WO1998005960A1 - Test method and test reagents for colon cancer - Google Patents
Test method and test reagents for colon cancer Download PDFInfo
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- 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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- G—PHYSICS
- G01—MEASURING; TESTING
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- 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
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- G—PHYSICS
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- 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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Abstract
A method for testing colon cancer characterized by determining the vascular endothelial cell growth factor/vascular permeability factor in human feces; and reagents for testing colon cancer characterized by containing a substance reacting specifically with the vascular endothelial cell growth factor/vascular permeability factor.
Description
明 細 書 大腸癌の検査方法及び検査薬 Description Colorectal cancer testing methods and drugs
技術分野 Technical field
本発明は、 糞便中の血管内皮細胞増殖因子/血管透過性因子の量を測定するこ とによる大腸癌の検査方法及びそれに使用する検査薬に関するものであり、 医療 の診断技術及び検査薬技術に関するものである。 背景技術 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
現在大腸癌患者は年々増加しており、 早期発見のための検査方法が必要とされ ている。 大腸癌は進行が比較的緩やかであり、 進行癌でも治癒切除が完全に行わ れれば予後は比較的良好であるが、 かなり進行するまで自覚症状が少なく確定診 断時にすでに転移や浸潤等をおこして切除不可能な場合も少なくない。 Currently, the number of colorectal cancer patients is increasing year by year, and testing methods for early detection are needed. The progression of colorectal cancer is relatively slow, and the prognosis is relatively good if curative resection is completely performed even for advanced cancer.However, subjective symptoms are few until progression is significant, and metastasis and invasion have already occurred at the time of definitive diagnosis. In many cases, resection is impossible.
大腸癌の検査として便の免疫潜血反応検査が現在広く用いられているが、 その 陽性率は 50~60%にすぎない。 また大腸癌の腫瘍マーカーとしては、 癌胎児 性抗原 (carcinoembryonic antigen, 以下 「CEA」 という) 、 CA 1 9— 9、 NCC - ST— 439、 ST N等があり、 治療効果の判定や再発のモニ夕一とし て用いられている力 早期大腸癌の発見に関しては十分な腫瘍マーカーであると はいいがたいものである。 なぜなら、 病期別腫瘍マ一カー陽性率は治癒切除可能 な Dukes C においても C EA、 CA 19— 9、 NCC- ST-439, STNで 各々 36%、 30%、 35%、 2 1 %にすぎないのである (大倉久直他 「大腸が んの腫瘍マーカー」 CRC, 1 (4), 42-47(1992)) 。 Stool immunooccult blood testing is currently widely used as a test for colorectal cancer, but the positive rate is only 50-60%. Tumor markers for colorectal cancer include carcinoembryonic antigen (hereinafter referred to as “CEA”), CA19-9, NCC-ST-439, STN, etc. The power used in the evening It is hard to say that it is a sufficient tumor marker for detecting early colorectal cancer. Because 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)).
—方、 血管新生、 即ち毛細血管内皮細胞の増殖、 移動及び組織への浸潤は 胎児の生長、 創傷治癒、 癌細胞の増殖等の生理的又は病理的現象において重 要な役割を果たしていることが知られている [Folkman 丄, Cancer Res. 46:467 (1986)] 。 On the other hand, angiogenesis, that is, proliferation, migration and invasion of tissues of capillary endothelial cells may play an important role in physiological or pathological phenomena such as fetal growth, wound healing, and cancer cell proliferation. Known [Folkman I, Cancer Res. 46: 467 (1986)].
血管新生を誘導する因子としては、 直接的に血管内皮細胞に作用する物質と して塩基性線維芽細胞増殖因子(basic fibroblast growth factor, b FGF) 、 酸性線維芽細胞增殖因子(acidic fibroblast growth factor, a F G F) 、 血
管内皮細胞增殖因子 Z血管透過性因子(vascular endothelial growth factor/ vascular permeability factor, 以下 「V E G F」 としヽぅ) 、 血小板由来内 皮細胞増殖因子(platelet— derived endothelial cell growth factor, P D - E C G F ) 等が、 また間接的に血管内皮細胞に作用する物質としてトランス フォーミング增殖因子 a (transforming growth factor- a , Ύ G F - a ) 、 トラ ンスフォーミング增殖因子 3 (transforming growth factor- β , TG F - β ) 、 アンギオゲニン (angiogenin)、 腫暴壊死因子 σ ( tumor necrosis factor- α , T N F— ひ) 等が見つけられている [Folkman J. & Shing Y. , J. Biol. Chem. , 267:10931 (1992) ] 。 Factors that induce angiogenesis include basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor, which act directly on vascular endothelial cells. , a FGF), blood Vascular endothelial growth factor / vascular permeability factor (hereinafter referred to as “VEGF”), platelet-derived endothelial cell growth factor (PD-ECGF), etc. But also indirectly act on vascular endothelial cells as transforming growth factor-a (ΎGF-a) and transforming growth factor-β (TGF-β) , Angiogenin, tumor necrosis factor-α (TNF-hi), etc. have been found [Folkman J. & Shing Y., J. Biol. Chem., 267: 10931 (1992) )].
V EG Fに関しては、 マウス、 ラッ 卜、 モルモッ ト、 ゥシ及びヒ卜の正常又は 腫瘍細胞株で分泌されており、 また組織別では脳、 下垂体、 腎臓、 卵巣に存在す ることが明らかにされている [Ferrara N. , et al., Endocrine Reviews, 13:18 (1992) ] 。 VEGF is secreted by normal or tumor cell lines in mice, rats, guinea pigs, mice and humans, and is apparently present in the brain, pituitary gland, kidney and ovary by tissue type. [Ferrara N., et al., Endocrine Reviews, 13:18 (1992)].
またヒト V EG Fは乳癌の血管新生と転移 [Weider N. , et al. , Ν. Engl. J. Med. , 324:1 (1991) ]ゃ腎細胞癌の血管新生 [医学のあゆみ 168:231 (1994) ] 、 あるいは網膜疾患における血管新生 [Adamis A. P. , et al. , Biochem. Biophys. Res. Co麵. , 193:631 (1993) ]に関与していること力報告されている。 Human VEGF is also angiogenesis and metastasis of breast cancer [Weider N., et al., Ν. Engl. J. Med., 324: 1 (1991)] ゃ 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)].
ヒト V E G Fの遺伝子についてはその c DN Αがすでに単離されて塩基配列が 決定され、 アミノ酸配列も推定されている。 この遺伝子では 1つの遺伝子からァ ミノ酸残基数の異なる 4種類の蛋白 (アミノ酸残基数が 1 2 1個、 1 6 5個、 1 89個、 2 0 6個の 4種類) が作られ、 それらの中で 1 2 1個のアミノ酸残 基数のもの (V E G F 121)と 1 6 5個のアミノ酸残基数のもの (V E G F 165) 力 '分泌蛋白であるといわれている [Ferrara N. , et al., Endocrine Reviews, 13:18 (1992)1 。 V EG F 121 は V EG F 165 のカルボキシル末端の 44個のァ ミノ酸が欠損したものである力 V EG F 121 と V EG F l 65 の間に、 血管内皮 細胞に対する作用の違いがあるかどうかについては明らかでない。 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. Or V EG F 121 between the force V EG F 121 and V EG F l 65 44 amino § amino acids of the carboxyl terminus of the V EG F 165 is obtained by lacking, there is a difference in the effect on vascular endothelial cells It is not clear how.
—方、 ヒ卜 V EG F 121 に対するモノクローナル抗体はすでに本発明者らによ り取得されている (特開平 8 - 1 69898号 「ぺプチド及びモノクローナル抗 体」 ) 。 更に、 そのモノクローナル抗体及びヒ卜 V E G F 121 に対するポリク
ローナル抗体を用いた酵素免疫測定法により、 数 pg/ml の VEGFが測定できる ことも明らかにされている (特開平 8— 313522号 「血管透過性因子の測定 方法」 ) 。 発明の開示 On the other hand, a monoclonal antibody against human VEGF 121 has already been obtained by the present inventors (Japanese Patent Application Laid-Open No. 8-1699898, "Peptides and Monoclonal Antibodies"). In addition, polyclones against the monoclonal antibody and human VEGF 121 It has also been clarified that several pg / ml of VEGF can be measured by an enzyme immunoassay using a lonal antibody (Japanese Patent Application Laid-Open No. 8-313522, "Method for measuring vascular permeability factor"). Disclosure of the invention
本発明者らは、 以上のような状況の中で、 大腸癌患者の糞便中の V EG Fの量 を測定することにより、 大腸癌の早期検査ができないか検討を行ったのである。 即ち、 本発明は大腸癌のスクリーニングとしての早期検査方法及びモニタリン グ Sを提供することを目的とするものである。 Under the circumstances described above, the present inventors examined whether early detection of colorectal cancer could be performed by measuring the amount of VEGF in feces of colorectal cancer patients. That is, an object of the present invention is to provide an early inspection method as a screening for colorectal cancer and monitoring S.
本発明者らは、 大腸癌患者の糞便を試料として用い、 糞便中の V EG F濃度を 測定することにより、 80 %以上の陽性率で大腸癌の検査ができることを見い出 し、 本発明を完成させたのである。 The present inventors have found that 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.
即ち、 本発明は、 ヒ ト糞便中の血管内皮細胞増殖因子 Z血管透過性因子 (VEGF) の量を測定することを特徴とする大腸癌の検査方法、 及び血管内皮 細胞増殖因子 Z血管透過性因子 (VEGF) と特異的に反応する物質を含むこと を特徴とする大腸癌検査薬を提供するものである。 That is, 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).
以下、 本発明について詳説する。 Hereinafter, the present invention will be described in detail.
血清中の VEGF濃度の測定には、 VEGFと特異的に反応する物質、 例えば 抗 V E G F抗体又は V E G F受容体等を用いる公知の標識免疫測定法が適してお り、 本発明においても好ましい方法である。 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. .
例えば、 抗 VEGF抗体や VEGF受容体等を用いる際には、 標識として放 射性同位元素、 酵素、 発光物質、 蛍光物質等を用いる標識免疫測定法等や、 抗 VEGF抗体や VEGF受容体等を結合した赤血球、 ラテックス、 金属粒子、 金 属ゲル、 パクテリオファージ等を用いる凝集反応やクロマト法等が適用される。 特に、 臨床で応用される場合は、 酵素免疫測定法が好適であり、 また実際に臨 床で酵素免疫測定法は広く用いられている。 酵素免疫測定法 (E I A法) は、 酵 素活性を指標として抗原抗体反応を追跡し、 これから抗原又は抗体の量を測定す る方法であり、 その詳細は、 例えば北川等による 「酵素免疫測定法 No.31蛋白質 核酸酵素別冊 1987年」 に明らかにされている。 この測定法は、 測定対象、 標識
物質、 抗原抗体反応の形式、 結合体 Z遊離体の分離方法 (B/ F分離法) 等の違 いにより、 競合法、 非競合法、 ホモジニァス法、 ヘテロジニアス法等に分類され ている力 いずれも本発明に適用することができる。 For example, when using an anti-VEGF antibody or a VEGF receptor, 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. In particular, the enzyme immunoassay is suitable for clinical application, and in practice, the enzyme immunoassay is widely used. Enzyme-linked immunosorbent assay (EIA) 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. For details, refer to Enzyme-linked immunosorbent assay by Kitagawa et al. No.31 Protein Nucleic Acid Enzyme Separate Volume 1987 ". This measurement method is based on Forces classified as competitive, non-competitive, homogenous, heterogeneous, etc. due to differences in substance, type of antigen-antibody reaction, method of separating conjugate Z free form (B / F separation method), etc. Can also be applied to the present invention.
本発明の大腸癌検査薬は、 前記の V EG Fと特異的に反応する物質、 例えば抗 The colorectal cancer test agent of the present invention is a substance that specifically reacts with the VEGF,
V EG F抗体又は V E G F受容体を検査薬の主要成分として含むことを特徴とす るものであり、 その他、 必要に応じて、 大腸癌検査薬において許容される担体又 は賦形剤等を適宜配合することができる。 図面の簡単な説明 It is characterized by containing VEGF antibody or VEGF receptor as a main component of the test drug.In addition, if necessary, carriers or excipients acceptable for colorectal cancer test drugs are appropriately used. Can be blended. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 大腸癌患者における糞便中 V E G F量の測定結果を示す。 FIG. 1 shows the measurement results of the amount of stool VEGF in colorectal cancer patients.
図 2は、 図 1に示した大腸癌患者を早期癌患者と進行癌患者に区別して糞便中 Figure 2 shows that the colorectal cancer patients shown in Figure 1 are separated into early cancer patients and advanced cancer patients
V EG F量の測定結果を示したものである。 It shows the measurement results of VEGF amount.
図 3は、 大腸癌患者の手術前後における糞便中 V E G F量の測定結果を示す。 発明を実施するための最良の形態 FIG. 3 shows the results of measuring the amount of fecal VEGF before and after surgery for colorectal cancer patients. BEST MODE FOR CARRYING OUT THE INVENTION
実施例 1 Example 1
( 1 ) 抗 V E G Fポリクロ一ナル抗体の作製 (1) Preparation of anti-VEGF polyclonal antibody
単離したヒ ト V E G F c D N Aをグル夕チオン S-トランスフェラ一ゼ (GST) との融合蛋白 (GST— VEGF) として大腸菌で産生させ、 得られ た蛋白を抗原として常法に従ってゥサギ抗 V E G Fポリクロ一ナル抗体を作製し た。 抗体価の上昇したゥサギの血清を分離し、 陰イオン交換カラムクロマ卜グラ フィ一によりゥサギ抗 V EG Fポリクローナル抗体の I gG画分を得た。 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. 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.
(2) 杭 V EG Fポリクローナル抗体の酵素標識 (2) Enzyme labeling of pile VEG F polyclonal antibody
I gG画分の一部をペプシンで消化して F(ab')2を調製後、 ヒンジ法によりべ ルォキシダ一ゼ (西洋わさび) と結合させ、 ペルォキシダ一ゼ標識したゥサギ抗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.
V E G Fポリクローナル抗体を得た。 A VEGF polyclonal antibody was obtained.
( 3 ) 酵素免疫測定法による大腸癌患者糞便中 V E G F量の測定 (3) Measurement of the amount of VEGF in feces of colorectal cancer patients by enzyme-linked immunosorbent assay
大腸癌患者及び健常者の糞便中 V EG F量の測定を、 以下に示すように、 酵素 免疫測定法により行った。
即ち、 ( 1 ) で得た抗 V EG Fポリクローナル抗体 (5 /Li g/m l ) を 100 U 1 Zwellずつ 96穴プレー卜にまき 4°Cでー晚放置した後、 0. 1 %ゥシ 血清アルブミン (B SA) を含むリン酸緩衝化生理食塩水 (P H = 7. 4) (PBS) で 4回洗浄した。 1 %BSA、 0. 1 M塩化ナトリウム及び 0. 1 % アジ化ナトリウムを含む 0. 1 M炭酸ナトリウム緩衝液 (pH= 6. 5) でブ ロッキング (37°Cで 4時間) した後、 1 %BSA、 0. 4%ゼラチン、 1 mM 塩化マグネシウム、 2 OmMエチレンジァミン四酢酸ナトリウム、 0. 1M塩化 ナトリゥム及び 0. 1 %アジ化ナ卜リゥムを含む 5 OmMリン酸ナ卜リウム緩衝 液 (PH= 7. 0) (検体希釈液) で 20倍に希釈した糞便抽出液あるいは同 検体希釈液に溶解した標準 V E G Fを入れ室温で 1時間放置した。 糞便抽出液 は、 糞便 l g (湿重量) に対して PBS 2m 1を加えてホモジナイズし、 穴径 0. 45/imのフィルタ一でろ過することにより調製した。 0. 1 %BSAを含 む P B Sで 6回洗浄後、 (2) で得たペルォキシダーゼ標識抗 V EG Fポリク 口一ナル抗体を 100 1 Zweilずつ入れ、 室温で 1時間反応させた。 再度、 0. 1 %B S Aを含む PB Sで 8回洗浄後、 0. 125 % (w/v) オルトフエニレ ンジァミン及び 0. 015%過酸化水素を含む 0. 2M卜リス (ヒドロキシメチ ル) ァミノメタン一クェン酸緩衝液 (pH=5. 2) を l OOw l Zwellずつ入 れ、 室温で 30分間反応させた。 2 N硫酸を 100 1 /wellずつ入れ、 反応を 停止させた後、 650 nmの吸光度に対する 490 nmの吸光度をブレー卜リー ^ - ( -Vraax, Molecular Devices社製) で測定した。 The measurement of the amount of VEGF in feces of colorectal cancer patients and healthy subjects was performed by an enzyme immunoassay as shown below. That is, the anti-VEGF polyclonal antibody (5 / Li g / ml) obtained in (1) was seeded on a 96-well plate in 100 U 1 Zwell at −4 ° C. and allowed to stand at 0.1 ° C. The plate was washed four times with phosphate-buffered saline (PH = 7.4) (PBS) containing serum albumin (BSA). After blocking with 0.1 M sodium carbonate buffer (pH = 6.5) containing 1% BSA, 0.1 M sodium chloride and 0.1% sodium azide (4 hours at 37 ° C), 5 OmM sodium phosphate buffer containing 5% BSA, 0.4% gelatin, 1 mM magnesium chloride, 2 OmM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride and 0.1% sodium azide (PH = 7.0) Fecal extract diluted 20-fold with (sample diluent) or standard VEGF dissolved in the same sample diluent was added and left at room temperature for 1 hour. 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. A citrate buffer solution (pH = 5.2) was added to each well, and the reaction was carried out at room temperature for 30 minutes. After stopping the reaction by adding 2 N sulfuric acid at a rate of 100 1 / well, the absorbance at 490 nm relative to the absorbance at 650 nm was measured using a Bratley ^-(-Vraax, manufactured by Molecular Devices).
大腸癌患者 26例及び健常者 10例の糞便抽出液中 V E G F量の測定結果を表 1及び図 1に ¾ ^した。 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.
表 1 大腸癌患者及び健常者の糞便中 V E G F量 Table 1 Fecal VEGF levels in colorectal cancer patients and healthy subjects
大腸癌患者 V EG F量 (平均 ±標準偏差値) は、 1 1 16. 9± 1566. 3
71111でぁったのに対し、 健常者は66. 3±36. 3 p gZmlであり、 健常 者と比較して大腸癌患者は有意に高値を示した。 また、 健常者 V EG F量の平均 値 +2倍の標準偏差値の値 (138. 9 pg/ml) をカッ トオフ値として陽性率 を算出すると、 大腸癌患者で 80. 8%であった。 既存の大腸癌検査である免疫 潜血反応検査や免疫へモグロビン検査の陽性率は、 大腸癌患者で 50〜 60 %で あることより、 本方法による大腸癌検査方法は非常に有用であるといえる。 前記の大腸癌患者を早期癌患者と進行癌患者に区別して糞便中 V E G F量を比 較したものが表 2及び図 2である。 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.
表 2 早期癌患者及び進行癌患者における糞便中 V E G F量 Table 2 Fecal VEGF levels in early and advanced cancer patients
早期癌患者 VEGF量 (平均 ±標準偏差値) は、 363. 4± 339. 7 pg /mlであり、 健常者に比べ有意に高値であった。 また、 進行癌患者 VEGF量 (平均土標準偏差値) は、 1451. 8± 1781. 7 P gZmlであり、 健常者 及び早期癌患者に比べ有意に高値であった。 健常者 V E G F量の平均値 + 2倍の 標準偏差値の値 ( 138. 9P g/ml) を力ッ 卜オフ値とした場合、 早期癌患者 の陽性率は 62. 5%、 進行癌患者の陽性率は 88. 9%であり、 既存の大腸癌 検査である免疫潜血反応検査や免疫へモグロビン検査に比べ、 本方法は早期大腸 癌検査方法にも非常に有用であるといえる。 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. When 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.
同一の大腸癌患者 5例において、 手術前後における糞便中 VEGF量を比較し たものが表 3及び図 3である。
表 3 手術前後における糞便中 V EG F量 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
V EG F量 (平均 ±標準偏差値) は、 手術前では 32 1 5. 2 ± 1 336. 9 P g/mlであり、 手術後では 535. 7± 1 24. 8 p gZmlであり、 手術前に 比べ、 手術後は有意に低値であった。 これは、 手術により大腸癌部位を取り除い たことにより、 糞便中 VEGF量が減少したことを示しており、 糞便中 VEGF 量の変動が手術後の経過を知る上で有用であることを示している。 産業上の利用の可能性 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
糞便中の V E G F量を測定することにより、 大腸癌の検査を行うという本発明 の検査方法による診断は、 大腸癌の早期発見、 大腸癌の病態の進行や治療効果の 判定、 再発の予測等に広く利用することができる優れたものであり、 また、 糞便 を用いることから、 これまで用いられてきた検査方法に加えて、 新たな検査方法 としても利用することができるものである。 更に、 本発明による大腸癌の検査 は、 既存の腫瘍マーカーや便の潜血反応を用いた場合よりも非常に高率に大腸癌 患者を発見することができるため、 臨床上非常に有用なものである。
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.
Claims
1 . ヒ卜糞便中の血管内皮細胞増殖因子 血管透過性因子の量を測定することを 特徴とする大腸癌の検査方法。 1. A method for examining colorectal cancer, comprising measuring the amount of vascular endothelial cell growth factor and vascular permeability factor in human feces.
2 . 血管内皮細胞増殖因子 Z血管透過性因子と特異的に反応する物質を含むこと を特徴とする大腸癌検査薬。 2. Vascular endothelial cell growth factor A colorectal cancer test agent containing a substance that specifically reacts with Z vascular permeability factor.
3 . 血管内皮細胞増殖因子 Z血管透過性因子と特異的に反応する物質が、 抗血管 内皮細胞増殖因子 Z血管透過性因子抗体又は血管内皮細胞増殖因子 血管透過性 因子受容体である請求の範囲第 2項記載の大腸癌検査薬。
3. The substance specifically reacting with vascular endothelial cell growth factor Z vascular permeability factor is an anti-vascular endothelial cell growth factor Z vascular permeability factor antibody or vascular endothelial cell growth factor vascular permeability factor receptor. 2. The colorectal cancer test agent according to item 2.
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