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WO2001079265A1 - Anticorps anti-facteur de croissance du keratinocyte humain - Google Patents

Anticorps anti-facteur de croissance du keratinocyte humain Download PDF

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Publication number
WO2001079265A1
WO2001079265A1 PCT/JP2000/008693 JP0008693W WO0179265A1 WO 2001079265 A1 WO2001079265 A1 WO 2001079265A1 JP 0008693 W JP0008693 W JP 0008693W WO 0179265 A1 WO0179265 A1 WO 0179265A1
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WO
WIPO (PCT)
Prior art keywords
kgf
antibody
peptide
human kgf
present
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2000/008693
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English (en)
Japanese (ja)
Inventor
Takehiko Koji
Mitsuru Nakamura
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nichirei Corp
Original Assignee
Nichirei Corp
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 Nichirei Corp filed Critical Nichirei Corp
Priority to AU17347/01A priority Critical patent/AU1734701A/en
Publication of WO2001079265A1 publication Critical patent/WO2001079265A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to an antibody that specifically binds to KGF (Keratinocyte Growth Factor) and its use.
  • KGF Keratinocyte Growth Factor
  • KGF is a cell growth factor classified as FGF 7 in the fibroblast growth factor (FGF) family 1 (FGF 1-9) due to the characteristics of the sequence of the mRNA that encodes it. That is, FGF7 is produced by fibroblasts and the like, and has an activity of specifically acting on epithelial cells to promote their proliferation. In addition, KGF may contribute not only to the development and growth of normal epithelial cells but also to the growth of tumors in various organs derived from epithelial tissue. Therefore, the antibody against KGF according to the present invention contributes to the diagnosis of various tumors and solid cancers by examining the presence or absence of KGF in various organs, tissues and cells.
  • FGF fibroblast growth factor
  • FIG. 2 is a drawing substitute photograph obtained by staining a sliced sample of cholesteatoma tissue embedded in formalin and embedded in paraffin with a polyclonal antibody.
  • FGF 7 FGF 7
  • the present invention has been made for the purpose of producing an antibody that can be stained in distinction from a cell growth factor belonging to another KGF family other than KGF).
  • the present invention has been made in order to achieve the above-mentioned object, and as a result of examination from various fields, has focused on an immunogen.
  • amino acids that are as short as possible, especially from an industrial standpoint of ease of production, It must have an acid sequence and must have good binding to the carrier protein for that purpose. Furthermore, it can smoothly immunize animals and efficiently produce antiserum or antibodies. From the standpoint of necessity, intensive screening was performed for peptides that could satisfy each of these requirements. As a result, we succeeded for the first time to screen the target peptide.
  • the rabbit was immunized with the complex, and it was confirmed for the first time that the obtained antiserum specifically bound to KGF.
  • the present invention has been completed as a result of further studies based on useful new findings.
  • the present invention has succeeded in creating an antibody that can analyze and measure the presence or absence and the amount of KGF at the level of the follicle follicles.
  • the antibody according to the present invention is an antibody that specifically recognizes KGF, and is a novel antibody that has hitherto been unknown.
  • human KGF is sometimes simply referred to as KGF, and immunoassay includes not only detection of KGF, confirmation and analysis of its presence, but also qualitative or quantitative analysis of KGF. Staining, immunoprecipitation and all other immunoassays are included.
  • the present invention provides numerous and extremely novel methods such as a novel immunogen comprising a specific peptide, a novel antibody obtained using the same, specific binding to KGF possessed by the novel antibody, and construction of a new assay system using the same.
  • Examples of the immunogen used for preparing the novel antibody according to the present invention include a peptide (1) having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing (FIG. 1), and / or Or a peptide (2) having the amino acid sequence shown in SEQ ID NO: 2 (FIG. 2).
  • These peptides (1) and (2) are novel as immunogens for preparing anti-KGF antibodies, but are appropriately produced by a conventionally known method such as using a peptide synthesizer.
  • Immunization may be performed according to a conventional method.
  • the peptide is conjugated to BSA, KLH thysiglobulin and other known carrier proteins, and this conjugate (complex) is used as an immunogen.
  • Freund's complete adjuvant or incomplete is used. This is done by injecting the animals as many times as necessary with various adjuvants such as adjuvants.
  • a mouse, a heron, a rat, a sheep, and the like can be used, and the injection can be performed according to a known method.
  • blood is collected from the injected animal to obtain antiserum.
  • any conventionally known method may be used.
  • protein purification methods such as affinity chromatography, ammonium sulfate salting-out, and ion exchange chromatography are suitably employed.
  • Antibodies prepared in this manner have extremely high specificity for human KGF, and have the property of specifically recognizing and reacting with KGF and binding to it. Immunization of human KGF can be performed.
  • immunoassays include radioimmunoassay (RIA), enzyme immunoassay (EIA :), fluorescence immunoassay, chemiluminescence immunoassay, immunohistochemical staining, immunoprecipitation, etc.
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • fluorescence immunoassay fluorescence immunoassay
  • chemiluminescence immunoassay chemiluminescence immunoassay
  • immunohistochemical staining immunoprecipitation, etc.
  • This antibody may be used as a first antibody and a second antibody that is labeled with Z or an enzyme.
  • KGF tissue sections
  • Anti-KGF antibody according to the present invention first antibody
  • KGF an enzyme is bound to the first antibody
  • KGF or a cell producing or containing KGF
  • staining is performed by the following operations: 1) reaction with the labeled first antibody, 2) reaction with the enzyme reagent, and 3) color development.
  • the reaction with the first antibody unlabeled
  • the second antibody labeleled
  • EI 1 an antibody that binds to a specific substance (KGF) (the anti-KGF antibody according to the present invention: the first antibody) is used in a micro-mouth plate or 2) Next, plate or beads were blocked with protein such as albumin, 3) A solution containing a specific substance (KGF) was added and reacted for a certain time, and 4) Enzyme-labeled An antibody that binds to a specific substance (enzyme-labeled anti-KGF antibody: second antibody) is added and allowed to react for a certain period of time. 5) An enzyme substrate is added, and the degree of color development is measured with a spectrophotometer. EIA will be implemented.
  • an antibody obtained using the peptide (1) as an immunogen is obtained as the first antibody, and a peptide (2) is obtained as the immunogen.
  • the second antibody can be used as the second antibody, and vice versa.
  • an avidin-biotin system in addition to using an enzyme-labeled antibody, an avidin-biotin system is used, an antibody labeled with biotin is used instead of the enzyme-labeled antibody, and an avidin-labeled enzyme is used as the enzyme.
  • the system may be assembled.
  • Avidin includes egg white-derived avidin, microorganism-derived avidin (For example, streptavidin) and the like can be used.
  • Labeling enzymes include enzymes commonly used in enzyme immunoassays (EIA), such as horseradish peroxidase (HRP), sea urchin small intestine alkaline phosphatase, yeast galactosidase, yeast urea, and glucose oxidase. Is used as appropriate, and a chromogenic substrate that is compatible with these enzymes and is commonly used in EIA is used.
  • EIA enzyme immunoassays
  • the chromogenic substrate in the case for example of HRP, 3, 3 ', 5 , 5' - tetramethyl-benzylidene Jin (TMBZ), TMBZ 'HC1, TMB Z ⁇ PS N ABTS, o- phenylene Renjiamin, p- hydroxyphenyl
  • p-ditrophenyl phosphate, 4-methylphenyl phenyl phosphate, etc. are used in the case of alkaline phosphatase, and o-in the case of ⁇ -galactosidase, in the case of alkaline phosphatase.
  • Nitrophenyl mono-D-galactovyranoside, 4-methylumbelliferyl /? — D-galactovyranoside, etc. are used.
  • the present invention also provides a novel immunogen as described above, a novel antibody obtained using the same, a method for detecting and measuring KGF using the same, and a method for staining, detecting and measuring cells expressing and producing the same.
  • examples of the present invention will be described.
  • An immunogen was prepared as described below, and a polyclonal antibody was prepared using the immunogen.
  • the peptide (2) having the amino acid sequence shown in SEQ ID NO: 2 was synthesized by a solid phase method using an automatic peptide synthesizer (manufactured by Beckman), and then purified by high-speed chromatography.
  • the amino acid composition of the obtained peptide was hydrolyzed with 1N hydrochloric acid at 120 ° C. overnight, and then measured using an amino acid analyzer. It was confirmed that the amino acid composition agreed with the theoretical value.
  • Peptide (1) was prepared in the same manner.
  • a carrier-protein was bound to this peptide as follows. That is, 10 mg of antigen-based peptide and 3 mg of dysthyroglobulin are dissolved in 1 ml of distilled water, and 3 Omg of 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride is added thereto, and light is shielded. After overnight reaction at room temperature, the mixture was dialyzed sufficiently against distilled water. It was confirmed by SDS-PAGE whether or not the antigen-based peptide bound to the carrier protein.
  • the resulting carrier-protein and peptide conjugate (corresponding to 100 ⁇ g of peptide) were injected into the back of a egret with Freund's complete adjuvant. Thereafter, each week, 100 ⁇ g of antigen-protein equivalent to bepidine was mixed with Freund's incomplete adjuvant and immunized a total of eight times in the same manner. On day 10 after the last immunization, whole blood was collected to separate antiserum. The IgG fraction was isolated from this antiserum by affinity chromatography using protein A. That is, 2 ml of protein A Sepharose CL4B (Pharmacia) was packed in a column, and equilibrated with 10 ml of a 1.5 M glycine solution (pH 8.7).
  • the paraffin-embedded cholesteatoma tissue block was sliced, attached to a slide glass, and fixed. Thereafter, deparaffinization treatment and deperoxidase treatment were performed.
  • the antibody against peptide (2) produced in Example 1 was labeled with peroxidase according to a commonly used labeling method. After adding the peroxidase-labeled anti-KGF polyclonal antibody to the above and reacting for 5 minutes, wash well, react with a substrate solution (diaminobenzidine, hydrogen peroxide) added dropwise and purified water. After washing and encapsulation, they were examined under a microscope.
  • epithelial cells (keratinocytes) were stained as shown in Fig. 3 (a photograph substituted for a drawing) (a positive image is shown at the site of the arrow).
  • this staining was not observed with the anti-KGF polyclonal antibody that had previously been reacted with the peptide as the immunogen.
  • KGF is involved in the proliferation of epithelial cells constituting cholesteatoma. Similar results were obtained when an antibody against peptide (1) was used.
  • the invention's effect has created an anti-KGF antibody.
  • This antibody is a novel antibody that has the property of specifically binding to KGF, and has high KGF-recognition properties. The use of this antibody has made it possible for the first time to perform an excellent immunoassay for KGF. .
  • This assay yields reproducible results and is very useful for diagnosing cancer and other diseases.
  • Examples of the assay or detection method using the antibody include EIA, RIA, immunohistochemical staining, immunoprecipitation, and Western blotting. It has become possible to accurately diagnose cancer and other diseases such as prostate cancer.
  • the present invention has made it possible for the first time to clarify the distribution of KGF-expressing cells in tumor tissue. With such a method, it is possible to analyze the presence or absence in various tumor tissues and the like, and the present invention plays an important role also in the field of pathology.

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

Abstract

L'invention concerne un anticorps polyclonal se liant spécifiquement à un facteur de croissance du kératinocyte humain. En raison d'une spécificité élevée vis-à-vis du facteur de croissance du kératinocyte, cet anticorps permet la détection et la détermination précises et rapides du facteur de croissance du kératinocyte dans divers organes, tissus et cellules. Ainsi, on peut l'utiliser pour détecter et doser sur le plan immunologique diverses tumeurs telles que le cholestéatome.
PCT/JP2000/008693 2000-04-17 2000-12-08 Anticorps anti-facteur de croissance du keratinocyte humain Ceased WO2001079265A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU17347/01A AU1734701A (en) 2000-04-17 2000-12-08 Antibody against human kgf

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-115167 2000-04-17
JP2000115167A JP2001302691A (ja) 2000-04-17 2000-04-17 ヒトｋｇｆに対する抗体

Publications (1)

Publication Number Publication Date
WO2001079265A1 true WO2001079265A1 (fr) 2001-10-25

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PCT/JP2000/008693 Ceased WO2001079265A1 (fr) 2000-04-17 2000-12-08 Anticorps anti-facteur de croissance du keratinocyte humain

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AU (1) AU1734701A (fr)
WO (1) WO2001079265A1 (fr)

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
US8278816B2 (en) 2004-09-30 2012-10-02 Global Tungsten & Powders Corp. High CRI electroluminescent lamp

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990008771A1 (fr) * 1989-01-31 1990-08-09 Rubin Jeffrey S Adn codant un facteur de croissance specifique contre des cellules epitheliales

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990008771A1 (fr) * 1989-01-31 1990-08-09 Rubin Jeffrey S Adn codant un facteur de croissance specifique contre des cellules epitheliales

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALARID ELAINE T. ET AL.: "Keratinocyte growth factor functions in epithelial induction during seminal vesicle development", PROC. NATL. ACAD. SCI. USA, vol. 91, no. 3, February 1994 (1994-02-01), pages 1074 - 1078, XP002937857 *
BAJAJ-ELLIOTT MONA ET AL.: "Interaction between stromal cell-derived keratinocyte growth factor and epithelial transforming growth factor in immune-mediated crypt cell hyperplasia", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 102, no. 8, 15 October 1998 (1998-10-15), pages 1473 - 1480, XP002937856 *
FINCH PAUL W. ET AL.: "Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth", SCIENCE, vol. 245, 18 August 1989 (1989-08-18), pages 752 - 754, XP002937855 *
PANOS RALPH J. ET AL.: "Keratinocyte growth factor and hepatocyte growth factor / scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 92, no. 2, August 1993 (1993-08-01), pages 969 - 977, XP002937858 *

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AU1734701A (en) 2001-10-30

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