WO2003051928A1

WO2003051928A1 - Antibody specific for detection of bradeion

Info

Publication number: WO2003051928A1
Application number: PCT/JP2001/011003
Authority: WO
Inventors: Manami Tanaka; Tomoo Tanaka; Kengo Sakaguchi
Original assignee: National Institute Of Advanced Industrial Science And Technology; Nippn, Co. Ltd.; Nippn Technocluster, Inc.
Priority date: 2001-12-14
Filing date: 2001-12-14
Publication date: 2003-06-26
Also published as: JPWO2003052102A1; AU2002222647A1; AU2002359980A1; WO2003052102A1

Abstract

A monoclonal antibody which reacts specifically with bradeion protein originating in colon cancer and/or prostatic cancer cells; a method of detecting cancer by using this antibody; and diagnostics for cancer containing this antibody.

Description

Description; Specific antibody for detecting bradion

The present invention relates to a monoclonal antibody that specifically reacts with a protein, a method for detecting cancer, and a diagnostic agent for cancer. Background art

In the 21st century, the revolution in molecular medicine aims to build a control monitor system tailored to individual characteristics by capturing the genetic and material basis of disease as a post-genome project. Specifically, based on the concept of "Quality of Life", detection of risk groups (diagnosis and gene monitoring) of disease groups that threaten social life, such as genetic diseases, cancer, and neurodegenerative diseases, and risk genes To establish a medical response system that matches the individual's genotype, such as the discovery of the disease and the search for susceptibility to treatment (eg, drug, gene therapy). Here, not only cancer but also many diseases are due to multi-gene effects, and environmental factors greatly influence it, so it cannot be asserted that no control will result in disease. However, it is possible to control disease control through the development of control technologies that control what has become ill.

Based on this concept, the development of technology to control canceration and immortalization of cells is being actively developed at the molecular level. Specifically, analysis of the signal transduction system (signal transduction) is often studied as control of cell life. Through such research, the molecular basis of various cell proliferation, division, and canceration has been elucidated. Already, the National Institute of Advanced Industrial Science and Technology (formerly the National Institute of Advanced Industrial Science and Technology) has filed a patent application for an invention relating to Bradion as such a cell life controlling factor (Japanese Patent No. 3141107, Japanese Patent Application Laid-Open No. 2001-161384). No. 09 / 440,936). Bradion, which is expressed only in cancer cells, especially colorectal cancer cells and skin cancer, is not only an early diagnosis It has become clear that it satisfies the conditions required as a different inhibitor and a target for gene therapy.

Prostate cancer, on the other hand, has seen a rapid increase in the patient population since its 60s, and is one of the high-risk cancers of the elderly in the 70s, including latent cancers, reaching the 30% range. At present, there is almost no case where a diagnosis is made at a screening site such as a medical check-up for the elderly, and a scrutiny of prostate enlargement or the like is scrutinized. Although there is a specific method of prostate cancer diagnosis by blood detection of prostate specific antigen, there are few sites where it can be performed, and in most cases, hematuria does not present symptoms that are easily detectable by primary examination. Often overlooked. Surgical resection of the primary tumor is as effective as other cancers as a root treatment, but prostate cancer is often difficult to diagnose at an early stage, and inoperable cases depend on age and progress of the cancer. In many cases, metastasis (bone metastasis) occurs at a high rate, resulting in poor prognosis. Therefore, there is an urgent need to develop simple and inexpensive technologies for early diagnosis and treatment of prostate cancer.

Disclosure of the invention

An object of the present invention is to provide a monoclonal antibody which specifically reacts with a bradion protein derived from colon cancer or prostate cancer cells, a method for detecting cancer using the antibody, and a diagnostic agent for cancer containing the antibody. I do.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, ascertained that the Bradion gene is involved in colorectal cancer and prostate cancer, and found that the Bradion protein derived from colon cancer or prostate cancer cells. By producing a monoclonal antibody that specifically reacts with the above and using it as a primary antibody for immunohistological staining, they found that the above-mentioned cancer cells could be detected, and thus completed the present invention.

That is, the present invention is a monoclonal antibody that specifically reacts with a bradion protein derived from colon cancer or prostate cancer cells.

The present invention is also a hybridoma that produces the above monoclonal antibody. In addition, the present invention provides a method of using the above monoclonal antibody to

'A cancer detection method characterized by detecting a protein. This Here, the cancer includes colon cancer and / or prostate cancer.

Further, the present invention is a diagnostic agent for cancer containing a monoclonal antibody. Cancers include colon cancer and Z or prostate cancer. Hereinafter, the present invention will be described in detail.

The present invention relates to a monoclonal antibody that specifically reacts with a bradion protein derived from colorectal cancer or prostate cancer cells and its use, and the monoclonal antibody is expressed only in colorectal cancer or prostate cancer cells It utilizes the property of reacting specifically with Bradion protein. The present invention has been completed by paying attention to this property. Since the cancer is detected by immunologically quantifying the above-mentioned bradion protein in a sample, the cancer can be detected quickly, with high sensitivity and easily.

1. Bradion protein

Bradion is a protein that is known to be specifically present in the human adult brain, etc., and has a structure similar to substances involved in cell division and growth control (Septin family). It also has the structure of determinants of cell lifespan (causing programmed cell death). The function has already been elucidated through preliminary experiments and other studies.Bradion is a cell division regulator called septin family that shows specific expression in cancer cells.

The AP kinase signaling cascade has been shown to serve as a modest pump for cell proliferators. Bradion protein is known to have two types of transcripts encoded by the same Bradion gene, namely, the type and the type), and the tissue-specific expression of the bradion protein is determined in colorectal cancer tissues and It is sneaked even in skin cancer tissues (Tanaka b, Biochemical and Biophysical Research Communications 286, 547-553 (2001)) ₀

The present inventors have recently shown that bradion is significantly involved in regulating the growth, division and metastatic potential of prostate cancer cells. Therefore, the present inventor has prepared a monoclonal antibody against the human blade ion protein, When the body was used in a test for detecting colon cancer cells and prostate cancer cells, it was confirmed that a monoclonal antibody against human bradion protein actually detected both colon cancer cells and prostate cancer cells.

2. Preparation of the monoclonal antibody of the present invention

The monoclonal antibody of the present invention has a feature of specifically reacting with bradion derived from colon cancer or prostate cancer cells. The globulin type of the monoclonal antibody of the present invention is not particularly limited as long as it has the above characteristics, and may be any of IgG, IgM, IgA, IgE, and IgD, but IgG and IgM are preferred. The monoclonal antibody of the present invention can be obtained according to the following procedure.

(1) Preparation of immunogen

In preparing the monoclonal antibody of the present invention, a protein to be used as an immunogen (antigen) is prepared. As the immunogenic protein, a human bradion protein is used. The amino acid sequence of the human bradion protein that can be used as an immunogen in the present invention and the cDNA sequence encoding the protein are disclosed in GenBank as accession numbers AB002110 and AB008753 for human bradion and j3, respectively. I have. Therefore, human bradion protein to be used as an immunogen can be synthesized by using publicly available amino acid sequence information by a method known in the art, for example, a solid phase peptide synthesis method.

It is also possible to produce a human bradion protein using information on cDNA encoding the human bradion protein by using a known gene recombination technique.

. Hereinafter, production of the human bradion protein using a recombinant technique will be described. A recombinant vector for producing bradion can be obtained by ligating the above-disclosed cDNA sequence to an appropriate vector, and a transformant is obtained by combining a recombinant vector for producing bradion with a bradion protein. It can be obtained by introducing it into a host so that it can be expressed.

A phage or plasmid capable of autonomous propagation in a host microorganism is used for the vector. Used. Plasmid DNAs include E. coli-derived plasmids (eg, pET21a, pGEX4T, pUC118, pUC119, pUC18, pUC19), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), and yeast-derived plasmids (eg, YEpl3, YEp24, YCp50, etc.) ), And the phage DNA includes λ phage (Agtl, λΖΑΡ, etc.). Furthermore, animal virus vectors such as vaccinia virus and insect virus vectors such as baculovirus can also be used.

To insert human bradion cDNA into a vector, first, the purified DNA is cut with an appropriate restriction enzyme, inserted into an appropriate vector-DNA restriction enzyme site or a multicloning site, and ligated to the vector. Is adopted.

In addition, recombinant vectors for producing bradion used in mammalian cells include promoters, human bradion cDNA, cis elements such as enhancers, splicing signals, poly-A addition signals, selection markers, and ribosomes, if desired. A binding sequence (SD sequence) or the like may be linked.

To ligate the DNA fragment and the vector fragment, a known DNA ligase is used. Then, the DNA fragment and the vector fragment are subjected to chaining and then ligated to prepare a recombinant vector for producing bradion.

The host used for the transformation is not particularly limited as long as it can express the human bradion protein. Examples include bacteria (such as Escherichia coli and Bacillus subtilis), yeast, animal cells (such as COS cells and CHO cells), and insect cells.

For example, when a bacterium is used as a host, the recombinant vector for producing bradion is capable of autonomous replication in the bacterium, and is composed of a promoter, a ribosome binding sequence, human bradion DNA, and a transcription termination sequence. Is preferred. Further, a gene that controls the promoter may be included. Escherichia coli includes, for example, Escherichia coli BRL, and Bacillus subtilis includes, for example, Bacillus subtilis. Any promoter may be used as long as it can be expressed in a host such as Escherichia coli. The method for introducing the recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions, Solution method and the like.

When a yeast, animal cell, insect cell, or the like is used as a host, a human bradion protein can be produced according to a method known in the art.

The human bradion protein used as an immunogen in the present invention can be obtained by culturing the above-prepared transformant and collecting it from the culture.

The term “culture” means any of a culture supernatant, a cultured cell, a cultured cell, or a cell or cell crushed product. The method of culturing the above transformant in a medium is performed according to a usual method used for culturing a host.

The medium for culturing the transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like, which can be used by the microorganisms, so that the cultivation of the transformants is efficient. Either a natural medium or a synthetic medium may be used as long as the medium can be performed in a controlled manner.

Cultivation is usually performed at 37 ° C for 6 to 24 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture period, the pH is kept near neutral. Adjustment of pH is performed using an inorganic or organic acid, an alkaline solution or the like. During the culture, an antibiotic such as ampicillin-tetracycline may be added to the medium as needed.

When the human bradion protein is produced in the cells or cells after culturing, the proteins are extracted by disrupting the cells or cells. When the human bradion protein is produced extracellularly or extracellularly, the culture solution is used as it is or the cells or cells are removed by centrifugation or the like. Then, by using general biochemical methods used for the isolation and purification of proteins, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in an appropriate combination. The human bradion protein can be isolated and purified from the culture.

Whether or not human bradion protein has been obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.

Next, the obtained protein is dissolved in a buffer to prepare an immunogen. If necessary, an adjuvant may be added for effective immunization. Adjuvant Examples thereof include commercially available complete Freund's adjuvant and incomplete Freund's adjuvant, and any of these may be mixed.

(2) Collection of immune and antibody-producing cells

The immunogen obtained as described above is administered to mammals, for example, rats, mice (eg, BALB / c of inbred mice), and egrets. The single dose of the immunogen is appropriately determined depending on the kind of the immunized animal, the administration route and the like, and is about 50 to 200 _§ per animal. Immunization is performed primarily by injecting the immunogen intravenously, subcutaneously, or intraperitoneally. The interval of immunization is not particularly limited. After the first immunization, booster immunization is performed 2 to 6 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 1 to 4 weeks. After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by the ELISA (Enzyme-Linked Immunosorbent Assay) method, etc., and when the antibody titer reaches a plateau, the immunogen is injected intravenously or intraperitoneally. Injection for final immunization. Then, 2 to 5 days, preferably 3 days after the final immunization, the antibody-producing cells are collected. Antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, with spleen cells or local lymph node cells being preferred.

(3) Cell fusion

To obtain hybridomas, cell fusion between antibody-producing cells obtained from the immunized animal and myeloma cells is performed as described above.

As the myeloma cells to be fused with the antibody-producing cells, generally available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in HAT selection medium (including hypoxanthine, aminopterin and thymine) in the unfused state, but can only survive in the state fused to antibody-producing cells. Those having properties are preferred. The cell line is preferably derived from an animal of the same strain as the immunized animal. Specific examples of myeloma cells include hypoxanthine, guanine, phospholiposyl, and transferase (HGPRT) deficient cell lines derived from BALB / c mice, such as P3X63-Ag.8 strain (ATCC TIB9) and P3X63-Ag. .8.Ul strain (cancer research Research Source Bank ( _JCRB ) 9085), P3 / NSI / l-Ag4-l strain (JCRB0009), P3x63Ag8.653 strain (JCRB0028) or Sp2 / 0-Agl4 strain (JCRB0029).

Next, the myeloma cells are fused with the antibody-producing cells. Cell fusion is performed by mixing antibody-producing cells and myeloma cells at a ratio of about 1: 1 to 20: 1 in an animal cell culture medium such as DMEM or RPMI-1640 medium that does not contain serum. The fusion reaction is performed in the presence of As a cell fusion promoter, polyethylene glycol having an average molecular weight of 1,500 to 4,000 daltons or the like can be used at a concentration of about 10 to 80%. In some cases, an auxiliary agent such as dimethyl sulfoxide may be used in combination to increase the fusion efficiency. Furthermore, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electoral poration).

(4) Selection and cloning of eight hybridomas

The target eight hybridomas are selected from the cells after the cell fusion treatment. As a method, the cell suspension is appropriately diluted, for example, with RPMI-1640 medium containing fetal calf serum, and then spread on a microplate at a rate of 2 × 10 ⁵ cells / well. Add the selected medium to the culture medium, and replace the medium as appropriate. The culture temperature is 20 to 40 ° C, preferably about 37 ° C. When the myeloma cells are HGPRT-deficient or thymidine kinase (TK) -deficient strains, cells having antibody-producing ability can be obtained by using a selective medium (HAT medium) containing hypoxanthine / aminopterin-thymidine. And myeloma cell hybridomas can be selectively cultured and proliferated. As a result, cells that grow about 14 days after the start of culture in the selection medium can be obtained as hybridomas.

Next, whether or not the target antibody is present in the culture supernatant of the proliferating hybridoma is screened. Screening for hybridomas may be performed according to a conventional method, and is not particularly limited. For example, a part of the culture supernatant contained in a well that grew as a hybridoma was collected and subjected to enzyme immunoassay (EIA; Enzyme Immuno Assav and ELISA) and radioimmunoassay (RIA; Radiolmmuno Assav). Yo Can be done.

Cloning of the fused cells is performed by limiting dilution, etc., and finally, a hybridoma, which is a monoclonal antibody-producing cell, is established. As described later, the hybridoma of the present invention is a monoclonal antibody that is stable in culture in a basic medium such as RPMI1640 or DMEM and reacts specifically with a bradion protein derived from colon cancer or prostate cancer cells. It is produced and secreted.

(5) Collection of monoclonal antibodies

As a method for collecting a monoclonal antibody from the established hybridoma, a normal cell culture method, an ascites formation method, or the like can be employed.

In the cell culture method, a hybridoma 10% © shea calf serum-containing RPMI-1640 medium, in an animal cell culture medium such as MEM medium or serum-free medium under conventional culture conditions (e.g. 37 ° C, 5% C0 ₂ ) For 2 to 10 days, and obtain antibody from the culture supernatant.

In the case of the ascites formation method, about 1 × 10 ⁷ hybridomas are administered intraperitoneally to mammals and allogeneic animals derived from myeoma cells, and the hybridomas are grown in large quantities. One to two weeks later, ascites or serum is collected.

When antibody purification is required in the above antibody collection method, a known method such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, or gel chromatography is appropriately selected. , Or by combining them, a purified monoclonal antibody of the present invention can be obtained.

3. Method of detecting cancer

The method for detecting cancer according to the present invention is characterized in that the monoclonal antibody of the present invention is used to immunologically detect or measure bradion protein derived from colon cancer or prostate cancer cells in a sample.

In the detection method of the present invention, any method using an antibody, that is, any immunoassay method may be used as the antibody of the present invention as the antibody used in the measurement method -Null antibodies can be used, such as enzyme immunoassay (ELISA, EIA), fluorescence immunoassay, radioimmunoassay (RIA), luminescence immunoassay, immunoturbidimetry, immunoassay, The detection method of the present invention is carried out by latex agglutination, latex turbidimetry, hemagglutination, particle agglutination, western blot, or the like.

The sample to be tested in the detection method of the present invention is not particularly limited as long as it is a biological sample that may contain a bradion protein derived from colon cancer or prostate cancer cells. Examples include blood, serum, plasma, lymphocyte culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, and the like, and cell or organ extracts can also be used. In particular, in a sample such as urine, the measured value of the bradion protein obtained by using the monoclonal antibody of the present invention is useful as an index for colorectal cancer or prostate cancer.

When the detection method of the present invention is performed by an immunoassay using a label such as an enzyme immunoassay, a fluorescence immunoassay, a radioimmunoassay, or a luminescence immunoassay, the monoclonal antibody of the present invention is immobilized on a solid phase. It is preferable to carry out the immunological reaction by immobilizing or immobilizing the components in the sample.

Solid carriers include polystyrene, polypropionate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, cellulose, sepharose, glass, metal, ceramics or magnetic Insoluble carriers in the form of beads, microplates, test tubes, sticks or test pieces made of materials such as bodies can be used. The solid phase can be formed by binding the solid phase carrier to the monoclonal antibody or the sample component of the present invention according to a known method such as a physical adsorption method, a chemical binding method, or a combination thereof.

In the present invention, in order to easily detect the reaction between the monoclonal antibody of the present invention and a bradion protein derived from colon cancer or prostate cancer cells in a sample, the reaction is carried out by labeling the monoclonal antibody of the present invention. Is detected directly or indirectly by using a labeled secondary antibody. The detection method of the present invention uses the latter indirect detection (for example, a sandwich method) in terms of sensitivity. Is preferred.

Labeling substances include peroxidase (POD), alkaline phosphatase, and enzyme-linked immunosorbent assay; 6-galactosidase, perease, lipase, glucose oxidase, and lactate dehydrogenase. , Amylase or a biotin-avidin complex, etc., in the case of a fluorescent immunoassay, fluorescein isothiocynate, tetramethyl mono-daminisothiocyanate, substituted rhodamine isotiosocyanate, dichlorotriazine For example, isothiocyanate, Alexa480 or AlexaFluor488, and in the case of radioimmunoassay, tritium, iodine- ¹²⁵ or iodine- ^131, etc., can be used. Further, for the luminescence immunoassay, a NADH-FMNH ₂ -luciferase system, a luminol-hydrogen peroxide-POD system, an acridinium ester system, a dioxetane compound system, or the like can be used.

The binding method between the labeling substance and the antibody may be a known method such as the dartalaldehyde method, the maleimide method, the pyridyl disulphide method or the periodate method in the case of an enzyme immunoassay, or the method of radioimmunoassay. Known methods such as the chloramine T method and the Porton Hunter method can be used.

The measurement operation method is a well-known method (Japanese Society of Clinical Pathology, “Special Issue on Clinical Pathology No. 53, Imnoassy Technology and Application for Clinical Examinations”, Clinical Pathology Publishing Association, 1983, Ei Ishikawa Ed. “Enzyme Immunoassay”, 3rd edition, Medical Shoin, 1987, Tsunehiro Kitagawa et al. “Protein Nucleic Acid Enzyme Supplement No. 31 Enzyme Immunoassay”, Kyoritsu Shuppan, 1988 1980, edited by Minoru Irie, "Radio Noatssey", Kodansha Syentifik, 1974, edited by Minoru Irie, "Continuing Radio Imnottsuy", Kodani Syentifik, 1979) It can be carried out.

For example, when directly labeling the monoclonal antibody of the present invention, the components in the sample are immobilized and contacted with the labeled monoclonal antibody of the present invention to form a complex of the bradion protein and the monoclonal antibody of the present invention. Is formed. Then, the unbound labeled monoclonal antibody is washed and separated, and the amount of bradion protein in the sample can be measured from the amount of the bound labeled monoclonal antibody or the amount of the unbound labeled monoclonal antibody. For example, when a labeled secondary antibody is used, the monoclonal antibody of the present invention is reacted with a sample (primary reaction), and further reacted with a labeled secondary antibody (secondary reaction). The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at a different time. The complex of the immobilized Bradion protein-monoclonal antibody-labeled secondary antibody of the present invention or the immobilized monoclonal antibody-Bradion protein-labeled secondary antibody of the present invention obtained by the first and second reactions An antibody complex forms. Then, unbound labeled secondary antibody is washed and separated, and the amount of bradion protein in the sample can be measured from the amount of bound labeled secondary antibody or the amount of unbound labeled secondary antibody.

Specifically, in the case of enzyme immunoassay, a labeling enzyme is reacted with a substrate under the optimal conditions, and the amount of the reaction product is measured by an optical method or the like. In the case of fluorescent immunoassay, the fluorescence intensity by the fluorescent substance label is measured, and in the case of radioimmunoassay, the amount of radioactivity by the radioactive substance label is measured. In the case of a luminescence immunoassay, the amount of luminescence by a luminescence reaction system is measured.

The detection method of the present invention comprises the steps of: producing an immunocomplex aggregate such as an immunoturbidimetry, latex agglutination reaction, latex nephelometry, hemagglutination reaction or particle agglutination reaction, and transmitting or scattering the light by an optical method. When the measurement is carried out by a measuring method that can be measured or visually measured, a phosphate buffer, a glycine buffer, a Tris buffer, a Good buffer, or the like can be used as a solvent, and a reaction accelerator such as polyethylene dalicol can be used. Or a non-specific reaction inhibitor.

Hereinafter, an example of a preferred embodiment of the detection method of the present invention will be described. First, the monoclonal antibody of the present invention is immobilized on an insoluble carrier as a primary monoclonal antibody. Preferably, the surface of the solid phase on which the antigen is not adsorbed is blocked by a protein unrelated to the antigen (such as pup serum, pac serum albumin, or gelatin). Subsequently, the test sample is brought into contact with the immobilized primary monoclonal antibody. Next, a labeled secondary antibody that reacts with the Bradion protein is brought into contact with a site different from the primary monoclonal antibody, and a signal from the label is detected.

As used here, "A site different from the primary monoclonal antibody, the Bradion protein The `` secondary antibody that reacts with '' is not particularly limited as long as it recognizes a site other than the binding site between the primary monoclonal antibody and the Bradion protein, regardless of the type of immunogen, polyclonal antibody, antiserum, Any of monoclonal antibodies may be used, and fragments of these antibodies (Fab, F (ab ') ₂ , Fab *, etc.) can also be used. Further, a plurality of types of monoclonal antibodies may be used as the secondary antibody.

Conversely, the monoclonal antibody of the present invention is labeled as a secondary antibody, and at a site different from the monoclonal antibody of the present invention, an antibody that reacts with the bradion protein is immobilized as a primary antibody on an insoluble carrier, The immobilized primary antibody may be brought into contact with a sample to be tested, and then contacted with the labeled monoclonal antibody of the present invention as a secondary antibody to detect a signal from the label. .

4. Cancer diagnostics

Further, as described above, the monoclonal antibody of the present invention can specifically be used as a diagnostic agent for cancer because it reacts specifically with the bradion protein derived from colon cancer or prostate cancer cells.

The diagnostic agent of the present invention contains the monoclonal antibody of the present invention. Therefore, using the diagnostic agent of the present invention, the large intestine contained in a sample collected from an individual suspected of having colon cancer or prostate cancer is obtained. By detecting a Bradion protein derived from a cancer or prostate cancer cell, the individual can be diagnosed with colorectal cancer or prostate cancer.

In addition, the diagnostic agent of the present invention can be used in any means as long as it is a means for performing an immunological measurement, but it can be used in combination with a simple means known in the art, such as a test strip for immunochromatography. By using these, cancer can be diagnosed more easily and quickly. The test strip for immunochromatography is, for example, a sample receiving portion made of a material that easily absorbs a sample, a reagent portion containing the diagnostic agent of the present invention, a developing portion in which a reactant between the sample and the diagnostic agent moves, and a developing portion. It is composed of a labeling part that shows the color of the reactant, a presenting part where the colored reactant unfolds, and the like, and can be in the same form as a pregnancy diagnostic drug. First, sump When a sample is supplied to the sample receiving section, the sample receiving section absorbs the sample and allows the sample to reach the reagent section. Subsequently, in the reagent part, a reaction between the colony cancer or prostate cancer cell-derived bradion protein in the sample and the monoclonal antibody of the present invention occurs, and only the reacted complex moves through the developing part and reaches the labeled part. . In the labeled part, a reaction between the above-mentioned reaction complex and the labeled secondary antibody occurs, and when the reaction product with the labeled secondary antibody spreads to the display part, coloration is recognized.

The immunochromatographic test strip described above does not pose any risk to the user due to the use of reagents or pains, and therefore can be used as a monitor at home, and the results can be analyzed at the level of each medical institution. Treatment (surgical resection, etc.) and metastasis * can be linked to prevention of recurrence. At present, this test strip can be mass-produced at low cost by a manufacturing method as described in, for example, Japanese Patent Application Laid-Open No. 10-54830. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1D are photographs showing the results of confirming the reaction specificity of the monoclonal antibody of the present invention for bradion protein derived from prostate cancer cells and colon cancer cells by immunohistological staining. The photographs show Fig. 1A: prostate cancer cell line DU145, Fig. 1B: prostate cancer cell line PC3, Fig. 1C: colon cancer cell line SW480, and Fig. 1D: breast cancer cell line MCF7 (control).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[Example 1] Preparation of recombinant bradion protein

A recombinant bradion protein was produced using the bradion gene integrated into the expression plasmid. That is, a BamHI / CE5B3 j3 (Bradion j3) / XhoI / pCRII plasmid DNA fragment containing the Bradion gene cDNA was digested with Bamffl / Xhol, and ligated to BamHiyXhoI digested expression vector pET21a to express the plasmid. De pET21a-bradeion was obtained (SEQ ID NO: 1). Escherichia coli obtained by transforming this pET21a-bradeion into Escherichia coli BRL was used in SOC medium (1 g of medium, 20 g of bactotryptone, 5 g of yeast extract, NaCl 0.5 g, MgCl ₂ 10 mM, 20 mM glucose). After the cells were grown to logarithmic growth phase, 0.2 mM IPTG was added, and culture was continued at 37 ° C. for 3 hours to induce the expression of Bradion protein. After the cells were collected by a centrifuge, the cells were lysed using an ultrasonic crusher, and the insoluble fraction containing the recombinant blade ion protein was separated by low-speed centrifugation. This insoluble fraction was solubilized with a buffer containing 8M urea, and the recombinant Bradion protein was purified by nickel chelate column chromatography. The recombinant bradion protein thus obtained was used as an immunogen in the following examples.

[Example 2] Immunization of mice and production of hybridomas

The Bradion protein immunogen solution (0.5 mg / mlX Ol ml) obtained in Example 1 was mixed with an equal amount of Freund's complete adjuvant until emulsified, and 0.1 ml of the mixture was intraperitoneally administered to mice (first time). Immunity). After 30 days, the mice were intraperitoneally administered with 0.05 ml of a mixed solution with Freund's incomplete adjuvant (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse.

The aseptic extirpated spleen is minced in MEM medium, loosened with forceps, centrifuged (500X g, 10 minutes), discarded supernatant, and washed with Tris-ammonium chloride buffer (pH7.65). The cells were treated for 1 to 2 minutes to remove red blood cells, and washed three times with MEM medium to obtain splenocytes for fusion.

The spleen cells and mouse myeloma cells that had been cultured in advance were mixed so that the number of cells became splenocyte: myeloma cells = 5 to 10: 1, and centrifuged (500 × g, 10 minutes). After removing the supernatant and thoroughly dissolving the precipitate, 0.5 mL of a 40% polyethylene glycol 4000 solution was added dropwise at 37 ° C, and mixed by gently rotating the tube for 1 minute. Next, 1 to 2 mL of MEM medium heated to 37 ° C. was added several times every 1 to 2 minutes, and MEM medium was added to bring the total volume to 50 mL. After centrifugation (500X g, 10 minutes), discard the supernatant, dissolve the precipitate well, add HAT medium and add 100mL And The cell suspension was dispensed by 100 H 1 / Ueru the plate for 96 Ueru culture in 5% C0 ₂ incubator one, and cultured for 7 to 14 days at 37 ° C.

[Example 3] Establishment of high Pridoma

The presence or absence of the antibody produced in the culture supernatant of the hybridoma was measured by enzyme immunoassay (ELISA). To each well of a 96-well ELISA plate, 50 L of the recombinant bradion protein solution (5 g / mL, diluted with PBS) obtained in Example 1 was dispensed and left at 25 ° C for 2 hours. Next, after washing three times with 0.05% Tween20-PBS, 50 L of the culture supernatant was added to each well and reacted at 25 ° C for 1 hour.

Next, after washing three times with 0.05% Tween20-PBS, 50 L of a peroxidase-labeled anti-mouse antibody diluted 200-fold with 0.05% Tween20-PBS was added to each well, and reacted at 25 ° C for 1 hour. After completion of the reaction, wash each well three times with 0.05% Tween20-PBS, add 100 L of a solution containing 0.015% hydrogen peroxide and 0.75 mg / mL tetramethylbenzidine to each well, and react at 25 ° C for 30 minutes. I let it. The enzyme reaction was stopped by adding 100 L of 2 M sulfuric acid, and the absorbance at 450 nm of each well was measured.

As a result, anti-Bradion monoclonal antibody production was observed in 4 out of 480 wells. Each hybridoma in the 4-well was transferred to a 24-well plate, cultured in an HT medium for 4 to 5 days, and cloned twice by the limiting dilution method. From the clones thus obtained, clones having good growth, high antibody secretion ability, and stable were selected, and the hybridomas A, B, C, and D that produce anti-Bradion monoclonal antibodies were established.

The subclass of the obtained monoclonal antibody was determined using a commercially available subcluster typing kit (manufactured by Tokushu Immune Laboratory) according to the instructions.

[Example 4] Preparation of monoclonal antibody

Pristane-treated (a Pris ne 0.5 _m L intraperitoneally administered 2 weeks of feeding) were 8 to 10 weeks old BALB / C mice, the embodiment Monoclonal antibody production obtained in 3 hybridoma 2 X 10 ⁶ cells Was administered intraperitoneally. Hypri-doma transforms into ascites cancer in 10-21 days did. Ascites was collected from the mice, and solids were removed by centrifugation, followed by salting with 50% ammonium sulfate.

For the antibody of subclass IgG, the precipitate was dissolved in 10 mM phosphate buffer (pH 8.0) and dialyzed against 1000 volumes of lOmM phosphate buffer. This was loaded on a protein A-Sepharose column which had been equilibrated with 10 mM phosphate buffer in advance. Elution of the monoclonal antibody was performed with 100 mM citrate buffer (pH 4.5). The eluted monoclonal antibody was concentrated by ultrafiltration, and dialyzed against PBS to obtain a purified antibody solution.

(Example 5) Immunofluorescence staining

Colorectal cancer cell lines colo205, Sw480 and DU145, prostate cancer cell lines DU145 and PC3, breast cancer cell line MCF7, cervical cancer cell line HeLa, and normal fibroblast MRC5, each independently on a slide glass with a gel , And fixed by treating with 2% paraformaldehyde for 5 minutes at room temperature. After washing with TBS containing 0.1% Triton X100 and blocking with 10% goat serum and 2% BSA, the purified monoclonal antibody (10 g / ml) obtained in Example 4 above was used as the primary antibody. The reaction was performed at 30 ° C for 1 hour. After washing with PBS, Alexa480-labeled anti-mouse IgG goat antibody (Molecular Probes) is added as a secondary antibody and reacted at room temperature for 1 hour. Was observed. As a result, the monoclonal antibody of the present invention was able to stain cancer cells positively. In addition, the monoclonal antibody did not show reactivity with the breast cancer cell line MCF7, the cervical cancer cell line HeLa, and the normal fibroblast MRC5 (FIG. 1). In contrast, in the colon cancer cell lines colo205, Sw480 and DU145 and the prostate cancer cell lines DU145 and PC3, clear fluorescence was observed in the cytoplasm (FIG. 1).

(Example 6) Immunohistochemical staining

From the paraffin-embedded specimen of colon cancer tissue, a 4 m section was prepared with mitochrome, taken on a silane-coated slide, deparaffinized with xylene, and then ethanol. It became hydrophilic. After washing with PBS and blocking with methanol containing 0.3% hydrogen peroxide and 10% egret serum, the purified monoclonal antibody (10 / ml) obtained in Example 4 above was used as a primary antibody at 4 ° C. The reaction was performed overnight. After washing with PBS, a secondary antibody (a biotin-labeled anti-mouse antibody) was applied, reacted at room temperature for 90 minutes, and then reacted with peroxidase-labeled streptavidin at room temperature for 5 minutes. After color development with a diaminobenzidine solution, nuclear staining was performed using methyl green, dehydration was performed, and mounted microscopy was performed. As a result, the anti-Bradion monoclonal antibody was able to stain cancer cells positively. The monoclonal antibody did not show reactivity with normal tissue cells.

Using a paraffin-embedded specimen of prostate cancer tissue, immunohistochemical staining was performed using the monoclonal antibody obtained in Example 4 as a primary antibody. The experiment was performed in the same manner as in the above colon cancer. As a result, the anti-Bradion monoclonal antibody stained cancer cells positively, as in colorectal cancer, and did not show reactivity with normal tissue cells. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial applicability

The present invention provides a monoclonal antibody useful for diagnosing cancer. The use of the monoclonal antibody of the present invention makes it possible to diagnose cancer easily and quickly, which is thought to contribute to early detection and prognosis of cancer. Sequence listing free text

Sequence number 1: Synthetic DNA

Claims

The scope of the claims

1. A monoclonal antibody that specifically reacts with the bradion protein derived from colon cancer or prostate cancer cells.

2. A hybridoma that produces the monoclonal antibody according to claim 1.

3. A method for detecting cancer, comprising detecting a bradion protein derived from a cancer cell in a sample using the monoclonal antibody according to claim 1.

4. The method according to claim 3, wherein the cancer is colon cancer and Z or prostate cancer.

5. A diagnostic agent for cancer comprising the monoclonal antibody according to claim 1.

6. The diagnostic agent according to claim 5, wherein the cancer is colon cancer and Z or prostate cancer.