drug is effective, no change or the expression amount does not increase, then the anti-tumor drug is ineffective.

 The preferred detection time is 12-120 hours.

 A ninth aspect of the present invention provides the use of the above monoclonal antibody for the preparation of a medicament for diagnosing or treating a neurodegenerative disease.

 A tenth aspect of the present invention provides the use of the above monoclonal antibody for the preparation of a kit for detecting apoptosis-related acetylcholinesterase expression levels in acute organ damage.

 The inventors have found an isoform of acetylcholinesterase (AChE) from apoptotic cells, named AR-AChE, and applied for a patent (a human acetylcholinesterase isoform protein (AR-AChE) And gene coding sequence. Application number: 01105781. 5, application date, 2001/3/23. ;). Later, the inventors found that many cell lines express AChE during apoptosis (Zhang XJ, 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 Various Cell Types. Cell Death and Differentiation 9 (8): 790-800, 2002.), due to the time of expression of the enzyme, cell type, intracellular distribution, post-translational modification, and functional characteristics are different from those on neurons. AChE on synaptic and motor endplates, AChE on erythrocyte membrane, and AChE in megakaryocytes. Therefore, the inventors referred to AChE expressed by apoptotic cells as apoptosis-related acetylcholinesterase (AR). -AChE). The inventors called an antibody against AR-AChE expressed by apoptotic cells, which is called an anti- apoptosis-related acetylcholinesterase antibody. For AR-AChE, the inventors were able to prepare monoclonal and polyclonal antibodies.

Anti-neurite AChE antibodies on the market today are also different from antibodies against AChE on erythrocyte membranes. The antibody of the present invention recognizes only AChE expressed by apoptotic cells, which the inventors call (apoptosis-related acetylcholinesterase, abbreviation AR-AChE), while anti-neurite AChE antibodies on the market may recognize AR-AChE. However, there is no specificity. They do not recognize the specificity of apoptotic cells and therefore cannot be used as markers of apoptosis. The inventors have obtained apoptosis-associated acetylcholinesterase (AR-AChE) as an antigen (Xue-Jun Zhang, Lei Yang, Qi-Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG) And Zi-Qing Jiang. Various apoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR-AChE) . Acta biochimica et biophysica sinica— 35 (2): pp213, 2003. On this basis, the inventors prepared monoclonal antibodies against AR-AChE. With the specific production of anti-AR-AChE monoclonal antibodies, AR-AChEo can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptosis, and for basic research. AD research Study, AD assisted diagnosis and treatment; judgment of chemotherapy effect and so on. This is the first monoclonal antibody in the world to specifically recognize AR-AChE expressed by apoptotic cells.

 Therefore, the inventors isolated AR-AChE from apoptotic cells using a Chinese patent (ZL 97 1 25216. 5, a method for obtaining brain-type AChE from mammalian cells), (Qi-Huang JIN, Yu-Fang SHI, Heng -Yi HE, Kelvin W Ng, Hua JIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun Zhang Isolation of AChE from Apoptotic Human Lung Fibroblast Cells by Antibody Affinity Chromatography. BioTechniques 33 (4): S92 - S97, 2002. ) . It was used to immunize BaLb/c mice, and three clones were screened to specifically produce monoclonal antibodies against apoptosis-associated acetylcholinesterase (AR-AChE).

 The monoclonal antibody of the present invention can be produced by the hybridoma of the present invention, and thus can be obtained from the culture solution for culturing the hybridoma of the present invention. However, the method of producing the monoclonal antibody of the present invention is not particularly limited, but if the genetically engineered antibody specifically binds to the AR-AChE protein, it is also within the scope of the present invention.

 The hybridoma of the present invention produces a monoclonal antibody recognizing AR-AChE, and can be obtained by fusing spleen cells or lymph node cells of an animal immunized with AR-AChE protein with myeloma cells.

 The hybridoma cell lines AM-Ml, ARA-M5, ARA-M7 of the present invention have been deposited with the China Center for Type Culture Collection (Wuhan) on September 21, 2004, in accordance with the Budapest Convention for the Conservation of Microorganisms for Patent Procedures. The deposit numbers are in turn CCTCC NO: - C200413, CCTCC NO. - -C200414, CCTCC NO : - C200415.

 Hybridomas of the invention can be produced using cell fusion techniques known in the art. Therefore, AR-AChE is used as an immunogen to immunize animals other than human, and the spleen cells or lymph node cells of the animal are fused with myeloma cells to produce a hybridoma, and a hybrid which produces a monoclonal antibody recognizing AR-AChE is selected therefrom. Tumor, thereby obtaining a hybridoma of the present invention.

 The immunogen for preparing the hybridoma described above is not particularly limited and may be recombinant AR-AChE, purified AR-AChE protein or tumor cells containing AR-AChE. For example, an engineered strain expressing an AR-AChE protein or a fusion protein containing an AR-AChE protein can be cultured on a suitable medium.

 When antibodies produced by non-human subjects or antibodies derived from expression of non-human antibody genes are therapeutically used in humans, they are recognized as foreign antibodies to varying degrees, and an immune response may be generated in the patient. One way to minimize or eliminate this immune response is to produce chimeric antibody derivatives, i.e., antibody molecules that bind non-human animal variable regions and human constant regions. Accordingly, the invention also encompasses the use of humanized antibodies, as well as humanized antibodies, in the manufacture of a medicament for the treatment of a neurodegenerative disease, such as AD. One skilled in the art can prepare humanized antibodies using the monoclonal antibodies of the present invention.

 These antibodies retain the epitope binding specificity of the original monoclonal antibody, but may be less plaque when administered to humans and are therefore more likely to be tolerated by the patient.

Chimeric monoclonal antibodies can be made using recombinant DNA techniques known in the art. For example, a gene encoding a constant region of a non-human antibody molecule is replaced with a gene encoding a human constant region (see, Robinson et al, PCT Patent Publication PCT / US86/02262; Akira, et al, European Patent Application 184, 187; or Taniguchi, M . European Patent Application 171, 496). It is possible to replace the variable region portion that does not involve binding to the antigen by replacing the equivalent portion of the human variable region with The chimeric antibody is "humanized" in one step.

 The field of application of the monoclonal antibody of the present invention is not particularly limited, and can be used for immunoassays, including quantitative or semi-quantitative detection. The monoclonal antibody of the present invention can detect the presence or absence of AR-AChE as a basis for judging the tumor chemotherapy results.

 An antigen-binding fragment of an antibody of the present invention refers to an antibody fragment which binds to an epitope of an apoptosis-associated acetylcholinesterase.

 The animal which is immunized to produce the hybridoma of the present invention is not particularly limited and includes, for example, goat, sheep, guinea pig, mouse, rat, and rabbit. Preferred among them are mice.

 The immunoassay of the present invention is carried out using at least one monoclonal antibody of the present invention, and the method can be carried out using only one antibody, or in combination with an AR-AChE polyclonal antibody, or simultaneously using two or more monoclonal antibodies (e.g., ARA - Ml and ARA - M5).

 As the immunoassay of the present invention, enzyme immunoassay (EIA:), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), Western blot, immunochromatography and the like are included. The various immunoassays described above can be used to measure target antigens or antibodies by labeling antigens or antibodies by competition or sandwich methods. Among the various immunoassays described above, ELISA and immunochromatography techniques are preferred.

 The above sandwich method includes, for example, sandwiching the AR-AChE between the monoclonal antibody of the present invention in a fixed subtype and the monoclonal antibody of the present invention or the anti-AR-AChE polyclonal antibody in another subtype labeled with the labeling agent. Then, a substrate or the like against a label (for example, an enzyme) is added, a color or the like is displayed, thereby detecting AR-AChE in the specimen. Monoclonal antibodies with high sensitivity are generally considered as preferred labeled antibodies because of their high specificity and low background noise, which makes detection more accurate, compared to polyclonal antibodies with low specificity and high background noise. '

 The above competitive methods are based, for example, on the quantitative competitive binding reaction of AR-AChE in a test specimen and a known amount of labeled AR-AChE to a monoclonal antibody of the present invention. In the above-mentioned competitive method specifically mentioned, a predetermined amount of the antibody immobilized on the carrier and a predetermined amount of AR-AChE labeled with the labeling agent are added to the sample solution containing AR-AChE. Then, the activity of the labeling agent remaining on the carrier or the activity of the labeling agent not remaining on the carrier is determined. In this regard, it is preferred to add the antibody and the labeled antigen almost simultaneously.

For the above-mentioned labeling agent, a radioactive isotope such as ¹²⁵ 1 , an enzyme, an enzyme substrate, a phosphorescent substance, a fluorescent substance, a biotin, and a coloring matter can be used. In the binding of these labeling agents to antigens or antibodies, the maleimide method can be used (J. Biochem. (1976), 79, 233), activated biotin method (J. Am. Chem. SOC. (1978) , 100, 3585) or hydrophobic bond method.

For the enzymes mentioned above, for example, peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase are included. For the substrate used in this case, a substrate suitable for the enzyme used in the case can be selected, including, for example: ABTS, luminol _3⁄40 ₂ , ₀ -phenylenediamine- 3⁄40 ₂ (for peroxidase), phosphoric acid p-Nitrophenyl ester, methyl formate phosphate, 3-(2'-spirocyclobutane)-4-methoxy-4-one (3'-phosphoryloxy)phenyl- 1,2- Dioxetane (for alkaline phosphatase), p-nitrophenyl-BETA-D-galactose (for beta-galactosidase).

It can be reacted at 4-40 °C for 1 minute and 18 hours, and then the result shows the color or fluorescence, phosphorescence or display The amount of color, the above test was carried out. Alternatively, a so-called rate test can be used, which is carried out at a temperature of 4 to 40 °C. Radiolabeling of the above antigens or antibodies can be conveniently carried out using a commercially available Bolton-Hunter reagent. For example, a Bolton-Hunter reagent can be added to a solution (prepared by dissolving an antigen or an antibody in 0.1 aqueous sodium hydrogencarbonate solution), followed by 1-2 hours, using a G-25 desalting column, etc. The unreacted Bolton-Hunter reagent fraction was removed.

In addition, the ¹²⁵ 1 radiolabel can be conveniently carried out using the Chlorphenamine T method, the iodine method, or the like.

 The above fluorescent substances, such as fluorescein and rhodamine, can be conveniently labeled using an activated ester method or an isocyanate method ("Enzyme Immunoassay", published by Igaku Shoin, 1987). For the above coloring matter, colored latex particles and colloidal gold can be used. ,

 The immunoassay of the present invention uses the monoclonal antibody of the present invention, and thus has outstanding characteristics, and recognizes only AR-AChE present in all apoptotic cells (including tumors or nerve cells) without erroneous detection due to cross-reaction Other substances and normal AChE allow for very specific assays.

In order to carry out the immunoassay of the present invention, the kit of the present invention can be used. The kit of the present invention contains at least one monoclonal antibody of the present invention and may contain only one monoclonal antibody. The monoclonal antibody of the present invention to be used in the kit of the present invention is not particularly limited, but may be any monoclonal antibody recognizing AR-AChE, and may also be a decomposition product of the monoclonal antibody of the present invention or an antigen-binding fragment thereof. Such as F (ab') ₂ , Fab\ Fab, etc.

 In the kit of the present invention, the monoclonal antibody of the present invention can be immobilized on a solid phase in advance, and the monoclonal antibody of the present invention can also be labeled with the above-mentioned labeling agent.

 The kit of the present invention can be used to detect a sample containing 'AR-AChE, such as blood, tissue or cerebrospinal fluid, by immunological methods, so that the presence or absence of AR-AChE and the change in expression amount can be judged.

 The solid phase of the kit used in the present invention is not particularly limited, but includes, for example, a polymer such as polystyrene, glass beads, magnetic particles, a microtiter plate, a filter paper for immunochromatography, a glass filter or other insoluble carrier.

 Kits of the invention may also contain other components. The above other components are not particularly limited, but include, for example, an enzyme for labeling, a substrate thereof, a radioactive isotope, a phosphor, a fluorescent substance, a coloring matter, a buffer, and a petri dish, and the above-mentioned components.

 The kit of the present invention may be a kit for carrying out the above competitive method or the above-described sandwich method. However, a kit using a sandwich method is preferred. The above sandwich method has the advantages of high sensitivity, short reaction time required, high accuracy, and convenient operation. Using the immunochromatography technique described above, a kit can be prepared which makes the test method convenient and simple, and which can be easily observed by visual observation. When the above-mentioned sandwich method is used by the ELISA technique, the formation of the enzyme reaction product is determined by the amount of the target substance to be tested, so that a system can be established which can easily determine the presence or absence of AR-AChE by visual observation through color development or the like. , can also be used to test its 0. D value, quantitative and semi-quantitative detection.

_: Kit of the present invention is used is not particularly limited samples, but includes, for example: blood and cerebrospinal fluid or tissue, preferably blood.

The monoclonal antibody of the invention and the kit of the invention can be used for screening anti-tumor drugs, and assisting in judging anti-tumor drugs Therapeutic effects, preparation of drugs for the diagnosis or treatment of neurodegenerative diseases, detection of apoptosis in acute organ damage

Expression levels and changes of AR-AChE.

 The above method for screening an antitumor drug comprises the following steps:

 (1) cultivating a human tumor cell line;

 (2) adding a candidate drug to the cell culture medium in the step (1), detecting the expression level of apoptosis-associated acetylcholinesterase using the above monoclonal antibody or the above kit; promoting the apoptosis-associated acetylcholinesterase or A drug candidate with an increased expression amount is a drug that promotes apoptosis of a tumor cell, and conversely, the drug candidate is ineffective.

 According to the present invention, the recognition of AR-AChE by a monoclonal antibody can eliminate the influence of other substances present in the sample, thereby detecting the presence of AR-AChE with very high sensitivity and specificity. Since hybridomas that produce monoclonal antibodies against AR-AChE have been established, the same monoclonal antibodies can now be produced almost permanently.

 The diagnostic kit of the present invention uses a monoclonal antibody with a high level of detection sensitivity and does not cause false negative or false positive problems, and there is no difference between batch and batch. If the labeled antibody is preferably a monoclonal antibody different from the monoclonal antibody on the solid phase, it is easier to control the quality of the product and to establish a uniform standard.

 The AR-AChE detection kit detects the principle of using anti-AR-AChE monoclonal antibody and anti-AR-AChE polyclonal antibody or another subtype anti-AR-AChE monoclonal antibody as the immobilized antibody and the enzyme-labeled antibody. If AR-AChE is present in the sample to be tested, an antibody-antigen-enzyme-labeled antibody complex is formed, and a substrate of TMB (enzyme-free color-developing series) is added to produce a color reaction, and vice versa, no color reaction occurs. Double antibody sandwich ELISA is a very sensitive detection technique, and the ELISA is highly sensitive and easy to operate. The development of supporting instruments and equipment has standardized and automated the operating procedures, thus further improving stability.

The present invention provides a hybridoma cell line which specifically produces an anti-AR-AChE monoclonal antibody, and an anti-human AR-AChE monoclonal antibody can be produced from these cell lines, and a kit can be prepared using the antibody for basic research, AD research, AD Auxiliary diagnosis and treatment; judgment of the effect of chemotherapy, etc. - So far, only the M30 antibody has been used to determine the effect of chemotherapy by serological methods. There are currently two companies, ALEXIS Biochemicals Corporation, and PolyPhqrma Group Inc., which launched the test kit U30-ApoptosenseTM ELISA Kite. The main component of this kit is M30 antibody, a mouse hybridoma monoclonal antibody, subtype As IgG2b, it recognizes a small fragment of the C-terminus of keratin (Cytokeratin) 18 (CK18). CK18 is a medium fiber of epithelial cells. When the epithelium is apoptotic, the activated kinase (Caspa _S e) 3, 6, 7, 9 cuts the C-terminus of CK18 into a small fragment. Since this small fragment appears only in apoptotic cells, it becomes a specific marker for epithelial cell apoptosis. With M30 antibody, apoptosis of tissue cells and cultured cells can be recognized, and changes in serum M30 can also be detected by ELISA to determine the effect of chemotherapy. But it is only for epithelial-related tumors and is expensive.

The use of the monoclonal antibodies of the invention is:

1. Screening anti-tumor drugs in vitro:

 To study new anti-tumor drugs, it is an important indicator to induce tumor cell apoptosis by screening among many drug candidates. The method of the invention can be used to screen new anti-tumor drugs in vitro. The human tumor cell strain cultured in vitro is added with different doses of the anti-tumor drug candidate to be detected, and after 18-20 hours, the cells or the culture solution are detected and passed.

s The presence and amount of AR-AChE changes to determine whether the drug being screened is effective or not. If the AR-AChE is increased, indicating that the drug has induced apoptosis, it is an effective anti-tumor drug.

2. Judging the effect of chemotherapy in clinical tumor patients

 For different tumors (including leukemia), different patients respond differently to various chemotherapeutic drugs. Some anti-tumor drugs work well in this patient, but not others. The method of the invention can help clinicians to selectively select tumor chemotherapy drugs, aim at finding the best drugs in the shortest time and bring the gospel to the patients.

 Chemotherapy drugs are mainly aimed at tumor cells (also normal cells), causing apoptosis to achieve therapeutic goals. The invention is based on new discoveries in scientific research. The principle is that cells that do not normally express AChE express AR-AChE in a large amount during apoptosis, and this enzyme is stably present in apoptotic cells. In normal people or tumor patients, although there is no apoptosis in the body, but the number is small, the apoptotic cells are quickly phagocytosed by the surrounding macrophages, and the AR-AChE in the apoptotic cells is not released into the bloodstream. in. However, if an effective chemotherapeutic drug causes a large amount of apoptosis of tumor cells, macrophages cannot rapidly phagocytose all apoptotic cells, or are affected by drugs, and the phagocytic ability of macrophages is reduced, and apoptotic cells cannot be rapidly phagocytized. A large amount of AR-AChE enters the bloodstream with apoptotic cells and apoptotic bodies. At this time, taking the patient's serum to determine the protein content of serum AR-AChE, it was found that the protein content of AR-AChE at 72 hours (the third day) after the start of treatment was more than twice as high as that before the treatment. Importantly, the inventors found that the hematopoietic stem cells of cord blood CD34+ did not express AR-AChE when apoptotic, and the determination of blood AR-AChE was more representative. Therefore, to determine the serum AR-AChE protein content before and after treatment, we know how the chemotherapy effect, help clinicians find the best targeted tumor chemotherapy drugs in the shortest time, and aim at saving patients' lives and gain valuable time. If the chemotherapy is not effective, the drug can be changed in time, thus providing the doctor with a quick and effective means of detecting the effect of chemotherapy, and truly achieving individualized treatment. Of course, the therapeutic effect of other antitumor drugs can also be analyzed by the monoclonal antibody of the present invention.

 Studying neurodegenerative diseases, especially AD, may be helpful in diagnosis and treatment

Evidence of brain cell apoptosis can be obtained by brain tissue sectioning and cerebrospinal fluid analysis. AChE accelerates the precipitation of amyloid beta peptide, which is independent of classical enzyme catalysis (Inestrosa NC, Alvarez A, Perez CA, Moreno RD, Vicente M, Linker C, Casanueva 01, Soto C, Garrido J, Acetylchol inesterase Accelerate assembly of amyloid-beta-protoss into Alzheimer's fibrils : possible role of the peripheral site of the enzyme, Neuron, 1996 16 (4); 881-891. ), and can form a complex with the beta amyloid peptide, and Complex toxicity is more toxic than a single amyloid beta peptide (Alvarez A, Alarcon R, Opazo C, Campos E0, Munoz FJ, Calderon FH, Dajas F, Gentry MK, Doctor BP, De Mello FG, Inestrosa NC, Stable complexes involving Acetylchol inesterase and amyloid-beta change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer* s fibrils, J Neurosci, 1998 18 (9); 3213-3223. Experiments on AChE antisense nucleic acids directly indicate that AChE plays a role in neuronal loss ( Shohami E, Kaufer D, Chen Y, Seidman S, Cohen 0, Ginzberg D, Melamed-Book N, Yirmiya R, Soreq H, Antisense prevention of neuronal damages following head injury in mice, J Mol Med, 2000 78 (4); 228-236. This monoclonal antibody, in addition to being used to identify evidence of apoptosis in brain tissue sections and cerebrospinal fluid, may also be used as a diagnostic reagent (especially after preparation of adult-derived antibodies), such as to prevent AChE acceleration. The precipitation of amyloid peptides, thereby achieving the effect of preventing the progressive development of AD. The medicament includes a monoclonal antibody of the invention and a pharmaceutically acceptable carrier and/or excipient.

 Detection of acute organ damage development and recovery

 For example, diseases such as outbreak hepatitis. These acute organ damages occur when there is a large amount of apoptosis, and the serum contains several times or even tens of times higher AR-AChE. By detecting its changes, it is possible to judge the development and prognosis of the disease.

 Advantages of the present invention: The method of the present invention detects apoptosis or apoptosis by detecting the AR-AChE activity expressed by the cells after the anti-tumor drug is administered to cells which do not express AChE in vitro, or determining the serum AR-AChE content. The cell AChE reaction is positive, indicating that the drug has induced apoptosis, and is an effective anti-tumor drug, which can conveniently and effectively screen anti-tumor drugs, and can also help doctors select chemotherapy drugs that are particularly sensitive to patients to improve the therapeutic effect. . The results of detecting the AR-AChE content by the method of the present invention to identify apoptotic cells cultured in vitro are consistent with the classical method. Moreover, the positioning is accurate, the method is simple and easy, no expensive equipment is needed, and too expensive reagents are not needed, and semi-quantitative and quantitative analysis can be performed. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Frozen sections of five-month human fetal brain tissue induced by labor (magnification 800 times, X 800)

 A. is immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, Rhodamine label, and 2 apoptotic cells are positive.

 B. is Hoechst No. 33258 staining showing all nuclei.

 C. is the overlap of A and B.

 D. is a visible light photo. This photograph shows that two apoptotic cells show positive and others are not.

 Figure 2. Photograph of cord blood smear (X 800)

 A. is immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, Rhodamin label, apoptotic nucleated cells show positive, red blood cells are negative;

 B. is Hoechst No. 33258 staining, showing nuclear material, only one nucleated cell in the whole field is red blood cells;

 C. It is a photo of visible light, showing a large number of red blood cells with one nucleated cell in the middle. This figure illustrates that the monoclonal antibody of the present invention specifically recognizes AR-AChE of apoptotic cells, but does not recognize AChE on the erythrocyte membrane. Figure 3. Apoptotic Apoptosis HELA Experiment

In the figure, from left to right, the apoptotic HELA lysate a. No substrate, but replaced with an equal amount of PBS; b. No antibody, substrate reaction, positive result; c. and d. Antibody, forming a complex, centrifuging, taking supernatant (c) and Precipitation (d) was used as a substrate reaction, and both were negative.

Figure 4. Detection of HELA cells with the monoclonal antibody of the invention

 The cultured HELA cells, the antibody is a mouse anti-human AR-AChE monoclonal antibody (ARA-M5) prepared by the inventors, 1 antibody is directly linked to HRP, DAB reaction, normal HELA cells are negative, and apoptotic cells are positive.

Figure 5. WESTERN BLOT

 A. Using the purchased antibody (BD Biosciences, San Jose, CA, USA) as the WESTERN BLOT, the antigen is derived from apoptotic HELA cells;

 B. Using ARA-M5 for WESTERN BLOT, the antigen is derived from apoptotic HELA cells;

 C. Using ARA-M5 for WESTERN BLOT, the antigen is derived from electroporation acetylcholinesterase (Sigma).

Figure 6. Photograph of umbilical cord blood nucleated cell smear (X 400)

 A. For visible light photos, black arrows point to apoptotic cells, white arrows point to normal cells;

 B. Is Hoechst No. 33258 staining, showing nuclear material, the apoptotic cell nucleus has formed apoptotic bodies, while the normal cell nucleus is intact;

 C. is immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, Rhodamin label, only apoptotic bodies show positive cells, while normal cells are negative, This monoclonal antibody specifically recognizes apoptotic cells.

Figure 7. Rat brain tissue section

 A. The antibody is a mouse anti-human AR-AChE monoclonal antibody (ARA-M1) prepared by the inventors, 1 antibody is directly linked to Rhodamine, red is positive cells (cross-reactive with rat AR-AChE); large pieces of nerve tissue There was no positive, there was cholinergic nerves present, and synaptic acetylcholinesterase did not show positive.

 B. TUNEL staining, positive for apoptotic cells.

 C. is the overlap of A and B. TUNEL staining was in good agreement with the anti-AR-AChE positive region, indicating that this monoclonal antibody specifically recognizes AR-AChE of apoptotic cells, but does not recognize the synaptic AChE expressed in rat brain tissue. Figure 8. Frozen sections of five-month human fetal brain tissue induced by induction (X 800)

 Photos are the same field of view

 A. is Hoechst No. 33258 staining, showing the nucleus;

 B. is cytochemical Ache staining (Konovsky and Root method), brownish yellow (dark gray in the figure) is AChE positive, can be seen more uniform and does not show the outline of cells or nuclei, indicating that the product is not concentrated in the cytoplasm or nucleus, This is completely different from the distribution found in apoptosis by the inventors;

C. is the result of simultaneous photographing of visible light photographs with Hoechst No. 33258 staining, further demonstrating that AChE positivity is not in the nucleus.

Figure 9. Photograph of human SK-N-SH cells cultured in vitro (X800)

 The photo is divided into five parts, A, B, C, D, and E, all of which are in the same field of view.

 A. TUNEL staining, positive for apoptotic cells;

B. is a photo of visible light, and there is a condensed cell with no cell protrusion on the lower right side of the cell mass; C is immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, Rhodamin label, and one apoptotic cell in the lower right shows positive;

 D. is hoechest staining, showing the nucleus;

 E. is the overlap of A, C, and D. Human SK-N-SH cells cultured in vitro can express a small amount of synaptic AChE normally. This photo shows that one apoptotic cell is positive and the other is not positive. TUNEL staining agrees well with anti-AR-AChE positive. , indicating that the monoclonal antibody specifically recognizes the AR-AChE of apoptotic cells, but does not recognize the synaptic AChE expressed by SK-N-SH cells.

Figure 10. Semi-quantitative detection of AR-AChE in serum of patients with cancer before and after chemotherapy

Figure 11. Photograph of rat PC12 cells

 A. Visible light photo. Apoptotic PC12 cells were positive for substrate and distributed in the cytoplasm and nucleus.

 B. Normal PC12 cells, confocal (C0NF0CAL) photos. FITC showed that AChE was expressed in normal living cells, distributed in the cytoplasm and membrane, and there was no positive in the nucleus. The blue color was H0ECHST staining, showing the nucleus.

 C. Photograph of visible light. Anti-AR-AChE antibody (ARA-M5), DAB reaction, brown positive, hematoxylin background staining, showing nuclei; adherent growth of live PC12 negative, pyknotic apoptotic cells positive.

Figure 12. Serum AR-AChE in 30 normal newborn cord blood and one tumor patient.

 Normal newborn cord blood AR-AChE 0D values are below 0.2, close to the blank control. In a tumor patient, the 0D value of serum AR-AChE on day 3 was 3.1. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be further described with reference to specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention. The reagents, instruments, and the like which are not particularly described in the present invention are commercially available conventional products. The experimental conditions in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions such as Sambrook, Russell et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2001), or According to the conditions recommended by the manufacturer. Real 1⁄4 case 1 Preparation of anti-apoptosis-related acetylcholinesterase monoclonal antibody and hybridoma

 I, preparation before cell fusion

 : (i) Immunization programme

A, 3⁄4 original: according to the method of the inventor's patent (patent number: ZL 97 1 25216. 5; Qi-Huang JIN, Yu-Fang SHI, Heng-Yi HE, Kelvin KW Ng, Hua JIANG, Lei YANG, Zi-Qing JIANG and Xue-Jun Zhang Isolation of AChE from Apoptotic Human Lung Fibroblast Cells by Antibody Affinity Chromatography. BioTechni _Q ues i33 (4): S92-S97, 2002) AR-AChE expressed by HELA cells.

B, mouse: BaLb I c mice, 3 males 6 to 10 weeks old. Human AR-AChE, Garfield's complete adjuvant (limited to the first use) or Freund's incomplete adjuvant, mixed together in equal volumes, with two syringes, through a three-way valve, repeated pushes to make the two into a fuel pack The chyle of water.

 Primary immunization antigen (Ag) l~50 g plus Freund's complete adjuvant subcutaneous injection

 I (0.8~lml 0.2ml/point)

 After 3 weeks

 Second immunization dose is the same as above, Jiafu's incomplete adjuvant subcutaneous

 After week

 The third dose is the same as above, without adjuvant, intraperitoneal injection (ip)

 I (5~7 days after blood sampling to measure its potency, detect immune effect)

 |2~3 weeks later

 Strengthen immunity, dose 100 g, ip

 3 days later

 Spleen fusion

 (2) Feeding cells Mouse peritoneal macrophages

 Preparation of mouse peritoneal macrophages on the day before fusion

 Using BaLb/c mice 6 to 10 weeks old

 1

 The neck is killed, soaked in 75% alcohol, disinfected for 3 to 5 minutes.

 I

 Cut the skin with sterile scissors and expose the peritoneum

 I

 Inject 6~8ml of culture solution with a sterile syringe

 I

 Rinse repeatedly, aspirate the rinse

' Put in a 10ml centrifuge tube, 1200 rpm / separate heart 5~6 minutes

 I

; Suspension with 20% calf serum (NCS) or fetal bovine serum (FCS), adjust the number of cells lX10 ⁵ /ml

 I

 Add 96-well plate, 100yl/?L

 1

Put in 37Ό C0 ₂ incubator culture

 (c) myeloma cells

The myeloma cells were resuscitated two weeks before the preparation for fusion. The inventors used the myeloma SP2/0 cell line, RPMI1640 medium, the concentration of calf serum was 20%, and the maximum density of cells did not exceed 10 ^s / ml. 1: 10 dilution passage, pass every 3 to 5 days.

 (four) immune spleen cells

 Preparation of spleen cell suspension: The spleen was taken out under aseptic conditions, washed once with an incomplete culture solution, placed on a stainless steel mesh in a dish, and ground to a cell suspension with a syringe needle and counted.

 II, cell fusion, selection of hybridoma

 (a) Cell fusion process

 (1) The logarithmic growth of myeloma cells was centrifuged at SP2 / 0, 1000 rpm for 5 minutes, the supernatant was discarded, the cells were suspended with incomplete culture, counted, the number of cells required was taken, and the cells were washed twice with incomplete broth. .

 (2) Simultaneous preparation of an immune spleen cell suspension, and washing with an incomplete culture solution twice.

 (3) Mix myeloma cells and spleen cells in a ratio of 1:10 or 1:5, and wash them in an incomplete medium in a 50 ml plastic centrifuge tube at 1200 rpm for 8 minutes.

 (4) Discard the supernatant and use a pipette to remove residual liquid to avoid affecting the concentration of PEG.

 (5) Gently hit the bottom of the centrifuge tube to loosen the cell pellet slightly.

 (6) Fusion at room temperature:

 1 Preheated lml45% PEG (Merek, molecular weight 4000) containing 5% DMS0 was added in 30 seconds while stirring.

2 Function 100 seconds.

 3 Add pre-heated incomplete culture solution, stop the action of PEG, add lml, 2ml, 3ml, 4ml, 5ml and 10ml every 2 minutes.

 (7) Centrifugation, 800 rpm, 6 minutes.

 (8) Discard the supernatant and gently suspend it with 6ml of 20% calf serum RPMI1640.

 (9) According to the number of 96-well culture plates used, add complete culture solution, 10 ml-block 96-well plate.

(10) The fused cell suspension was added to a 96-well plate containing feeder cells, 100 μl / ί, 37 ° C 5 C0 ₂ incubator.

A 96-well plate contains 1 X 10 ⁷ spleen cells.

 (B) HAT selects hybridomas.

 After 24 hours of fusion, the HAT was selected by adding HAT. Store the 50XHT and HAT reagents in lml

50 ml of 20% calf serum in complete medium.

 50 X HAT

; H: 5X 10" ³ M

A: 2X 10" ⁵ M

T: 8 X 10— ⁴ Μ

 After the HAT selection medium was maintained for two weeks, the HT medium was used instead, and the culture was maintained for two weeks, and the general culture solution was used instead.

 III. Detection of antibodies

The inventors used an ELISA screening method. IV. Cloning and cryopreservation of hybridomas

 (i) Cloning scheme

 Clone by limiting dilution, the procedure is as follows:

 1 Preparation of feeder cell suspension (prepared before fusion)

2 count the number of positive well cells, and adjust the number of cells in l~5 X 10 ³ / ml

 3 Take 130 cells into 6. 5ml complete culture medium containing feeder cells, ie 20 cells / ml, 100 μ ΐ / well plus A, B, C three rows for 2 cells per well. The remaining 2. 9ml cell suspension is supplemented with 2. 9ml of complete culture medium containing feeder cells, the number of cells is 10 / ml, 100 μ 1 /well plus D, E, F three rows, one cell per well. 2. 2 ml of cell suspension supplement 2. 2 ml of complete culture medium containing feeder cells, the number of cells is 5 / ml, 100 u l / ?L, plus G, H two rows, 0.5 cells per well.

 4 After 4 to 5 days of culture, a small cell clone was observed on an inverted microscope, and a complete culture solution of 200 μM / well was added.

 5 On days 8 to 9, cell clones were visible to the naked eye and antibody detection was performed in time.

 (ii) Cryopreservation of hybridoma cells

The hybridoma cells contain 1 X 10 ⁶ or more per ampule.

 Cell cryopreservation: 50% calf serum; 40% incomplete medium; 10% DMS0 (dimethyl sulfoxide)

 V, mass production of monoclonal antibodies

 1. Inoculate hybridoma cells in vivo to prepare ascites.

The ascites was prepared by intraperitoneal injection of 0.5 ml of Pristane or planting paraffin in BaLb / c mice. After 1 to 2 weeks, IX 10 ⁶ hybridoma cells were intraperitoneally injected. After inoculation for 7 to 10 days, the mice were sacrificed. , collect ascites with a dropper. Example 2 Identification of AR-AChE antibodies

 1. Identification of antibody specificity: In addition to the detection of antibodies by immunogen (antigen) by ELISA, cross-test with human albumin, actin, and electroporation AChE was performed, and the results were correlated with human AR-AChE. Strongly, weakly positive with AChE, negative for human albumin, actin (see Figure 5), immunohistochemistry and cytochemical detection, no recognition of human brain AChE (see Figure 1) and human erythrocyte AChE (see figure 2).

 2 Identification of Ig and subclasses of McAb: Screening with an enzyme-labeled secondary antibody has largely established the antibody type Ig. Analyzed by (BioType) kit (Mouse Typer sub-isotyping Kit catalog number 1 _2051), ARA-Ml (Accession No. CCTCC-C200413) is IgG; ARA-M5 (Accession No. CCTCC-C200414) is IgG2a; ARA-M7 (Accession No. CCTCC-C200415) is IgGl.

3. Identification of McAb neutralization activity: The addition of antibodies to AR_AChE can block the catalytic activity of the enzyme. In order to identify whether the inventor's monoclonal antibody specifically recognizes AR-AChE expressed by apoptotic cells, and whether the esterase activity changes after the antibody forms a complex with the antigen, the inventors performed immunosuppressive experiments, and the inventors used Karnovsky, MJ Roots, L. A method for detecting acetylcholinesterase activity (Karnovsky, MJRoots, L. A "Direct-Coloring" Thiocholine Method for Cholinesterases. J Histochem C ytochem 1964 ; 12 : 219- 21. ), and then in 96 wells, the enzyme activity was detected at 405 nm using a microplate reader.

Using lytic apoptotic HELA, divided into 3 groups

 A. Apoptosis HELA lysate, without substrate, but replaced with an equivalent amount of PBS

 B. No antibody, substrate reaction, positive result

 C. Add excess antibody, form a complex, centrifuge, and make a substrate reaction between the supernatant and the precipitate, respectively, and the results are both negative (Fig. 3). Inventor C performed the supernatant and precipitation as WESTERN BLOT, indicating that there was no AChE in the supernatant, and the precipitated antigen complex had an AChE positive band. Example 3 Comparison of AR-AChE Antibodies with Commercially Available Antibodies

Common: 1. Identify AChE (common to common commercial AChE antibodies)

 AR-AChE antibody: normal HELA cells were negative and apoptotic cells were positive (the results of the product are shown in the next paragraph, and the monoclonal antibody is shown in Figure 4). The AR-AChE antibody recognizes the same band as the commercial AChE antibody (see Figure 5).

 The inventors have reported (Zhang XJ, 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 Various Cell Types. Cell Death and Differentiation 9 (8): 790-800, 2002.), normal live HELA cells do not express AChE, whereas apoptotic HELA cells express AChE instead of butyrylcholinesterase (BuChE). The inventors used two affinity columns (concanavalin A - Sepharose and edrophonium - Sepharose) (Ji Young Son, Sook Shin, Kwang Ho Choi, and In Kook Park. Purification of soluble acetylcholinesterase from Japanese quai l brain by affinity chromatography. The international Journal of biochemistry- & cell biology. 34 (2) : 204- 210, 2002) AChE was isolated from apoptotic HELA cells. The inventors used the isolated protein as the WESTERN BLOT, and the results showed that the purchased antibody and the inventor's monoclonal antibody recognized the same band (66 kDa). (Figure 5 )

 2. Inhibition of enzyme activity, AChE activity of apoptotic HELA can be blocked by the inventor's monoclonal antibody (identification of McAb neutralization activity, Figure 3), the inventors will perform supernatant and precipitation as WESTERN BLOT, showing no in the supernatant AChE, while in the pellet, there is an AChE positive band.

Different from existing antibodies: specificity.

 The antibody of the present invention recognizes only AR-AChE expressed by apoptotic cells, and is different from the commercially available antibody, 绚JS immunoassay, which recognizes only AR-AChE expressed by apoptotic cells, and specific evidence (Fig. 1, 6). Figure 6 shows that some nucleated cells isolated from blood were placed at room temperature for 18 hours, and some cells were apoptotic. The photos are all the same field of view.

1. Human and rat synaptic (brain) AChE are not recognized (Fig. 1, Fig. 7). Figure 7 shows rat brain tissue sections, AR-AChE immunohistochemistry and TUNEL double staining. The rat is subjected to ischemic treatment of the middle cerebral artery (Longa EZ, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989 Jan; 20 (l): 84- 91.) From the damage surrounding group ^ only. photo A, B, and C are all the same field of view.

 2. Human red blood cell membrane AChE is not recognized (Fig. 2C). The cord blood was allowed to stand at room temperature for 18 hours, and some cells developed apoptosis. The pictures of A, B, and C in Figure 2 are all the same field of view.

 Anti-neuronal AChE antibodies on the market today are also different from antibodies against AChE on erythrocyte membranes. The antibody of the present invention recognizes only AChE expressed by apoptotic cells, which the inventors call (apoptosis-related acetylcholinesterase, abbreviation AR-AChE), and the anti-neuronal AChE antibody on the market can recognize AR-AChE. However, there is no specificity, they do not recognize the specificity of apoptotic cells, and therefore cannot be used as markers of apoptosis. The inventors have obtained apoptosis-associated acetylcholinesterase (AR-AChE) as an antigen (Xue-Jun Zhang, Lei Yang, Qi-Huang Jin, Yu-Fang Shi*, Hua Jiang, Heng-Yi He, Kelvin NG) And Zi-Qing Jiang. Various apoptotic mammalian cells express apoptosis-related acetylcholinesterase (AR-AChE) . Acta biochimica et biophysica sinica 35 (2): pp213, 2003. On this basis, the inventors prepared monoclonal antibodies against AR-AChE. With specific anti-AR-AChE monoclonal antibodies, AR-AChE can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptotic phenomena, for basic research, AD research, AD-assisted diagnosis and treatment, and the judgment of chemotherapy effects. This is the first monoclonal antibody in the world to specifically recognize AR-AChE expressed by apoptotic cells. Example 4 Observing the distribution of normal brain type AChE and the relationship between enzyme and nucleus

 The five-month human fetal brain tissue induced by labor (Red House Hospital, with the consent of the family members) was frozen and sectioned, fixed with 4% paraformaldehyde, and stained for acetylcholinesterase activity. Preparation of acetylcholinesterase substrate reaction solution (0.1 M phosphate buffer Ph 6. 0 150 ml, acetylthiocholine 100 mg / 200 ml, 0.1 M sodium citrate llml, 30 mM copper sulfate 20 ml, 5 mM ferric cyanide Potassium (20 ml), the enzyme reaction was carried out. When detecting the activity of acetylcholinesterase in cells, the cells were incubated in the reaction solution for 3-4 hours at room temperature, and the reaction was yellow-brown (dark gray in the figure). (See S 8B) o It was more uniform and showed no outline of cells or nuclei. , indicating that the product is not concentrated in the cytoplasm or fine nucleus, which is completely different from the distribution in apoptosis found by the inventors. Figure 8C further demonstrates that AChE positivity is not in the nucleus. Example 5 Observing a small amount of neuronal apoptosis during development

The five-month human fetal brain tissue was inoculated, frozen in 4% paraformaldehyde, immunohistochemical reaction and Hoechst staining, showing nuclear and AR-AChE positive cells, respectively (see Figure 1), indicating that the antibody of the present invention does not recognize large pieces. The presence of acetylcholinesterase recognizes only apoptosis-associated AChE. Example 6 Using human SK-N-SH cells cultured in vitro, it was confirmed that the monoclonal antibody of the present invention specifically utilizes human SK-N-SH cells cultured in vitro, indicating that the antibody of the present invention recognizes only apoptotic cells, but does not recognize normal cells. Acetylcholinesterase, because SK-N-SH cells express acetylcholinesterase and are distributed on the cell membrane. See Figure 9, the photo is divided into five parts, A, B, C, D, and E, all of which are the same field of view. C-1 antibody is a mouse anti-human AR-AChE monoclonal antibody (ARA-M7), 2 anti-mouse anti-mouse IgG antibody (1:100 dilution, Rhodamine conjugated anti-mouse IgG-, Santa Cruz), and an apoptotic cell on the lower right side showed positive. Human SK-N-SH cells cultured in vitro can express a small amount of synaptic AChE normally. In this photo, one apoptotic cell showed positive, others were not positive, and TUNEL staining was in good agreement with anti-AR-AChE positive. This monoclonal antibody specifically recognizes AR-AChE of apoptotic cells, but does not recognize the synaptic AChE expressed by SK-N-SH cells. Example 7 Using the monoclonal antibody of the present invention to determine the tumor chemotherapy effect

 The semi-quantitative analysis of acetylcholinesterase in human serum using the antibody of the present invention can be used to judge the effect of tumor chemotherapy (including leukemia). This was carried out by using the monoclonal antibody against human AR-AChE (ARA-M5 and ARA-Ml), and the sandwich bread method was used for ELISA to detect the AR-AChE content in serum of patients with tumors before and after PAD, TZH and GB chemotherapy. It can be seen that the levels of AR-AChE in the serum of patients with PAD and TZH 72 hours after chemotherapy were more than 1 times higher than those before treatment, indicating that the curative effect is better. Patients with serum AR-ACHE levels before and after GB treatment did not change, the efficacy was poor (see Figure 10). Example 8 Specificity of the monoclonal antibody of the present invention for identifying apoptotic cells

 Using rat PC12 cells cultured in vitro, it was demonstrated that the monoclonal antibody of the present invention recognizes only apoptotic cells, but does not recognize normal acetylcholinesterase, because rat PC12 cells express acetylcholinesterase and are distributed on the cell membrane (see Fig. 11B). . Normal PC12 cells, C0NF0CAL photo FITC showed that AChE was expressed in normal living cells, distributed in cytoplasm and membrane, and there was no positive in the nucleus; apoptotic PC12 cells, positive for substrate reaction, distributed in cytoplasm and nucleus (see figure 11). Example 9 Semi-quantitative detection using monoclonal antibodies of the present invention 30 normal newborn cord blood and one tumor patient serum AR-AChE

 Semi-quantitative analysis of acetylcholinesterase in serum of 30 normal newborns with cord blood was performed using the antibody of the present invention, and the 0D values were all below 0.2, which was close to the blank control. The 0D value of serum AR-AChE on the third day of chemotherapy in a tumor patient was above 3.1. This is the use of the monoclonal antibody against human AR-AChE of the present invention (paired ARA-M5 and ARA-M1), and the sandwich bread method was used for ELISA to detect the AR-AChE content in the cord blood serum of tumor patients and normal newborns, respectively. It can be seen that the serum AR-AChE content of the patient at the 72 hours after chemotherapy is more than 20 times that of normal newborn cord blood. The content of AR-AChE in the serum of normal newborn cord blood is very low, and the 0D value is below 0.2, which is close to the blank control (see Figure 12). All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the equivalents of the scope of the present invention.

Claims

 Rights request

A monoclonal antibody, which specifically recognizes apoptosis-associated acetylcholinesterase.

The antibody according to claim 1, wherein the antibody is secreted by a mouse hybridoma cell line having the accession number CCTCC N0: -C200413, CCTCC NO: -C200414 or CCTCC NO: - C200415.

 The monoclonal antibody according to claim 2, wherein the antibody is a humanized antibody.

 The monoclonal antibody according to claim 2, wherein the antibody is a chimeric monoclonal antibody comprising a non-human variable region and a human light chain and heavy chain constant region.

 A hybridoma cell line characterized in that the hybridoma cell line produces the monoclonal antibody of claim 1.

 The hybridoma cell line according to claim 5, wherein the hybridoma cell line has the accession number CCTCC NO: -C200413, CCTCC NO: - C200414 or CCTCC NO: - C200415.

 7. An immunoassay characterized in that the method is carried out using at least one monoclonal antibody of claim 1.

 8. The immunoassay of claim 7, wherein the specimen is blood, tissue or cerebrospinal fluid.

 The immunoassay according to claim 7, wherein the assay is carried out by an ELISA technique.

 The immunoassay according to claim 7, wherein the assay is carried out by immunochromatography.

A kit comprising at least one monoclonal antibody of claim 1.

The kit according to claim 11, wherein the kit comprises any one of the monoclonal antibodies of claim 2.

 13. A composition comprising at least one monoclonal antibody of claim 1 and a pharmaceutically acceptable carrier and/or excipient.

 14. Use of the monoclonal antibody of claim 1 for screening an antitumor drug. .

 15. A method of screening for an anti-tumor drug, characterized in that it comprises the steps of:

 (!) cultivating human tumor cell lines;

 (2) adding a drug candidate to the cell culture medium in the step (1), detecting the expression level of apoptosis-associated acetylcholinesterase by the monoclonal antibody of claim 1, and promoting the apoptosis-associated acetylcholinesterase The candidate drug with increased expression is a drug that promotes apoptosis of tumor cells, and conversely, the drug candidate is ineffective.

Id The use of the monoclonal antibody of claim 1 for determining the therapeutic effect of an antitumor drug.

 17; a method for judging the therapeutic effect of an anti-tumor drug, characterized in that it comprises the steps of: detecting the expression level of apoptosis-associated acetylcholinesterase in peripheral blood of a tumor patient after 8 to 168 hours after anti-tumor treatment, and treating Pre-comparison; Fine: Increased expression of apoptosis-associated acetylcholinesterase, indicating that anti-tumor drugs are effective, no change or expression is not increased ii, anti-tumor drugs are ineffective.

18. Use of the monoclonal antibody of claim 1 for the manufacture of a medicament for the diagnosis and/or treatment of a neurodegenerative disease:

19. Use of the monoclonal antibody of claim 1 for the preparation of a kit for detecting the expression level of apoptosis in acute organ damage.