WO2003051928A1 - Anticorps spécifique pour la détection du bradéion - Google Patents

Anticorps spécifique pour la détection du bradéion Download PDF

Info

Publication number
WO2003051928A1
WO2003051928A1 PCT/JP2001/011003 JP0111003W WO03051928A1 WO 2003051928 A1 WO2003051928 A1 WO 2003051928A1 JP 0111003 W JP0111003 W JP 0111003W WO 03051928 A1 WO03051928 A1 WO 03051928A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
monoclonal antibody
antibody
bradion
cells
Prior art date
Application number
PCT/JP2001/011003
Other languages
English (en)
Japanese (ja)
Inventor
Manami Tanaka
Tomoo Tanaka
Kengo Sakaguchi
Original Assignee
National Institute Of Advanced Industrial Science And Technology
Nippn, Co. Ltd.
Nippn Technocluster, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National Institute Of Advanced Industrial Science And Technology, Nippn, Co. Ltd., Nippn Technocluster, Inc. filed Critical National Institute Of Advanced Industrial Science And Technology
Priority to AU2002222647A priority Critical patent/AU2002222647A1/en
Priority to PCT/JP2001/011003 priority patent/WO2003051928A1/fr
Priority to JP2003552969A priority patent/JPWO2003052102A1/ja
Priority to AU2002359980A priority patent/AU2002359980A1/en
Priority to PCT/JP2002/013075 priority patent/WO2003052102A1/fr
Publication of WO2003051928A1 publication Critical patent/WO2003051928A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate

Definitions

  • 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.
  • Prostate cancer 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.
  • 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.
  • the Bradion gene is involved in colorectal cancer and prostate cancer
  • 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.
  • 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.
  • the present invention provides a method of using the above monoclonal antibody to
  • the cancer detection method characterized by detecting a protein.
  • the cancer includes colon cancer and / or prostate cancer.
  • the present invention is a diagnostic agent for cancer containing a monoclonal antibody.
  • Cancers include colon cancer and Z or prostate cancer.
  • 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.
  • 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.
  • 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.
  • a protein to be used as an immunogen is prepared.
  • a human bradion protein is used as the immunogenic protein.
  • 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.
  • 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.
  • 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 ⁇ phage (Agtl, ⁇ , etc.). Furthermore, animal virus vectors such as vaccinia virus and insect virus vectors such as baculovirus can also be used.
  • E. coli-derived plasmids eg, pET21a, pGEX4T, pUC118, pUC119, pUC18, pUC19
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, etc.
  • 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.
  • 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.
  • a known DNA ligase is used to ligate the DNA fragment and the vector fragment. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the human bradion protein 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • hybridomas cell fusion between antibody-producing cells obtained from the immunized animal and myeloma cells is performed as described above.
  • 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.
  • 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).
  • P3X63-Ag.8 strain ATCC TIB9
  • P3X63-Ag. .8.Ul strain cancer research Research Source Bank ( JCRB ) 9085
  • P3 / NSI / l-Ag4-l strain JCRB0009
  • P3x63Ag8.653 strain JCRB0028
  • 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%.
  • an auxiliary agent such as dimethyl sulfoxide may be used in combination to increase the fusion efficiency.
  • antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electoral poration).
  • the target eight hybridomas are selected from the cells after the cell fusion treatment.
  • 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.
  • TK thymidine kinase
  • 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.
  • 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.
  • EIA Enzyme Immuno Assav and ELISA
  • RIA Radiolmmuno Assav
  • 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.
  • a normal cell culture method 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • ELISA enzyme immunoassay
  • EIA fluorescence immunoassay
  • RIA radioimmunoassay
  • luminescence immunoassay immunoturbidimetry
  • immunoassay 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the reaction 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.
  • POD peroxidase
  • alkaline phosphatase alkaline phosphatase
  • enzyme-linked immunosorbent assay 6-galactosidase, perease, lipase, glucose oxidase, and lactate dehydrogenase.
  • 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.
  • 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.
  • 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.
  • 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.
  • fluorescent immunoassay the fluorescence intensity by the fluorescent substance label is measured
  • radioimmunoassay the amount of radioactivity by the radioactive substance label is measured.
  • 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.
  • an immunocomplex aggregate such as an immunoturbidimetry, latex agglutination reaction, latex nephelometry, hemagglutination reaction or particle agglutination reaction
  • transmitting or scattering the light by an optical method.
  • 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.
  • the monoclonal antibody of the present invention is immobilized on an insoluble carrier as a primary monoclonal antibody.
  • 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).
  • a protein unrelated to the antigen such as pup serum, pac serum albumin, or gelatin.
  • the test sample is brought into contact with the immobilized primary monoclonal antibody.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 presence or absence of the antibody produced in the culture supernatant of the hybridoma was measured by enzyme immunoassay (ELISA).
  • ELISA enzyme immunoassay
  • 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.
  • 50 L of the culture supernatant was added to each well and reacted at 25 ° C for 1 hour.
  • 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.
  • 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.
  • 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.
  • the monoclonal antibody of the present invention was able to stain cancer cells positively.
  • 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).
  • 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).
  • the anti-Bradion monoclonal antibody was able to stain cancer cells positively.
  • the monoclonal antibody did not show reactivity with normal tissue cells.
  • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Reproductive Health (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un anticorps monoclonal qui réagit de manière spécifique à la protéine du bradéion, provenant de cellules cancéreuses de la prostate et/ou du colon. L'invention a pour objet un procédé de détection du cancer mettant en oeuvre cet anticorps et des outils de diagnostique du cancer contenant cet anticorps.
PCT/JP2001/011003 2001-12-14 2001-12-14 Anticorps spécifique pour la détection du bradéion WO2003051928A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU2002222647A AU2002222647A1 (en) 2001-12-14 2001-12-14 Antibody specific for detection of bradeion
PCT/JP2001/011003 WO2003051928A1 (fr) 2001-12-14 2001-12-14 Anticorps spécifique pour la détection du bradéion
JP2003552969A JPWO2003052102A1 (ja) 2001-12-14 2002-12-13 ブラディオン検出用特異的抗体
AU2002359980A AU2002359980A1 (en) 2001-12-14 2002-12-13 Specific antibody for detecting bradeion
PCT/JP2002/013075 WO2003052102A1 (fr) 2001-12-14 2002-12-13 Anticorps specifique destine a la detection de proteines de bradeion

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2001/011003 WO2003051928A1 (fr) 2001-12-14 2001-12-14 Anticorps spécifique pour la détection du bradéion

Publications (1)

Publication Number Publication Date
WO2003051928A1 true WO2003051928A1 (fr) 2003-06-26

Family

ID=11738043

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/JP2001/011003 WO2003051928A1 (fr) 2001-12-14 2001-12-14 Anticorps spécifique pour la détection du bradéion
PCT/JP2002/013075 WO2003052102A1 (fr) 2001-12-14 2002-12-13 Anticorps specifique destine a la detection de proteines de bradeion

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/013075 WO2003052102A1 (fr) 2001-12-14 2002-12-13 Anticorps specifique destine a la detection de proteines de bradeion

Country Status (3)

Country Link
JP (1) JPWO2003052102A1 (fr)
AU (2) AU2002222647A1 (fr)
WO (2) WO2003051928A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000139470A (ja) * 1998-11-16 2000-05-23 Agency Of Ind Science & Technol ヒト由来ブラディオン蛋白質、それをコードするｄｎａ及びそれらの使用
JP2001161384A (ja) * 2000-10-10 2001-06-19 Natl Inst Of Advanced Industrial Science & Technology Meti 癌の検出法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000139470A (ja) * 1998-11-16 2000-05-23 Agency Of Ind Science & Technol ヒト由来ブラディオン蛋白質、それをコードするｄｎａ及びそれらの使用
JP2001161384A (ja) * 2000-10-10 2001-06-19 Natl Inst Of Advanced Industrial Science & Technology Meti 癌の検出法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LARISCH-BLOCH ET AL.: "Selective loss of the transforming growth factor-beta apoptotic signaling pathway in mutant NRP-154 rat prostatic epithelial cells", CELL GROWTH DIFFER., vol. 11, no. 1, 2000, pages 1 - 10, XP002908884 *
TANAKA ET AL.: "Characterization of tissue- and cell-type-specific expression of a novel human septin family gene, bradeion", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 286, no. 3, August 2001 (2001-08-01), pages 547 - 553, XP002908883 *

Also Published As

Publication number Publication date
AU2002222647A1 (en) 2003-06-30
WO2003052102A1 (fr) 2003-06-26
AU2002359980A1 (en) 2003-06-30
JPWO2003052102A1 (ja) 2005-04-28

Similar Documents

Publication Publication Date Title
US7183385B2 (en) Phospho-specific antibodies to Flt3 and uses thereof
US9309312B2 (en) Immunoassay method for human CXCL1 protein
JP6324970B2 (ja) 抗ウロプラキンｉｉ抗体システムおよび方法
JP3626177B2 (ja) 尿路上皮癌腫瘍マーカー
CN108794630A (zh) 鼠抗人tim3蛋白单克隆抗体制备及其免疫组化用途
KR101777259B1 (ko) En2 단백질을 특이적으로 인식하는 단클론 항체 또는 이를 함유하는 전립선암 진단용 조성물
KR101777254B1 (ko) En2 단백질을 특이적으로 인식하는 특정 항원으로부터 얻어진 단클론 항체 또는 이를 함유하는 전립선암 진단용 조성물
US20050214301A1 (en) Antibodies specific for BCR-ABL fusion protein and uses thereof
US7105642B2 (en) Monoclonal antibodies specific for phosphorylated estrogen receptor alpha (Ser118) and uses thereof
JP2001502669A (ja) ヒトａｃｔおよびセリンプロテアーゼからなる複合体に対するモノクローナル抗体
US20100093008A1 (en) Phospho-specific antibodies to flt3 (tyr969) and uses thereof
US20040142400A1 (en) High affinity monoclonal antibody for recognizing the estrogen receptor (ER) and method for creating the antibody
WO2003051928A1 (fr) Anticorps spécifique pour la détection du bradéion
JP4451784B2 (ja) 前立腺癌腫瘍マーカー
US9127054B2 (en) Immunoassay of cofilin 1 protein
CN116003567A (zh) En2蛋白的免疫原性片段肽或特异性识别其的抗体组合物
US7569675B2 (en) High affinity monoclonal antibody for recognizing the progesterone receptor (PR) and method for creating the antibody
JP2012018119A (ja) 大腸癌検出用マーカーおよびそれを用いた大腸癌検出方法
US7157286B2 (en) Method for immunologically measuring RCAS1 and kit for measuring the same
US7141435B2 (en) Monoclonal antibodies against human apoptosis inhibitory protein NAIP and method for assaying the NAIP
JP2021519284A (ja) 治療薬による処置から利益を受けうる被験体を特定するためのサンドウィッチｅｌｉｓａ
JP2007008844A (ja) セプチン２タンパク質と特異的に反応するモノクローナル抗体、それを産生するハイブリドーマ、癌の検出方法及び癌の検出用キット
MXPA97005161A (es) La proteina"a"como diagnostico de cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP