WO1998005960A1 - Methode et reactifs pour detecter le cancer du colon - Google Patents

Methode et reactifs pour detecter le cancer du colon Download PDF

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Publication number
WO1998005960A1
WO1998005960A1 PCT/JP1997/002690 JP9702690W WO9805960A1 WO 1998005960 A1 WO1998005960 A1 WO 1998005960A1 JP 9702690 W JP9702690 W JP 9702690W WO 9805960 A1 WO9805960 A1 WO 9805960A1
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WIPO (PCT)
Prior art keywords
vegf
colorectal cancer
test
growth factor
factor
Prior art date
Application number
PCT/JP1997/002690
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English (en)
Japanese (ja)
Inventor
Keiichi Mitsuyama
Kyuichi Tanikawa
Mitsuya Hanatani
Isamu Matsubara
Katsuhiko Matsuo
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Toagosei Co. Ltd.
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 Toagosei Co. Ltd. filed Critical Toagosei Co. Ltd.
Publication of WO1998005960A1 publication Critical patent/WO1998005960A1/fr

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a method for examining colorectal cancer by measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in feces, and a test drug used therefor, and relates to a medical diagnostic technique and a test drug technique. Things. Background art
  • Tumor markers for colorectal cancer include carcinoembryonic antigen (hereinafter referred to as “CEA”), CA19-9, NCC-ST-439, STN, etc.
  • CEA carcinoembryonic antigen
  • the power used in the evening It is hard to say that it is a sufficient tumor marker for detecting early colorectal cancer.
  • the tumor marker positive rate by stage was 36%, 30%, 35%, and 21% for CEA, CA 19-9, NCC-ST-439, and STN, respectively, even for curable resectable Dukes C. (Hisao Okura et al., “Tumor Markers for Colorectal Cancer” CRC, 1 (4), 42-47 (1992)).
  • vascular endothelial growth factor a FGF
  • VEGF blood Vascular endothelial growth factor / vascular permeability factor
  • PD-ECGF platelet-derived endothelial cell growth factor
  • ⁇ GF-a transforming growth factor-a
  • TGF- ⁇ transforming growth factor- ⁇
  • TGF-hi tumor necrosis factor- ⁇
  • Human VEGF is also angiogenesis and metastasis of breast cancer [Weider N., et al., ⁇ . Engl. J. Med., 324: 1 (1991)] ⁇ Angiogenesis of renal cell carcinoma [Ayumi of Medicine 168: 231 (1994)] or angiogenesis in retinal diseases [Adamis AP, et al., Biochem. Biophys. Res. Co., 193: 631 (1993)].
  • VEGF For the human VEGF gene, its cDNA has already been isolated, its nucleotide sequence has been determined, and its amino acid sequence has been deduced. In this gene, four types of proteins with four different amino acid residues (four types of amino acid residues: 121, 165, 189, and 206) are produced from one gene. Among them, those with 121 amino acid residues (VEGF 121 ) and those with 16 5 amino acid residues (VEGF 165 ) are said to be power'secretory proteins [Ferrara N., et al., Endocrine Reviews, 13:18 (1992) 1.
  • an object of the present invention is to provide an early inspection method as a screening for colorectal cancer and monitoring S.
  • colorectal cancer can be tested at a positive rate of 80% or more by measuring the VEGF concentration in feces using the feces of colorectal cancer patients as a sample. Was completed.
  • the present invention provides a method for examining colorectal cancer, comprising measuring the amount of vascular endothelial cell growth factor Z vascular permeability factor (VEGF) in human feces, and vascular endothelial cell growth factor Z vascular permeability. It is intended to provide a colorectal cancer test agent containing a substance that specifically reacts with a factor (VEGF).
  • VEGF vascular endothelial cell growth factor Z vascular permeability factor
  • a well-known labeling immunoassay using a substance that specifically reacts with VEGF for example, an anti-VEGF antibody or a VEGF receptor, is suitable for measuring the VEGF concentration in serum, and is also a preferable method in the present invention. .
  • a labeled immunoassay using a radioisotope, an enzyme, a luminescent substance, a fluorescent substance, or the like as a label, or an anti-VEGF antibody or a VEGF receptor, etc.
  • An agglutination reaction using bound erythrocytes, latex, metal particles, metal gel, pateriophage, or the like, a chromatography method, or the like is applied.
  • the enzyme immunoassay is suitable for clinical application, and in practice, the enzyme immunoassay is widely used.
  • Enzyme-linked immunosorbent assay is a method of tracking the antigen-antibody reaction using enzyme activity as an index, and measuring the amount of antigen or antibody from this.
  • EIA Enzyme-linked immunosorbent assay
  • the colorectal cancer test agent of the present invention is a substance that specifically reacts with the VEGF,
  • VEGF antibody or VEGF receptor as a main component of the test drug.
  • carriers or excipients acceptable for colorectal cancer test drugs are appropriately used. Can be blended.
  • FIG. 1 shows the measurement results of the amount of stool VEGF in colorectal cancer patients.
  • FIG 2 shows that the colorectal cancer patients shown in Figure 1 are separated into early cancer patients and advanced cancer patients
  • FIG. 3 shows the results of measuring the amount of fecal VEGF before and after surgery for colorectal cancer patients.
  • the isolated human VEGF cDNA was produced in Escherichia coli as a fusion protein with glutathione S-transferase (GST) (GST-VEGF), and the obtained protein was used as an antigen in accordance with a conventional method to prepare a egret anti-VEGF polyclonal.
  • GST-VEGF glutathione S-transferase
  • One null antibody was prepared. Egret serum with an increased antibody titer was separated, and an IgG fraction of an egret anti-VEGF polyclonal antibody was obtained by anion exchange column chromatography.
  • a portion of the IgG fraction was digested with pepsin to prepare F (ab ') 2, which was then ligated with peroxidase (horseradish) by the hinge method, and a peroxidase-labeled mouse egret was prepared.
  • a VEGF polyclonal antibody was obtained.
  • PH 7.4
  • BSA serum albumin
  • the stool extract was prepared by adding 2 ml of PBS to stool lg (wet weight), homogenizing, and filtering through a filter having a pore size of 0.45 / im. After washing 6 times with PBS containing 0.1% BSA, 1001 Zweil of the peroxidase-labeled anti-VEGF polyclonal antibody obtained in (2) was added thereto and reacted at room temperature for 1 hour. After washing again 8 times with PBS containing 0.1% BSA, 0.2M tris (hydroxymethyl) aminomethane containing 0.125% (w / v) orthophenylenediamine and 0.015% hydrogen peroxide was added.
  • Table 1 and FIG. 1 show the measurement results of the amount of VEGF in the fecal extract of 26 colorectal cancer patients and 10 healthy subjects.
  • Colorectal cancer patients had a VEGF level (mean ⁇ standard deviation) of 1 1 16.9 ⁇ 1566. 3 In contrast to 71111, the value for healthy subjects was 66.3 ⁇ 36.3 pgZml, and colorectal cancer patients showed significantly higher values than healthy subjects.
  • the positive rate was calculated as 80.8% in colorectal cancer patients using the mean value of the VEGF level in healthy volunteers + the standard deviation of 2 times (138.9 pg / ml) as the cutoff value. .
  • the colorectal cancer test method according to this method can be said to be very useful, as the positive rates of the existing colorectal cancer tests, immunological occult blood test and immunohemoglobin test, are 50 to 60% in colorectal cancer patients.
  • Table 2 and FIG. 2 show a comparison of the amount of VEGF in feces by distinguishing the colorectal cancer patients into early cancer patients and advanced cancer patients.
  • the amount of VEGF (mean ⁇ standard deviation) in early cancer patients was 363.4 ⁇ 339.7 pg / ml, which was significantly higher than that in healthy subjects.
  • the amount of VEGF (average soil standard deviation) in advanced cancer patients was 141.8 ⁇ 1781.7 PgZml, which was significantly higher than that in healthy subjects and early cancer patients.
  • the power-off value is the mean value of the VEGF amount + 2 times the standard deviation of healthy subjects (138.9 Pg / ml)
  • the positive rate for early-stage cancer patients is 62.5%, and that for advanced cancer patients is 62.5%.
  • the positive rate was 88.9%, indicating that this method is very useful for early colorectal cancer testing, as compared to the existing colorectal cancer tests, such as the immune occult blood test and the immunohemoglobin test.
  • Table 3 and FIG. 3 show a comparison of the amount of VEGF in feces before and after surgery in the same five patients with colorectal cancer.
  • Table 3 VEGF levels in feces before and after surgery
  • the amount of VEGF (mean ⁇ standard deviation) was 32 15.2 ⁇ 1 336.9 Pg / ml before surgery, and 535.7 ⁇ 1 24.8 pgZml after surgery. The value was significantly lower after surgery than before. This indicates that the amount of VEGF in feces was reduced by removing the colorectal cancer site by surgery, and that fluctuations in the amount of VEGF in feces are useful in knowing the progress after surgery. . Industrial applicability
  • Diagnosis by the test method of the present invention in which colorectal cancer is tested by measuring the amount of VEGF in feces, is useful for early detection of colorectal cancer, judgment of progression of colorectal cancer pathology and treatment effect, prediction of recurrence, etc. It is an excellent product that can be widely used, and because it uses feces, it can be used as a new test method in addition to the test methods used so far. Furthermore, the colorectal cancer test according to the present invention is very clinically useful because it can detect colorectal cancer patients at a much higher rate than when existing tumor markers or stool occult blood reactions are used. is there.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une méthode permettant de détecter le cancer du colon, selon laquelle on dose le facteur de croissance de l'endothélium vasculaire/le facteur de perméabilité vasculaire dans les selles humaines. L'invention concerne également des réactifs permettant de détecter le cancer du colon, caractérisés par le fait qu'ils contiennent une substance réagissant sélectivement avec le facteur de croissance de l'endothélium vasculaire/le facteur de perméabilité vasculaire.
PCT/JP1997/002690 1996-08-02 1997-08-01 Methode et reactifs pour detecter le cancer du colon WO1998005960A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP22057596 1996-08-02
JP8/220575 1996-08-02

Publications (1)

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WO1998005960A1 true WO1998005960A1 (fr) 1998-02-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034938A1 (fr) * 2000-10-26 2002-05-02 Eisai C0. Ltd. Procede de diagnostic et d'examen du cancer du colon utilisant tannase en tant qu'indication

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06281649A (ja) * 1993-02-01 1994-10-07 Toagosei Chem Ind Co Ltd 腫瘍診断方法及び診断薬
JPH0843384A (ja) * 1994-07-27 1996-02-16 Toagosei Co Ltd 大腸癌の診断方法および診断薬
JPH0843398A (ja) * 1994-08-03 1996-02-16 Toagosei Co Ltd 癌の診断方法
JPH08313522A (ja) * 1995-05-16 1996-11-29 Toagosei Co Ltd 血管透過性因子の測定方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06281649A (ja) * 1993-02-01 1994-10-07 Toagosei Chem Ind Co Ltd 腫瘍診断方法及び診断薬
JPH0843384A (ja) * 1994-07-27 1996-02-16 Toagosei Co Ltd 大腸癌の診断方法および診断薬
JPH0843398A (ja) * 1994-08-03 1996-02-16 Toagosei Co Ltd 癌の診断方法
JPH08313522A (ja) * 1995-05-16 1996-11-29 Toagosei Co Ltd 血管透過性因子の測定方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002034938A1 (fr) * 2000-10-26 2002-05-02 Eisai C0. Ltd. Procede de diagnostic et d'examen du cancer du colon utilisant tannase en tant qu'indication
US7090997B2 (en) 2000-10-26 2006-08-15 Eisai Co., Ltd. Diagnostic agent and test method for colon cancer using tannase as index

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