CN1772766A - Acetylcholinesterase monoclonal antibody related to resisting apoptosis and its use - Google Patents

Abstract

Based on the discovery of apoptosis-related acetylcholinesterase (AR-AChE), the present invention uses purified human AR-AChE as an antigen to immunize mice and perform cell fusion to obtain hybridomas, especially to obtain expression and secretion anti-apoptosis The relevant acetylcholinesterase (AR-AChE) monoclonal antibody and its hybridoma cell lines ARA-M1, ARA-M5 and ARA-M7 are amplified to produce antibodies ARA-M1, ARA-M5 and ARA-M7. These antibodies can be used for anti-tumor drug screening, judging the effect of chemotherapy for cancer patients, preparing drugs for the diagnosis and/or treatment of degenerative neurological diseases, such as apoptosis analysis of AD and basic and clinical research and diagnosis of cell apoptosis, and detection of acute organ damage Expression level of apoptosis in middle cells. The invention also provides an immunoassay method and kit for semi-quantitative and/or quantitative analysis of AR-AChE.

Description

Anti-apoptosis-related acetylcholinesterase monoclonal antibody and use thereof

technical field

The invention relates to the field of biotechnology, in particular to an anti-apoptosis-related acetylcholinesterase (AR-AChE) monoclonal antibody and application thereof.

Background technique

Acetylcholinesterase (EC 3.1.17, acetylcholinesterase, AChE) is a hydrolase of neurotransmitter acetylcholine, which belongs to glycoprotein. It mainly exists in the electrical organs of electric rays, cholinergic nerves of mammals, nerve-muscle junctions, red blood cell membranes (Zakut-H; et al: J-Clin-Invest.1990, 86(3): 900-8) and In rodent megakaryocyte platelets (A. Shafferman and B. Velan, Multidisplinary approaches to cholinesterase functions. 1992 Plenum Press, New York). AChE can be divided into neuromuscular type and erythrocyte type according to its location. According to the molecular structure, it can be divided into heteromeric class with different subunits and homomeric class with the same subunit. Catalytic subunits of different subunit multimeric types are linked to triple helical collagen subunits or linked to lipids. The same subunit polymer type is divided into hydrophilic (hydrophilic) and lipophilic (glycophospholipid-linked) and other subtypes. The molecular weight of the AChE tetramer on the red blood cell membrane is about 260KDa. The function of AChE in the cholinergic nervous system is to hydrolyze the neurotransmitter acetylcholine to generate choline and acetic acid. It has also been reported that AChE has the proteolytic activity of serine protease. In recent years, since AChE mRNA transcription was also detected in tumor cell lines cultured in vitro and AChE activity was detected in the serum of ovarian cancer patients after chemotherapy, some authors believed that AChE was related to tumorigenesis (Lev-Lehman-E, et al. al: Blood. 1997, 89(10): 3644-53).

The inventors first discovered that mammalian cells express AChE during apoptosis (Zhang XJ, Yang L, Zhao Q, CaenJP, 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.), cell lines derived from nervous system (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.) also has the same phenomenon. In order to further study the role of AChE in apoptosis, the inventor established a method for isolating 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-YiHE, 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. BioTechniques 33(4): S92- S97, 2002.). Due to the cell where the enzyme is expressed, the time of appearance, the function performed, the post-translational modification and even the cutting of mRNA (“A human acetylcholinesterase isoform protein (AR-AChE) and gene coding sequence”, application number: 01105781.5 , date of application, 2001/3/23.), completely different from the reported neuromuscular and erythrocyte AChE, the inventor calls the AChE expressed by apoptotic cells apoptosis-related acetylcholinesterase (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 sinica 35(2):pp213, 2003.). Utilizing the characteristics of apoptotic cells expressing AR-AChE, the inventors can use the method of detecting the enzyme activity to screen anti-tumor drugs ("anti-tumor drug screening method", CN 1186859 has been authorized. Patent No.: ZL 97125220.3) and used as an apoptotic cell markers.

For the neuromuscular type, erythrocyte type, and electric ray AChE, people have prepared polyclonal and monoclonal antibodies (polyclonal anti-AChE antibody (Institute of Pharmacology and Toxicology, Academy of 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)), which recognizes neuromuscular Type antibodies can also recognize AchE expressed by apoptotic cells (Zhang XJ, YangL, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF, Induction of Acetylchol inesterase 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 (it does not recognize AChE on normal synapses), it is still difficult to identify apoptotic cells by recognizing AChE using immunological methods.

The inventors found that cells that do not express AChE express a large amount of AChE during the process of cell apoptosis, mainly concentrated in the nucleus, and as the nucleus is formed into apoptotic bodies, AChE exists in the apoptotic bodies. The inventor established a method for detecting AChE activity, identifying apoptotic cells and judging the effect of chemotherapy (the method for screening anti-tumor drugs and judging the effect of clinical chemotherapy, Chinese Patent No. 97 1 25220.3). Due to the activity detection, only the protein can be qualitatively analyzed, and the presence of butyrylcholinesterase (BuChE) in the serum will interfere with the activity of AChE and affect the detection of the activity of AChE.

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

Contents of the invention

The purpose of the present invention is to provide a monoclonal antibody against apoptosis-related acetylcholinesterase (AR-AChE) and a mouse hybridoma cell line producing the antibody.

Another object of the present invention is to provide the application of anti-AR-AChE monoclonal antibody, and the antibody of the present invention can be used to measure the activity and expression level of AR-AChE during cell apoptosis. Furthermore, the antibody can be prepared into a kit to screen anti-tumor drugs, determine the effect of anti-tumor drug treatment, assist in the diagnosis of neurodegenerative diseases such as AD, and detect the expression level of apoptosis in acute organ damage, providing doctors with information on the incidence of patients with disease. and later reference data.

Another object of the present invention is to provide a method for screening anti-tumor chemotherapeutic drugs and an immunoassay for detecting apoptosis-related acetylcholinesterase.

The first aspect of the present invention provides a monoclonal antibody, which specifically recognizes apoptosis-related acetylcholinesterase.

Preferably, the above-mentioned monoclonal antibody is secreted by mouse hybridoma cell lines with deposit numbers CCTCC NO: -C200413, CCTCC NO: -C200414, and CCTCC NO: -C200415.

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

The second aspect of the present invention provides a hybridoma cell line, characterized in that the hybridoma cell line produces the above-mentioned monoclonal antibody. Preferably, the aforementioned hybridoma cell line is a mouse hybridoma cell line. Preferably, the aforementioned hybridoma cell lines are mouse hybridoma cell lines with preservation numbers CCTCC NO:-C200413, CCTCC NO:-C200414, and CCTCC NO:-C200415. The present invention also provides the monoclonal antibody secreted by the above-mentioned hybridoma cell line.

A third aspect of the present invention provides an immunoassay performed with at least one monoclonal antibody as described above. The sample in the above immunoassay is blood, tissue or cerebrospinal fluid, preferably blood supernatant. The immunoassays described above can be performed using ELISA techniques or immunochromatographic techniques. Preferably, the above-mentioned monoclonal antibodies are monoclonal antibodies of one or more subtypes secreted by mouse hybridoma cell lines with deposit numbers CCTCC NO:-C200413, CCTCC NO:-C200414, and CCTCC NO:-C200415.

The fourth aspect of the present invention provides a kit containing at least one of the above-mentioned monoclonal antibodies. Preferably, the above-mentioned monoclonal antibody is selected from any one of ARA-M1, ARA-M5, and ARA-M7 (hybridoma cell lines, see 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 acetylcholinesterase related to cell apoptosis, especially the expression level of AR-AChE in acute organ damage and the like.

The fifth aspect of the present invention provides a composition comprising at least one monoclonal antibody mentioned above and a pharmaceutically acceptable carrier and/or excipient. Preferably, the above composition includes monoclonal antibodies of one or more subtypes secreted by mouse hybridoma cell lines with deposit numbers CCTCC NO:-C200413, CCTCCNO:-C200414, and CCTCC NO:-C200415. The composition of the invention is used for treating diseases caused by abnormal expression of apoptosis-related acetylcholinesterase, including neurodegenerative diseases.

The fifth aspect of the present invention provides the application of the above-mentioned monoclonal antibody in screening anti-tumor drugs.

The sixth aspect of the present invention provides a method for screening antitumor drugs, which includes the following steps:

(1) Cultivate human tumor cell lines;

(2) Add candidate drugs to the cell line culture medium in step (1), and use the above-mentioned monoclonal antibody to detect the expression level of apoptosis-related acetylcholinesterase; Candidate drugs are drugs that promote tumor apoptosis, otherwise, the candidate drugs are ineffective.

Preferably, the above-mentioned 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 adding the candidate drug.

The seventh aspect of the present invention provides the application of the above-mentioned monoclonal antibody in judging the therapeutic effect of anti-tumor drugs.

The eighth aspect of the present invention provides a method for judging the therapeutic effect of anti-tumor drugs, which includes the steps of: detecting the expression level of acetylcholinesterase related to apoptosis in the peripheral blood of tumor patients 8-168 hours after being treated with anti-tumor drugs, And compared with before treatment; the expression of apoptosis-related acetylcholinesterase increases, indicating that the anti-tumor drug is effective, and if there is no change or the expression does not increase, the anti-tumor drug is ineffective.

The preferred detection time is 12-120 hours.

The ninth aspect of this aspect provides the use of the above-mentioned monoclonal antibody in the preparation of a drug for diagnosing or treating neurodegenerative diseases.

The tenth aspect of this aspect provides the application of the above-mentioned monoclonal antibody in the preparation of a kit for detecting the expression level of acetylcholinesterase related to apoptosis in acute organ damage.

The inventor once discovered the isoform (isoform) of acetylcholinesterase (AChE) from apoptotic cells, named it 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 inventors found that many cell lines expressed 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.), because the expression time of the enzyme, cell type, intracellular distribution, post-translational modification and functional characteristics are different from those of the synapse on neurons AChE on haptic and motor endplates, AChE on erythrocyte membrane, and AChE in megakaryocytes, therefore, the inventors called the AChE expressed by apoptotic cells apoptosis-related acetylcholinesterase (apoptosis-related acetylcholinesterase, AR- AChE). The inventors call the antibody against AR-AChE expressed by apoptotic cells anti-apoptosis-related acetylcholinesterase antibody (antiapoptosis-related acetylcholinesterase antibody). For AR-AChE, the inventors can prepare monoclonal antibodies and polyclonal antibodies.

The anti-neurite AChE antibody on the market is also different from the antibody against AChE on the red blood cell membrane. The antibody of the present invention only recognizes AChE expressed by apoptotic cells, which the inventors call (apoptosis-related acetylcholinesterase, abbreviated as AR-AChE), while anti-neurite AChE antibodies on the market may recognize AR- AChE, but without specificity, they do not have the specificity to recognize apoptotic cells, so they cannot be used as a marker of apoptosis. The inventors have obtained apoptosis-related acetylcholinesterase (AR-AChE) as an antigen (Xue-Jun Zhang, Lei Yang, Qi-HuangJin, 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 a monoclonal antibody against AR-AChE. With the specific production of anti-AR-AChE monoclonal antibody, AR-AChE can be distinguished from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptosis phenomena, basic research, AD research, auxiliary diagnosis and treatment of AD, judgment of chemotherapy effect, etc. This is the first monoclonal antibody invented in the world that specifically recognizes AR-AChE expressed by apoptotic cells.

Therefore, the inventor used the Chinese patent (ZL 97 1 25216.5, method for obtaining brain-type AChE from mammalian cells) to isolate AR-AChE from apoptotic cells, (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 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 out, which specifically produced anti-apoptosis-related acetylcholinesterase (AR-AChE) monoclonal antibody.

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

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

The hybridoma cell lines ARA-M1 of the present invention, ARA-M5, and ARA-M7 have been preserved in the China Type Culture Collection Center (Wuhan) on September 21, 2004 according to the "Budapest Convention on the Preservation of Microorganisms for Patent Procedures", The deposit numbers are CCTCC NO: -C200413, CCTCC NO: -C200414, CCTCC NO: -C200415 respectively.

Hybridomas of the invention can be produced using cell fusion techniques known in the art. Therefore, animals other than humans are immunized with AR-AChE as an immunogen, spleen cells or lymph node cells of the animals are fused with myeloma cells to generate hybridomas, and hybrids that produce monoclonal antibodies that recognize AR-AChE are selected from them. tumor, thereby obtaining the hybridoma of the present invention.

The above-mentioned immunogen for preparing hybridomas 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 AR-AChE protein or a fusion protein containing AR-AChE protein can be cultivated on a suitable medium.

When antibodies produced by non-human subjects or antibodies derived from the expression of non-human antibody genes are used in the human body, they are recognized as foreign antibodies to varying degrees, and immune responses may occur in patients. One approach to minimizing or eliminating this immune response is to generate chimeric antibody derivatives, antibody molecules that combine non-human animal variable regions with human constant regions. Therefore, the present invention also includes humanized antibodies, and the application of humanized antibodies in the preparation of drugs for treating neurodegenerative diseases (such as AD). Those skilled in the art can use the monoclonal antibody of the present invention to prepare humanized antibodies.

These antibodies retain the epitope-binding specificity of the original monoclonal antibody, but may be less immunogenic when administered to humans and thus more likely to be tolerated by patients.

Chimeric monoclonal antibodies can be prepared using recombinant DNA techniques known in the art. For example, genes encoding constant regions of non-human antibody molecules are replaced with genes encoding human constant regions (see, Robinson et al., PCT Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; or Taniguchi, M. ., European Patent Application 171,496). Chimeric antibodies can be further "humanized" by replacing portions of the variable region not involved in binding antigen with human equivalent portions of the variable region.

The application field of the monoclonal antibody of the present invention is not particularly limited, such as it can be used in immunoassay, including quantitative or semi-quantitative detection. The monoclonal antibody of the present invention can detect the presence or absence of AR-AChE as the basis for judging the result of tumor chemotherapy.

The antigen-binding fragment of the antibody of the present invention refers to an antibody fragment that can bind to the antigenic epitope of apoptosis-related acetylcholinesterase.

Animals immunized to produce the hybridoma of the present invention are not particularly limited, and include, for example, goats, sheep, guinea pigs, mice, rats and rabbits. Of these, mice are preferred.

The immunoassay method of the present invention is carried out with at least one monoclonal antibody of the present invention, and the method can be carried out with only one kind of antibody, also can carry out in combination with AR-AChE polyclonal antibody, or use two or more monoclonal antibodies simultaneously (such as ARA-M1 and ARA-M5).

Immunoassay methods of the present invention include techniques such as enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, and immunochromatography. The various immunoassays described above can be used to detect target antigens or antibodies using antigens or antibodies labeled with markers in a competition method or a sandwich method. Among the various immunoassays described above, ELISA and immunochromatographic techniques are preferred.

The above-mentioned sandwich method includes, for example, sandwiching AR-AChE between immobilized one subtype of the present monoclonal antibody and another subtype of the present monoclonal antibody or anti-AR-AChE polyclonal antibody labeled with a labeling agent. , and then add a substrate for a marker (such as an enzyme), display a color, etc., thereby detecting AR-AChE in the sample. Monoclonal antibodies with high sensitivity are usually considered as the preferred labeled antibodies, because compared with polyclonal antibodies with low specificity and high background noise, monoclonal antibodies have high specificity and low background noise, making the detection more accurate.

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

For the above labeling agent, radioactive isotopes such as ¹²⁵ I, enzymes, enzyme substrates, phosphorescent substances, fluorescent substances, biotin, and coloring substances can be used. In binding these labeling agents to antigens or antibodies, the maleimide method (J.Biochem.(1976), 79, 233), the activated biotin method (J.Am.Chem.SOC.(1978) , 100, 3585) or hydrophobic bond method.

As the above-mentioned enzymes, there are included, for example, peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase. For the substrate used in this occasion, substrates suitable for the enzyme used in this occasion can be selected, including for example: ABTS, luminol-H ₂ O ₂ , o-phenylenediamine-H ₂ O ₂ (for peroxide enzyme), p-nitrophenyl phosphate, methyl phosphonate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1 ,, 2-dioxetane (for alkaline phosphatase), p-nitrophenyl-BETA-D-galactose (for β-galactosidase).

It can be reacted at 4-40°C for 1 minute to 18 hours, and then the color or fluorescence, phosphorescence or color development displayed by the result is measured, and the above test is carried out. Alternatively, a so-called rate test can be used, which is carried out with an incubator in the range 4-40°C.

Radiolabeling of the above-mentioned antigens or antibodies can be conveniently performed using commercially available Bolton-Hunter reagents. For example, it can be removed by adding Bolton-Hunter reagent to a solution (the solvent is prepared by dissolving antigen or antibody in 0.1M aqueous sodium bicarbonate solution), and then removing it with G-25 desalting column etc. after 1-2 hours Unreacted Bolton-Hunter reagent fraction.

In addition, ¹²⁵ I radiolabeling can be conveniently carried out by using the chloramine T method, the iodine method, and the like.

The above-mentioned fluorescent substances, such as fluorescein and rhodamine, can be conveniently labeled using the activated ester method or the isocyanate method ("Enzyme immunoassay technique", published by Igaku Shoin, 1987). As the above-mentioned coloring substances, 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 the outstanding property of recognizing only AR-AChE present in all apoptotic cells, including tumor or neuronal cells, without false detection due to cross-reactivity Other substances and normal AChE, so a very specific assay is possible.

To carry out the immunoassay of the invention, the kits of the 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 that recognizes AR-AChE, and may also be a breakdown 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 may be immobilized on a solid phase in advance, and the monoclonal antibody of the present invention may also be labeled with the above-mentioned labeling agent.

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

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

The kits of the invention may also contain other components. The above-mentioned other components are not particularly limited, but include, for example, enzymes for labeling, substrates thereof, radioactive isotopes, phosphorescent substances, fluorescent substances, coloring substances, buffers, and petri dishes, as well as the above-mentioned components.

The kit of the present invention may be a kit for carrying out the above-mentioned competitive method or the above-mentioned sandwich method. Preference is however given to kits using the sandwich method. The above-mentioned sandwich method has the advantages of high sensitivity, short reaction time, high accuracy and convenient operation. Using the above-mentioned immunochromatographic technique, a kit can be prepared, which makes the test method convenient and simple, and with which the result can be judged simply by visual observation. When using ELISA technology and using the above-mentioned sandwich method, the formation of the enzyme reaction product is determined by the amount of the test target substance, so a system can be established that can easily determine the presence or absence of AR-AChE by visual observation through color development, etc. , It can also be used to test its O.D value with instruments for quantitative and semi-quantitative detection.

The specimens used in the kit of the present invention are not particularly limited, but include, for example: blood and cerebrospinal fluid or tissue, preferably blood.

The monoclonal antibody of the present invention and the kit of the present invention can be used to screen anti-tumor drugs, assist in judging the therapeutic effect of anti-tumor drugs, prepare drugs for diagnosing or treating neurodegenerative diseases, and detect the expression of AR-AChE produced by apoptosis in acute organ damage. Expression levels and changes.

The above-mentioned method for screening antineoplastic drugs comprises the following steps:

(1) Cultivate human tumor cell lines;

(2) Add candidate drugs to the cell line culture medium in step (1), and detect the expression level of apoptosis-related acetylcholinesterase with the above-mentioned monoclonal antibody or the above-mentioned kit; promote the appearance of apoptosis-related acetylcholinesterase or The candidate drug whose expression level is increased is the drug that promotes tumor cell apoptosis; otherwise, the candidate drug is ineffective.

According to the present invention, using the monoclonal antibody to recognize AR-AChE can eliminate the influence of other substances present in the sample, so that the existence of AR-AChE can be detected with very high sensitivity and specificity. Since hybridomas producing monoclonal antibodies against AR-AChE have been established, it is now possible to produce the same monoclonal antibodies almost permanently.

The diagnostic kit of the present invention uses monoclonal antibodies, has a high level of detection sensitivity, does not generate false negatives or false positives, and does not have any difference between batches. If the labeled antibody is preferably a monoclonal antibody of a different subtype from the monoclonal antibody on the solid phase carrier, it will be easier to control product quality and formulate a unified standard.

The detection principle of the AR-AChE detection kit uses anti-AR-AChE monoclonal antibodies and anti-AR-AChE polyclonal antibodies or another subtype of anti-AR-AChE monoclonal antibodies as solid-phase antibodies and enzyme-labeled antibodies, If there is AR-AChE in the sample to be tested, an antibody-antigen-enzyme-labeled antibody complex will be formed, and TMB (enzyme immunochromogenic series product) substrate will be added to produce a color reaction, otherwise there will be no color reaction. The double-antibody sandwich ELISA method is a very sensitive detection technology, and the ELISA has high sensitivity and is easy to operate. The development of supporting equipment has standardized and automated the operating procedures, thereby further improving the stability.

The present invention provides hybridoma cell lines that specifically produce anti-AR-AChE monoclonal antibodies. These cell lines can produce anti-human AR-AChE monoclonal antibodies. The antibodies can be used to prepare kits for basic research, AD research, and AD Auxiliary diagnosis and treatment; judgment of chemotherapy effect, etc.

So far, only the M30 antibody has been used to judge the effect of chemotherapy by serological methods. Currently, two companies, ALEXIS Biochemicals Corporation and DiaPhqrma Group Inc., have launched the kit-M30-Apoptosense ^TM ELISA Kit. The main component of this kit is M30 antibody, a mouse hybridoma monoclonal antibody with subtype IgG2b, which 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 kinases (Caspase) 3, 6, 7, 9 will cut a small fragment of the C-terminus of CK18. Since this small fragment only appears in apoptotic cells, it becomes a specific marker of epithelial cell apoptosis. Using M30 antibody can recognize the apoptosis of tissue cells and cultured cells. ELISA can also be used to detect the change of M30 in serum to judge the effect of chemotherapy. But it only targets epithelial-associated tumors and is expensive.

The purposes of monoclonal antibody of the present invention are:

1. In vitro experiments to screen antitumor drugs:

To study new anti-tumor drugs, it is necessary to screen many candidate drugs, and whether it can induce tumor cell apoptosis is an important indicator. The method of the invention can be used for screening new anti-tumor drugs in vitro. Human tumor cell lines cultured in vitro, add different doses of anti-tumor candidate drugs to be tested, and detect the cells or culture medium after 18-20 hours, and judge the efficacy of the drug to be screened by the change of the appearance and amount of AR-AChE Whether it is effective or not, if AR-AChE increases, it means that the drug has induced apoptosis, and it is an effective anti-tumor drug.

2. Judgment of chemotherapy effect in clinical cancer patients

For different tumors (including leukemia), different patients respond differently to various chemotherapy drugs. Some anti-tumor drugs have a good effect on one patient, but not on another patient. The method of the invention can help clinicians to select tumor chemotherapeutic drugs in a targeted manner, find the best drug in the shortest time, and bring good news to patients.

Chemotherapy drugs are mainly aimed at tumor cells (and normal cells) to cause apoptosis, so as to achieve the purpose of treatment. The present invention is based on new discoveries of scientific research. The principle is that under normal circumstances, cells that do not express AChE express a large amount of AR-AChE during apoptosis, and this enzyme exists relatively stably in apoptotic cells. In the absence of treatment in normal people or cancer patients, although the body continues to have apoptosis, the number is small, and the apoptotic cells are quickly phagocytized by the surrounding macrophages, and the AR-AChE in the apoptotic cells is not released into the blood stream middle. However, if effective chemotherapeutic drugs cause massive apoptosis of tumor cells, macrophages cannot phagocytize all apoptotic cells quickly, or the phagocytic ability of macrophages is reduced due to the influence of drugs, and they cannot phagocytize apoptotic cells quickly. A large amount of AR-AChE enters the blood along with apoptotic cells and apoptotic bodies. At this time, the patient's serum was taken to measure the protein content of serum AR-AChE, and it was found that the protein content of AR-AChE 72 hours after the start of treatment (the third day) was more than 1 times higher than that before treatment. What is important is that the inventors found that cord blood CD34+ hematopoietic stem cells do not express AR-AChE during apoptosis, and the measurement of blood AR-AChE is more representative. Therefore, by measuring the serum AR-AChE protein levels before and after treatment, we can know how effective the chemotherapy is, and help clinicians find the best targeted tumor chemotherapy drugs in the shortest possible time. If the chemotherapy is ineffective, the drug can be replaced in time, thus providing doctors with a fast and effective means of detecting the effect of chemotherapy and truly realizing individualized treatment. Of course, the therapeutic effect of other antitumor drugs can also be analyzed by using the monoclonal antibody of the present invention.

3. Research on neurodegenerative diseases, especially AD, may benefit diagnosis and treatment

Evidence of brain cell apoptosis can be obtained through brain tissue sections and cerebrospinal fluid analysis. AChE accelerates the precipitation of β-amyloid peptide independently of classical enzymatic catalysis (Inestrosa NC, Alvarez A, Perez CA, Moreno RD, Vicente M, Linker C, Casanueva OI, Soto C, Garrido J, Acetylcholinesterase accelerates 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 a complex with β-amyloid peptide, and the complex is more toxic than a single β Amyloid peptides are more toxic (Alvarez A, Alarcon R, Opazo C, Campos EO, Munoz FJ, Calderon FH, Dajas F, Gentry MK, Doctor BP, De MelloFG, Inestrosa NC, Stable complexes involving acetylchol esterase and amyloid-betapeptide change the biochemical properties of the enzyme and increase theneurotoxicity of Alzheimer's fibrils, J Neurosci, 199818(9); 3213-3223.). Experiments with AChE antisense nucleic acids directly implicate AChE in neuronal loss (Shohami E, Kaufer D, Chen Y, Seidman S, Cohen O, Ginzberg D, Melamed-Book N, Yirmiya R, Soreq H, Antisense prevention of neuronal damages following head injury in mice, J Mol Med, 200078(4); 228-236.). This monoclonal antibody, in addition to being used to identify evidence of apoptosis in brain tissue sections and cerebrospinal fluid, as a diagnostic reagent, may also be used as a drug (especially after the humanized antibody is prepared), such as preventing AChE from accelerating β The precipitation of amyloid peptide can prevent the progressive development of AD. The medicine includes the monoclonal antibody of the present invention and pharmaceutically acceptable carriers and/or excipients.

4. Detection of the development and prognosis of acute organ damage

Diseases such as fulminant hepatitis. When these acute organ damages occur, there are a large number of apoptosis cells, and the serum contains several times or even dozens of times higher AR-AChE. By detecting its changes, the development and prognosis of the disease can be judged.

Advantages of the present invention: the method of the present invention is to identify whether apoptosis occurs by detecting the AR-AChE activity expressed by the cells after the antitumor drugs act on cells that do not express AChE in vitro, or measuring the serum AR-AChE content. The AChE reaction of the cells is positive, indicating that the drug has induced apoptosis and is an effective anti-tumor drug. It can conveniently and effectively screen anti-tumor drugs, and it can also help doctors choose chemotherapy drugs that are particularly sensitive to patients, so as to improve the therapeutic effect . The result of detecting the content of AR-AChE by the method of the present invention to identify the apoptotic cells cultured in vitro is consistent with the classical method. Moreover, the positioning is accurate, the method is simple and easy, and does not require expensive equipment or expensive reagents, and semi-quantitative and quantitative analysis can be performed.

Description of drawings

Figure 1. Frozen section of brain tissue of five-month-old human fetus induced by labor induction (800 times magnification, ×800)

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

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

C. is the overlap of A and B.

D. is a photo of visible light. This photo shows that 2 apoptotic cells are positive, and the others are not positive.

Figure 2. Photographs of umbilical cord blood smears (×800)

A. Immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, labeled with rhodamine, apoptotic nucleated cells are positive, and red blood cells are negative;

B. It is Hoechst No.33258 staining, which shows the nuclear material, and there is only one nucleated cell in the whole field of view, and the rest are red blood cells;

C. It is a photo of visible light, a large number of red blood cells can be seen, and there is a nucleated cell in the middle. This figure shows that the monoclonal antibody of the present invention specifically recognizes AR-AChE of apoptotic cells, but does not recognize AChE on the red blood cell membrane.

Figure 3. Lysed Apoptotic HELA Experiment

From left to right in the figure, the apoptotic HELA lysate respectively a. No substrate was added, but replaced by the same amount of PBS; b. No antibody was added, the substrate reaction was performed, and the result was positive; c. and d. Excessive addition Antibodies form a complex, centrifuge, take the supernatant (c) and the precipitate (d) for substrate reaction respectively, and the results of both are negative.

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

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

Figure 5. WESTERN BLOT

A. Use the purchased antibody (BD Biosciences, San Jose, CA, USA) to do WESTERN BLOT, and the antigen comes from apoptotic HELA cells;

B. Use ARA-M5 to do WESTERN BLOT, and the antigen comes from apoptotic HELA cells;

C. ARA-M5 was used to do WESTERN BLOT, and the antigen was from ray acetylcholinesterase (Sigma).

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

A. It is a photo of visible light, the black arrow points to the apoptotic cell, and the white arrow points to the normal cell;

B. It is Hoechst No.33258 staining, which shows the nuclear material. The nucleus of apoptotic cells has formed apoptotic bodies, while the nucleus of normal cells is intact;

C. is immunofluorescence, 1 antibody is mouse anti-human AR-AChE monoclonal antibody, 2 antibody is goat anti-mouse IgG antibody, labeled with rhodamine, only cells with apoptotic bodies are positive, while normal cells are negative, It shows that the 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 inventor. 1 antibody is directly connected to Rhodamine, and red is a positive cell (cross-reacting with rat AR-AChE); a large piece of nerve tissue There is no positive, there are cholinergic nerves there, and the synaptic acetylcholinesterase does 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 area, indicating that the monoclonal antibody specifically recognizes AR-AChE in apoptotic cells, but does not recognize synaptic AChE expressed in rat brain tissue.

Figure 8. Frozen sections (×800) of five-month-old human fetal brain tissue induced labor are all in the same field of view

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

B. It is cytochemical Ache staining (Konovsky and Root method), brownish yellow (dark gray in the figure) is positive for AChE, and it can be seen that it is relatively uniform without showing 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 in apoptosis found by the inventors;

C. is the result of visible light photo and Hoechst No.33258 staining at the same time, which further proves that AChE positive is not in the nucleus.

Figure 9. Photos of human SK-N-SH cells cultured in vitro (×800)

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

A. TUNEL staining, positive for apoptotic cells;

B. is a photo of visible light, there is a pyknotic cell without cell protrusions in the lower right of the cell mass;

C. is immunofluorescence, 1 antibody is a mouse anti-human AR-AChE monoclonal antibody, 2 antibody is a goat anti-mouse IgG antibody, labeled with rhodamine, and there is an apoptotic cell in the lower right showing positive;

D. is hoechest staining, showing the nucleus;

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

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

Figure 11. Photographs of rat PC12 cells

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

B. Normal PC12 cells, confocal (CONFOCAL) photograph. FITC shows that AChE is expressed in normal living cells, distributed in the cytoplasm and membrane, and there is no positive in the nucleus. The blue color is HOECHST staining, showing the nucleus.

C. Visible light photo. Anti-AR-AChE antibody (ARA-M5), DAB reaction, positive in brown, hematoxylin background staining, showing nuclei; living PC12 attached to the wall is negative, and pyknotic apoptotic cells are positive.

Figure 12. Cord blood of 30 normal neonates and serum AR-AChE of a tumor patient.

The OD values of AR-AChE in cord blood of normal newborns were all below 0.2, close to the blank control. The OD value of serum AR-AChE in a tumor patient on the third day of chemotherapy was 3.1.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Reagents, instruments, etc. that are not specifically described in the present invention are commercially available conventional products. The experimental method that does not indicate specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, Russell, molecular cloning: the conditions described in the laboratory manual III (New York: Cold Spring Harbor Laboratory Press, 2001), or Follow the conditions recommended by the manufacturer.

Example 1 Preparation of anti-apoptosis-related acetylcholinesterase monoclonal antibody and hybridoma

I. Preparation before cell fusion

(1) Immunization program

A, Antigen: According to the inventor's patent method (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 ZhangIsolation of AChE from Apoptotic Human Lung Fibroblast Cells by Antibody AffinityChromatography. BioTechniques i33(4):S92-S97, 2002) to obtain AR-AChE expressed by HELA cells.

B, Mice: BaLb/c mice, 3 male mice aged 6-10 weeks.

Human AR-AChE, plus Freund's complete adjuvant (limited to the first use) or Freund's incomplete adjuvant, mixed together in equal volumes, used two syringes, passed the three-way valve, and injected repeatedly to make the two form an oil package Watery chylus.

Primary immunization Antigen (Ag) 1～50μg Subcutaneous multi-point injection with Freund’s complete adjuvant

| (0.8～1ml 0.2ml/point)

↓After 3 weeks

The second immunization dose is the same as above, add Freund's incomplete adjuvant subcutaneously

↓After 3 weeks

The third immunization dose is the same as above, no adjuvant, intraperitoneal injection (ip)

|(After 5-7 days, take blood to test its potency and detect the immune effect)

↓After 2-3 weeks

Booster immunization, dose 100μg, ip

↓3 days later

Spleen extraction and fusion

(2) Feeder cell mouse peritoneal macrophages

Prepare mouse peritoneal macrophages the day before fusion

BaLb/c mice aged 6-10 weeks were used

↓

Put to death by pulling the neck, soak in 75% alcohol, and disinfect for 3 to 5 minutes

↓

Cut the skin with sterile scissors to expose the peritoneum

↓

Inject 6-8ml culture solution with a sterile syringe

↓

Rinse repeatedly, aspirate rinse

↓

Put into a 10ml centrifuge tube, centrifuge at 1200 rpm for 5-6 minutes

↓

Suspend with 20% calf serum (NCS) or fetal calf serum (FCS) culture medium, adjust the cell number to 1×10 ⁵ /ml

↓

Add to 96-well plate, 100μl/well

↓

Placed in a 37°C CO ₂ incubator for culture

(3) Myeloma cells

Two weeks before preparation for fusion, myeloma cells were thawed. The inventor used the myeloma SP2/0 cell line, RPMI1640 medium, the concentration of calf serum was 20%, and the maximum density of the cells was not more than 10 ⁶ /ml. The expanded culture was diluted and passaged at 1:10, once every 3 to 5 days .

(4) Immune splenocytes

Preparation of splenocyte suspension: take out the spleen under aseptic conditions, wash it once with incomplete culture medium, put it on a stainless steel screen in a plate, grind it into a cell suspension with a syringe needle core, and count it.

II. Cell Fusion, Selection of Hybridomas

(1) Cell fusion process

(1) Take logarithmically grown myeloma cells SP2/0, centrifuge at 1000rpm for 5 minutes, discard the supernatant, suspend the cells with incomplete culture medium and count them, get the required number of cells, wash twice with incomplete culture medium .

(2) Simultaneously prepare immune splenocyte suspension and wash twice with incomplete culture medium.

(3) Myeloma cells and splenocytes were mixed together at a ratio of 1:10 or 1:5, washed once with incomplete culture medium in a 50ml plastic centrifuge tube, 1200rpm, 8 minutes.

(4) Discard the supernatant, and suck up the residual liquid with a dropper, so as not to affect the concentration of PEG.

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

(6) Fusion at room temperature:

① Add 1ml of preheated 45% PEG (Merek, molecular weight 4000) containing 5% DMSO within 30 seconds, and stir while adding.

②The effect is 100 seconds.

③ Add preheated incomplete culture medium to terminate the PEG effect, and add 1ml, 2ml, 3ml, 4ml, 5ml and 10ml every 2 minutes.

(7) Centrifuge at 800 rpm for 6 minutes.

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

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

(10) Add the fused cell suspension into a 96-well plate containing feeder cells, 100 μl/well, and culture in a 37° C., 5% CO ₂ incubator.

One 96-well plate contains 1 x ¹⁰⁷ splenocytes.

(2) HAT selects hybridomas

After 24 hours of fusion, HAT selection medium was added. Add 1 ml of stored 50×HT and HAT reagents to 50 ml of 20% calf serum complete culture medium.

50×HAT

H: 5×10 ^-3 M

A: 2×10 ^-5 M

T: 8×10 ^-4 M

After the HAT selection medium was maintained for two weeks, the HT medium was changed to the HT medium, and after another two weeks of maintenance, the normal medium was used.

III. Antibody detection

The inventors employed an ELISA screening method.

IV. Cloning and cryopreservation of hybridomas

(1) Cloning scheme

Cloning by limiting dilution, the procedure is as follows:

① Preparation of feeder cell suspension (same as preparation before fusion)

②Count the cells in the positive wells and adjust the cell number to 1～5×10 ³ /ml

③Take 130 cells and put them into 6.5ml of complete culture medium containing feeder cells, that is, 20 cells/ml, 100μl/well and add three rows of A, B, and C to make 2 cells per well. To the remaining 2.9ml of cell suspension, add 2.9ml of complete culture medium containing feeder cells, the number of cells is 10/ml, and 100μl/well is added to three rows of D, E, and F, which is 1 cell per well. Add 2.2ml of complete culture solution containing feeder cells to the remaining 2.2ml of cell suspension, the number of cells is 5/ml, 100μl/well, add two rows of G and H, 0.5 cells per well.

④ After 4-5 days of culture, small cell clones can be seen on the inverted microscope, add 200 μl/well of complete culture solution.

⑤ On the 8th to 9th day, cell clones can be seen with the naked eye, and antibody detection should be carried out in time.

(2) Cryopreservation of hybridoma cells

Hybridoma cells contain more than 1×10 ⁶ in each ampoule.

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

V. Mass production of monoclonal antibodies

1. Hybridoma cells were inoculated in vivo to prepare ascites.

Preparation of ascites: Inject 0.5ml Pristane (pristane) or liquid paraffin into BaLb/c mice intraperitoneally, inject 1× ¹⁰⁶ hybridoma cells intraperitoneally after 1 to 2 weeks, and kill the mice 7 to 10 days after inoculating the cells , collect ascites with a dropper.

Example 2 Identification of AR-AChE Antibody

1. Identification of antibody specificity: use ELISA method, in addition to immunogen (antigen) for antibody detection, also apply cross test with human albumin, actin, electric ray AChE, the result reacts with human AR-AChE Strongly, weakly positive to ray AChE, negative to human albumin and actin (see Figure 5), immunohistochemical and cytochemical tests did not recognize human brain AChE (see Figure 1) and human erythrocyte AChE (see figure 2).

2. Identification of Ig class and subclass of McAb: screening with enzyme-labeled secondary antibody has basically determined the Ig type of the antibody. Using the kit (Mouse Typer sub-isotyping Kit catalog number 172-2051) of (BioRed) company to analyze, ARA-M1 number (preservation number CCTCC-C200413) is IgG; ARA-M5 number (preservation number CCTCC-C200414) is IgG2α , No. ARA-M7 (Deposit No. CCTCC-C200415) is IgG1.

3. Identification of McAb neutralizing activity: Adding 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 inventor conducted an immunosuppression experiment. The inventor used Karnovsky, MJRoots, L.A method detects acetylcholinesterase activity (Karnovsky, MJRoots, L.A "Direct-Coloring" Thiocholine Method for Cholinesterases.J Histochem Cytochem 1964; 12:219-21.), and then in 96 wells, use a microplate reader to detect the enzyme at 405nm active.

With lysed apoptotic HELA, divided into 3 groups

A. Apoptotic HELA lysate, no substrate, but replaced by an equal amount of PBS

B. Do not add antibody, do substrate reaction, the result is positive

C. Add excess antibody to form a complex, centrifuge, supernatant and precipitate react with substrates respectively, and the results are all negative (Figure 3). Inventor C performed Western blotting of the supernatant and the precipitate, which showed that there was no AChE in the supernatant, but there was an AChE-positive band in the precipitated antigen-antibody complex.

Example 3 Comparison of the performance of AR-AChE antibody and commercially available antibodies

Commonality: 1. Recognize AChE (common with commercially available AChE antibodies)

AR-AChE antibody: negative for normal HELA cells, positive for apoptotic cells (see the paper cited in the next paragraph for the results of commercial products, and see Figure 4 for monoclonal antibodies). 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, AlemanyM, Zhang LY, Shi YF., Induction of Acetylcholinesterase Expression during Apoptosis in Various Cell Types. Cell Death and Differentiation 9 (8): 790-800, 2002.), normal living HELA cells do not express AChE, while 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 solubleacetylcholinesterase from Japanese quail brain by affinity chromatography. The international journal of biochemistry & cell biology.34(2):204-210, 2002) isolated AChE from apoptotic HELA cells. The inventor used the isolated protein to do Western blot, and the results showed that the purchased antibody and the inventor's monoclonal antibody recognized the same band (66kDa). (Figure 5)

2. Inhibition of enzyme activity, the AChE activity of apoptotic HELA can be blocked by the inventor's monoclonal antibody (identification of McAb neutralization activity, Figure 3), the inventor made the supernatant and the precipitate for WESTERN BLOT, showing that there is no AChE in the supernatant, In the precipitate, there is an AChE positive band. Different from existing antibodies: specificity.

The antibody of the present invention only recognizes AR-AChE expressed by apoptotic cells, which is different from existing commercialized antibodies. According to cellular immune analysis, the monoclonal antibody only recognizes AR-AChE expressed by apoptotic cells, as shown in concrete evidence (Fig. 1, 6). Figure 6 shows that the nucleated cells isolated from cord blood were left at room temperature for 18 hours, and some cells underwent apoptosis. The photos are all from the same field of view.

1. Human and rat synaptic (brain) AChE is not recognized (Figure 1, Figure 7). Rat brain tissue section in Figure 7, AR-AChE immunohistochemistry and TUNEL double staining. The rat was treated with middle cerebral artery ischemia (Longa EZ, Weinstein PR, CarlsonS.Cummins R.Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke.1989 Jan; 20(1):84-91.), taken from damage surrounding tissues. Photos A, B, and C are all of the same field of view.

2. Does not recognize human erythrocyte membrane AChE (Fig. 2C). The umbilical cord blood was kept at room temperature for 18 hours, and some cells underwent apoptosis. Photos A, B, and C in Figure 2 are all from the same field of view.

The anti-neurite AChE antibody on the market is also different from the antibody against AChE on the red blood cell membrane. The antibody of the present invention only recognizes AChE expressed by apoptotic cells, which the inventors call (apoptosis-related acetylcholinesterase, abbreviated as AR-AChE), while the anti-neurite AChE antibody on the market can recognize AR-AChE, However, without specificity, they do not have the specificity to recognize apoptotic cells and thus cannot be used as markers of apoptosis. The inventors have obtained apoptosis-related 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 a monoclonal antibody against AR-AChE. With the specific anti-AR-AChE monoclonal antibody, it is possible to distinguish AR-AChE from various types of AChE. With this specificity, it can be used to identify apoptotic cells, apoptosis phenomena, basic research, AD research, auxiliary diagnosis and treatment of AD, judgment of chemotherapy effect, etc. This is the first monoclonal antibody invented in the world that specifically recognizes AR-AChE expressed by apoptotic cells.

Example 4 Observing the distribution of normal brain AChE and the relationship between the enzyme and the nucleus

Five-month-old human fetal brain tissue induced by labor (with the consent of the family members) was frozen and sectioned, fixed with 4% paraformaldehyde, and then stained for acetylcholinesterase activity. Prepare acetylcholinesterase substrate reaction solution (0.1M phosphate buffer Ph 6.0150ml, acetylthiocholine 100mg/200ml, 0.1M sodium citrate 11ml, 30mM copper sulfate 20ml, 5mM potassium ferricyanide 20ml) for enzyme reaction . When detecting cell acetylcholinesterase activity, the cells were incubated in the reaction solution at room temperature for 3-4 hours, and a yellowish brown color (dark gray in the figure) was a positive reaction (see FIG. 8B ). It can be seen that it is relatively uniform without showing the outline of cells or nuclei, indicating that the products are not concentrated in the cytoplasm or nuclei, 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 Observation of a small amount of nerve cell apoptosis during development

Frozen sections of human fetal brain tissue of five months induced labor were fixed with 4% paraformaldehyde, and immunohistochemical reaction and Hoechst staining were performed, respectively showing nuclei and AR-AChE positive cells (see Figure 1), indicating that the antibody of the present invention does not recognize large pieces Acetylcholinesterase present, but only AChE associated with apoptosis is recognized.

Example 6 Use of human SK-N-SH cells cultured in vitro to demonstrate the specificity of the monoclonal antibody of the present invention

The use of human SK-N-SH cells cultured in vitro shows that the antibody of the present invention only recognizes apoptotic cells, but not normal acetylcholinesterase, because SK-N-SH cells express acetylcholinesterase and distribute on the cell membrane. See Figure 9, the photo is divided into five parts A, B, C, D, and E, all of which have the same field of view. Antibody 1 in C is mouse anti-human AR-AChE monoclonal antibody (ARA-M7), antibody 2 is goat anti-mouse IgG antibody (1:100 dilution, Rhodamine conjugated anti-mouse IgG-R, Santa Cruz), right One apoptotic cell below is positive. Human SK-N-SH cells cultured in vitro can normally express a small amount of neurosynaptic AChE. In this photo, one apoptotic cell is positive, and the others are not positive. TUNEL staining is in good agreement with anti-AR-AChE positive. It shows that this monoclonal antibody specifically recognizes AR-AChE of apoptotic cells, but does not recognize synaptic AChE expressed by SK-N-SH cells.

Example 7 Use the monoclonal antibody of the present invention to judge the effect of tumor chemotherapy

The semi-quantitative analysis of acetylcholinesterase in human serum by using the antibody of the present invention can be used to judge the effect of tumor chemotherapy (including leukemia). This is to use the anti-human AR-AChE monoclonal antibody (ARA-M5 and ARA-M1 pairing) of the present invention, and use the sandwich bread method to do ELISA, and detect the AR-AChE content in serum of tumor patients PAD, TZH and GB before and after chemotherapy respectively. It can be seen that the content of AR-AChE in the serum of patients with PAD and TZH 72 hours after chemotherapy was more than 1 times higher than that before treatment, indicating that the curative effect is better. If the content of AR-ACHE in the serum of the patient does not change before and after GB treatment, the curative effect is poor (see Figure 10).

Example 8 The specificity experiment of the monoclonal antibody of the present invention to recognize apoptotic cells

Using rat PC12 cells cultured in vitro, it shows that the monoclonal antibody of the present invention only recognizes apoptotic cells, but not normal acetylcholinesterase, because rat PC12 cells can express acetylcholinesterase and distribute on the cell membrane (see Figure 11B) . Normal PC12 cells, CONFOCAL photo FITC shows that AChE is expressed in normal living cells, distributed in the cytoplasm and membrane, and no positive in the nucleus; in apoptotic PC12 cells, the substrate reaction is positive, distributed in the cytoplasm and nucleus (see Figure 11).

Example 9 Using the monoclonal antibody of the present invention to semi-quantitatively detect umbilical cord blood of 30 normal neonates and a tumor patient's serum AR-AChE

The semi-quantitative analysis of acetylcholinesterase in the umbilical cord blood serum of 30 normal neonates with the antibody of the present invention shows that the OD values are all below 0.2, which is close to the blank control. The OD value of serum AR-AChE in a tumor patient on the third day of chemotherapy was above 3.1. This is to use the anti-human AR-AChE monoclonal antibody (paired ARA-M5 and ARA-M1) of the present invention to do ELISA with the sandwich bread method to detect the AR-AChE content in the umbilical cord blood serum of tumor patients and normal newborns respectively. It can be seen that the content of AR-AChE in serum of patients 72 hours after chemotherapy is more than 20 times higher than that of normal neonatal umbilical cord blood. The content of AR-AChE in normal neonatal umbilical cord blood serum is very low, and the OD value is below 0.2, which is close to the blank control (see Figure 12).

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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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