JPS6067860A - Diagnosing/examination agent - Google Patents
Diagnosing/examination agentInfo
- Publication number
- JPS6067860A JPS6067860A JP17677483A JP17677483A JPS6067860A JP S6067860 A JPS6067860 A JP S6067860A JP 17677483 A JP17677483 A JP 17677483A JP 17677483 A JP17677483 A JP 17677483A JP S6067860 A JPS6067860 A JP S6067860A
- Authority
- JP
- Japan
- Prior art keywords
- afp
- antibody
- monoclonal antibody
- present
- cell line
- 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.)
- Pending
Links
- 210000000170 cell membrane Anatomy 0.000 claims description 4
- 102000013529 alpha-Fetoproteins Human genes 0.000 claims 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 claims 2
- 239000000439 tumor marker Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 19
- 238000000034 method Methods 0.000 description 19
- 239000003795 chemical substances by application Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 239000012528 membrane Substances 0.000 description 9
- 210000004408 hybridoma Anatomy 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 208000014018 liver neoplasm Diseases 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 239000012228 culture supernatant Substances 0.000 description 4
- 201000007270 liver cancer Diseases 0.000 description 4
- 102000013415 peroxidase activity proteins Human genes 0.000 description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 101000848653 Homo sapiens Tripartite motif-containing protein 26 Proteins 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 102000046101 human AFP Human genes 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000512259 Ascophyllum nodosum Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000283986 Lepus Species 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010049190 Red blood cell agglutination Diseases 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Biotechnology (AREA)
- Hospice & Palliative Care (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
(57)【要約】本公報は電子出願前の出願データであるた
め要約のデータは記録されません。(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.
Description
【発明の詳細な説明】
ン（’Ａ Ｆ Ｐ ）及び胎児性抗原（ＣＢＡＪ力玉、
よく知られている。ＣＥＡは、消化器系のガン細胞の膜
表面に存在するとされている。他方、ＡＦＰは、多くの
肝ガン及びセルラインで産生ずることが認められている
が、細胞質に存在することが確かめられているにすぎず
、膜表面に存在するか否かは必ずしも明らかではない。[Detailed description of the invention]
well known. CEA is said to exist on the membrane surface of cancer cells in the digestive system. On the other hand, although it has been recognized that AFP is produced in many liver cancers and cell lines, it has only been confirmed that it exists in the cytoplasm, and it is not necessarily clear whether it exists on the membrane surface or not. .
本発明者らは、診断・検査薬として好適なＡＦＰに対す
るモノクローナル抗体を見出すべく、種々検討を行ない
、膜表面に存在するＡＦＰ角１に朱七ル坊箋すＡモノク
ローナル抗体を見出し、本発明に到達した。The present inventors conducted various studies in order to find a monoclonal antibody against AFP suitable as a diagnostic/testing agent, and discovered a monoclonal antibody A that binds to the AFP angle 1 present on the membrane surface. Reached.
すなわち、本発明の要旨は、細胞膜表面に存在するＡ’
ＦＰを認識するモノクローナル抗体を使用して分る診断
・検査薬にある。That is, the gist of the present invention is that A' present on the cell membrane surface
It is a diagnostic/testing agent that uses a monoclonal antibody that recognizes FP.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
まず、本発明において用いられるモノクローナル抗体は
、次のような方法で得られる。First, the monoclonal antibody used in the present invention can be obtained by the following method.
すなわち、まず、ヒト胎盤由来のＡＦＰ’ｉ−たとえば
Ｂ Ａ Ｌ Ｂ ／ Ｃマウス等に免疫した後、肺臓ヲ
摘出し、ポリエチレングリコールＹ用い。That is, first, after immunizing AFP'i derived from human placenta, such as BAL B/C mice, the lungs were removed and polyethylene glycol Y was used.
Ｐ３−ｖｌ等のマウスミエローマ細胞と融合し、常法に
よりハイブリドーマを得ろ。そしてハイプリドーマ上清
よりモノクローナル抗体を得る。Fuse with mouse myeloma cells such as P3-vl and obtain hybridomas by standard methods. Monoclonal antibodies are then obtained from the hybridoma supernatant.
つぎに、常法により得られたこれらの抗体を用いて、肝
ガンセルライン（たとえば、ＰＬＯ。Next, using these antibodies obtained by conventional methods, a liver cancer cell line (eg, PLO.
ＫＮ、ＮｕＥ）７免疫組織化学的に染色し、陽性を示す
抗体を選択する。この検出は、アピオシンコピオチン化
ワザビペルオキシダーゼコンブし・マウス（ＡＢＣ）キ
ットを用い、ホースラデイシュ・ペルオキシダーゼの酵
素活性により。KN, NuE) 7 immunohistochemically stained and select antibodies showing positive. This detection is based on the enzymatic activity of horseradish peroxidase using the apiocincopyotinated horseradish peroxidase kelp/mouse (ABC) kit.
ジアミノベンジジンを基質として用いて行なわれる。It is carried out using diaminobenzidine as a substrate.
陽性を示す抗体を大量に入手するには、この選択された
抗体ヶ産生するハイブリドーマをＢＡＴ、Ｂ１０ マウ
ス腹腔内に注射し、増殖させ、腹水を採取することによ
り行なうことができる。In order to obtain a large amount of antibodies that show positive results, hybridomas producing the selected antibodies can be intraperitoneally injected into BAT, B10 mice, allowed to proliferate, and ascites fluid collected.
また、上記ハイプリドーマを培養タンクで大量培養する
方法によることもできる。Alternatively, the above hybridoma may be cultured in large quantities in a culture tank.
このようにして得られるモノクローナル抗体は次のよう
な性質を有する。The monoclonal antibody thus obtained has the following properties.
ｊ） Ｉ４０ラベル化した本抗体を用いて、胎盤由来Ａ
ＦＰｌ：Ｊ″−ＰＬＣ肝ガンセルラインの膜に対する結
合を阻害するか否かをみると、ＡＦＰが抗体とセルライ
ン膜との結合？競合的に阻害することがわかる。j) Using this I40-labeled antibody, placenta-derived A
When examining whether binding to the membrane of FPl:J''-PLC liver cancer cell line is inhibited, it is found that AFP competitively inhibits the binding of antibodies to the cell line membrane.
ＩＩ）ｐＬｃセルラインを１４０−ロイシンでラベルし
、その膜成分’＆ ” ） ＩＪ ）ンＸ”′で可溶化
し、本抗体との結合をみろと、明らかな結合性が認めら
れろ。II) Label the pLc cell line with 140-leucine, solubilize it with its membrane component '&'')IJ)' and examine its binding to the antibody. Clear binding is observed.
また、培養土清中の分泌蛋白にも結合性が確認される。In addition, binding to secreted proteins in the culture medium was also confirmed.
町 ｐＬａセルラインの可溶化膜成分ならびに培養土清
中の抗体反応物を、オファレル（０’Ｆａｒｒｅｌ ）
うの方法に準じて二次元電気泳動を行なうと、膜由来、
培養上清由来のいずれの結合物もＡ Ｆ−Ｐの性質（等
電１点、分子量）火有することがわかる。The solubilized membrane components of the Machi pLa cell line and antibody reactants in the culture medium were collected using O'Farrel.
When two-dimensional electrophoresis is performed according to the method described above, membrane-derived,
It can be seen that all the bound substances derived from the culture supernatant have the properties of AFP (isoelectricity 1 point, molecular weight).
ＩＶＪＰＬＯセルラインの膜分画をトリプシン、プロテ
アーゼ、チモトリプシン等の蛋白質分解酵素で消化する
と、本抗体との結合性は低下又は低下傾向を示す。When the membrane fraction of the IVJPLO cell line is digested with a proteolytic enzyme such as trypsin, protease, or thymotrypsin, the binding property with the present antibody decreases or tends to decrease.
また、” ｌ−ＩＪ ）ンｘ ”処理で低下傾向を示し
、リパーゼ処理により、その結合性は増加する。In addition, the binding property showed a decreasing tendency with the "l-IJ)" treatment, and the binding property increased with the lipase treatment.
本発明に係る診断・検査薬は上記モノクローナル抗体を
使用してなる。The diagnostic/testing agent according to the present invention uses the above monoclonal antibody.
この診断・検査薬は、ＡＦＰの免疫学的な測定に広く用
いることができる。たとえば、寒天ゲル内二重拡散法〔
Ｍ、０法〕、−元免疫拡散法、免疫電気泳動法（Ｌａｕ
ｒｅｌｌ法〕、ラテックス凝集反応、赤血球凝集反応、
ラジオ・イムノアセイ／ｆ：ＣＲＩ Ａ法〕、酵素イム
ノアセイ法等が挙げられる。This diagnostic/testing agent can be widely used for immunological measurement of AFP. For example, double diffusion method in agar gel [
M, 0 method], - original immunodiffusion method, immunoelectrophoresis method (Lau
rell method], latex agglutination reaction, red blood cell agglutination reaction,
Radio immunoassay/f: CRI A method], enzyme immunoassay method, and the like.
これらの方法夕月いて、血清中のＡＦＰ含有量を測定す
ることができるが、本発明に係る診断・検査薬は、細胞
膜表面に存在するＡＦＰを認識するモノクローナル抗体
を用いるので、特に細胞診に好適である。Although these methods can measure the AFP content in serum, the diagnostic/testing agent according to the present invention uses a monoclonal antibody that recognizes AFP present on the cell membrane surface, so it is particularly difficult to use for cytodiagnosis. suitable.
すなわち、たとえば、遊離９７７４球におけろ検出、組
織切片における検出等に適用される。That is, it is applied to, for example, detection of free 9774 spheres, detection of tissue sections, and the like.
これらの場合においても、検出法自体は常法を用いるこ
とができ、たとえば、ＥＬ：５ＳＡ法、ＲＩＡ法、螢光
抗体法、酵素抗体法、ビオチン−アビジン−ホースラデ
ィツシュ・ペルオキシダーゼ法等が挙げられる。Even in these cases, conventional detection methods can be used, such as the EL:5SA method, RIA method, fluorescent antibody method, enzyme antibody method, biotin-avidin-horseradish peroxidase method, etc. It will be done.
たとえば、−例としてＥＬＩＳＡ法による場合を説明す
ると、まず標的細胞ケプレートに固定シ、各穴（ｗｅｌ
ｌ ）にモノクローナル抗体を加え、室温でインキュベ
ートした後、緩衝液で洗浄スる。次に−ベルオキシダー
ゼで標識した抗マウス■ｇを加え、室温でインキュベー
トシ、洗浄後、ｏ−フェニルジアミンを加えて反応させ
た後、ＮａＦを加えて反応を停止させ、顕微鏡下又はス
ペクトロフォトメーターで判定する。For example, to explain the case using the ELISA method, first, target cells are immobilized on a plate, each well is
Add the monoclonal antibody to 1), incubate at room temperature, and then wash with buffer. Next, add anti-mouse labeled with -peroxidase, incubate at room temperature, wash, add o-phenyldiamine to react, add NaF to stop the reaction, and use a microscope or spectrophotometer. Judge by.
この方法において、酵素標識抗体の代わりに、ペルオキ
シダーゼマウスアンチペルオキシダーゼ（ＰＡＰ Ｊ複
合体を用いて、標的細胞ｔモノクローナル抗体及び抗マ
ウスエｇと反応させた後に、さらにこれ乞反応させ、基
質により発色させることもできる。In this method, peroxidase mouse antiperoxidase (PAP J complex) is used instead of an enzyme-labeled antibody, and after reacting with a target cell t monoclonal antibody and an anti-mouse egg, this is further reacted and colored with a substrate. You can also do it.
また、本発明に係る診断・検査薬は、放射性同位元素（
ＲＩ ）標識化したモノクローナル抗体を用いて、体内
に投与することにより、ＲＩの局在や動態欠γカメラで
測定する、いわゆる″イメージング″にも用いることが
できる。In addition, the diagnostic/testing agent according to the present invention contains a radioisotope (
By administering a monoclonal antibody labeled with RI) into the body, it can also be used for so-called "imaging," which measures the localization and dynamics of RI with a gamma camera.
なお、本発明において用いられるモノクローナル抗体は
、測定に際し、その目的に応じて、常法により標識化す
る（ＲＩ、酵素、螢光団、金属ゾル等〕ことができる。In addition, the monoclonal antibody used in the present invention can be labeled by a conventional method (RI, enzyme, fluorophore, metal sol, etc.) depending on the purpose at the time of measurement.
本発明に係る診断・検査薬は、細胞質のみならず、細胞
膜表面に存在するＡＦＰ’％−認識するモノクローナル
抗体を使用するので、解析能を上げ、診断の精度を向上
させることができ、特に肝ガン等の肝疾患の早期発見に
有用である。Since the diagnostic/testing agent according to the present invention uses a monoclonal antibody that recognizes AFP'% present not only in the cytoplasm but also on the surface of the cell membrane, it is possible to increase the analytical ability and improve the accuracy of diagnosis, especially in the liver. It is useful for early detection of liver diseases such as cancer.
以下、実施例によりさらに本発明の詳細な説明する。Hereinafter, the present invention will be further explained in detail with reference to Examples.
参考例／
（１） マウスモノクローナル抗体の作製：ヒトＡＦＰ
として、胎盤より精製された純度ｑｑ％以上で、免疫化
学的にヒトアルブミン（Ｈ８Ａ）と反応しないもの（■
森永生科研製）を用いた。Reference example/ (1) Production of mouse monoclonal antibody: human AFP
A substance purified from placenta with a purity of qq% or higher and which does not immunochemically react with human albumin (H8A) (■
(manufactured by Morinaga Seikaken) was used.
このヒトＡＦＰを、ＢＡＬＢ／Ｃマウスに１０μｊｉ、
ｔ１回、フロイントの完全アジュバントとともに感作し
、最終免疫は静脈より注射し、３日後に肺臓を摘出し、
ポリエチレングリコール＋り００を用いＰ ３− ＴＩ
’ ／マウスミエローマと融合し、常法によりノ、・イ
ブリＭ−マを作製した。クローニングは限界希釈法を用
い、同法’ｆ１１回以上行なった。なお、大量の抗体は
、ＢＡＬＢ／Ｃ！マウス腹水系により採取した。10μji of this human AFP was administered to BALB/C mice.
The animals were sensitized once with Freund's complete adjuvant, the final immunization was given by intravenous injection, and the lungs were removed 3 days later.
P3-TI using polyethylene glycol + 00
'/mouse myeloma was fused with the mouse myeloma, and the IbriM-ma was produced by a conventional method. Cloning was carried out using the limiting dilution method, which was repeated 11 times or more. In addition, a large amount of antibodies are BALB/C! Collected by mouse ascites system.
抗ＡＦＰ抗体を産生するハイブリドーマは、６回の融合
により、約ｙｏｏクローンが選別された。そのうち、／
／クローンの培養上清の抗体価ＩｇＧ量を表１に示す（
上清欠１０倍濃縮した後、測定。）。About yoo clones of hybridomas producing anti-AFP antibodies were selected through six fusions. One of these days, /
The antibody titer IgG amount of the culture supernatant of /clone is shown in Table 1 (
Measure after removing the supernatant and concentrating it 10 times. ).
表 ／
クローナル抗体
（２）抗体の選択
ａ）セルラインは、−週間以上、イーグルＭ Ｅ Ｍ
７％基本とした培地に代えて培養後、リン酸緩衝液で７
回洗浄し、１）直ちにバイブリドーマ培養上清と反応さ
せる方法、゛及び、１ｉ）ｙ ％パラホルムアルデヒド
固定後、メタノール、Ｈ２Ｏ２溶液で、内因性酵素を不
活化し、抗体と反応させろ方法、７用いた。Table / Clonal Antibodies (2) Antibody Selection a) Cell line: - weeks or more, Eagle MEM
After culturing in 7% basic medium, incubate with 7% phosphate buffer.
1) Immediately react with hybridoma culture supernatant; and 1i) After fixation with y% paraformaldehyde, inactivate the endogenous enzyme with methanol and H2O2 solution and react with antibody. there was.
抗体の検出はベクタスタイン（ｖｅｃｔａｓｔａｉｎ）
社のＡＢＣキットを用い、ホースラディツシュ・ベルオ
キシダーゼの酵素活性により、基質としてジアミノベン
ジジンＹ用いて行なった。Antibody detection is done using Vectastain.
The enzyme activity of horseradish peroxidase was carried out using diaminobenzidine Y as a substrate using the ABC kit manufactured by Kasei.
ｂ）セルラインと維持
ヒト肝ガンのセルラインとして＆ｉ、ＫＮｌ、’ ＰＬ
Ｏ，また、胎児性肝細胞由来のガン細胞株ＮｕＥ４、そ
の他コントロールとして一ヒト胃ガン培養株Ｋ Ａ Ｔ
Ｏ■ｌ、ＭＫＮり３１大腸ガンＣＩの各細胞株を用い
た。なお、培養細胞は、ユＯ％牛脂児血清含有ＲＰＭ工
／Ａ’ＩＯの培養液で、３７．０℃、ｓ％ｃｏ２．９５
％Ａｉｒの条件で維持、増殖させた。b) Cell line and maintenance human liver cancer cell line &i,KNl,' PL
O, also a cancer cell line NuE4 derived from fetal liver cells, and a human gastric cancer culture line KAT as a control.
Cell lines of O, MKN, and 31 colon cancer CI were used. The cultured cells were cultured in RPM/A'IO culture medium containing 0% tallow serum at 37.0°C and s%CO2.95.
The cells were maintained and grown under conditions of % Air.
リ 上記（１）の抗ＡＦＰモノクローナル抗体ヲ含む培
養上清及びその希釈物（２８倍まで〕７肝ガンセルライ
ンＰ、ＬＣ！、ＫＮならびに胎児肝細胞ＮｕＫと免疫組
織化学的に反応させたところ、／９Ｆ／、２に強い反応
ケ認めた。この反応は無固定標本でも、固定、メタノー
ル、Ｈ２Ｏ２処理法のいずれでも同様であった。Culture supernatant containing the anti-AFP monoclonal antibody in (1) above and its dilution (up to 28 times) immunohistochemically reacted with 7 liver cancer cell lines P, LC!, KN and fetal liver cells NuK A strong reaction was observed for , /9F/, 2. This reaction was the same for unfixed specimens, fixation, methanol, and H2O2 treatment methods.
また、コントロールとして市販の抗ＡＦＰモノクローナ
ル抗体（ハイブリチック社製）を、抗体価として１０３
にあわせて反応させたが、他のハイブリドーマクローン
と同様に陽性反応は認められなかった。In addition, as a control, a commercially available anti-AFP monoclonal antibody (manufactured by Hybritic) was used with an antibody titer of 103.
However, as with other hybridoma clones, no positive reaction was observed.
／ ９Ｆ／ｕは表／に示すように抗体価が特に強いもの
でもなく、またＩｇＧ量が多いというものでもないが、
上記のように、他と異なり陽性を示した。/9F/u does not have a particularly strong antibody titer, nor does it have a large amount of IgG, as shown in Table/.
As mentioned above, unlike the others, it was positive.
一方、ＡＦＰの産生が認められないセルラインＭＫＮ
＋ ｈ、ＫＡＴＯ■、Ｃ−／にばこの／９Ｆ１２は反応
しなかった。On the other hand, cell line MKN in which AFP production is not observed.
+h, KATO■, C-/Nibako/9F12 did not react.
実施例／
参考例で得られたモノクローナル抗体を用いＶＯＩ ３
’７．３０乙、７９ｇ／）に準じて行なった。VOI 3 using the monoclonal antibody obtained in Example/Reference Example
'7.30, 79g/).
９乙穴イムノプレートに精製した市販抗ＡＦＰ抗体（ウ
サギ）ヲ夕θμｌづつ添加し１．７７℃でユ時間インキ
ュベートした。つぎに、５％ウシ血清アルブミンで過剰
の結合部位をおおった後、種々の濃度のＡＦＰを含む試
料ｙ、４ｓｏμｇづつ添加し、３７℃にて、一時間反応
させた。θ μl of purified commercially available anti-AFP antibody (rabbit) was added to a 9-well immunoplate and incubated at 1.77° C. for 1 hour. Next, after covering the excess binding site with 5% bovine serum albumin, 4 so μg of samples y containing various concentrations of AFP were added and allowed to react at 37° C. for 1 hour.
生理食塩水で十分に洗浄したのち、上記のペルオキシダ
ーゼ標識抗ＡＦＰモノクローナル抗体を添加し、ス時間
反応？行なった。After thorough washing with physiological saline, the above peroxidase-labeled anti-AFP monoclonal antibody was added and a time reaction was performed. I did it.
競合したペルオキシダーゼの活性を過酸化水素７基質と
して、シーアミノアンチピリンフェノール法で検出し、
図１に示す検量線が得られた０
この検量線７用いて、濃度未知の試料のＡ Ｆ、Ｐ濃度
ン求めることができる。Competing peroxidase activity was detected using the sea aminoantipyrine phenol method using hydrogen peroxide 7 as a substrate.
The calibration curve shown in FIG. 1 was obtained.Using this calibration curve 7, the AF and P concentrations of a sample with unknown concentrations can be determined.
なお、図７において、縦軸ばｓｏｏｎｍにおける吸光度
、横軸はＡＦＰ濃度を表わす。In FIG. 7, the vertical axis represents the absorbance at soonm, and the horizontal axis represents the AFP concentration.
図／は、本発明に係る検査・診断薬を用いて、試料中の
ＡＦｐ濃度を測定するための検量線の例を示す。
出願人 三菱化成工業株式会社
代理人 弁理士 長谷用 −
（ほか７名）
突Ｌｎｊ”のｎ１ζＦ（Ｆ勺′ど１１こ変更なしン図
１
ＡＦＰ （／Ｌｔ９７ｍｌ ）
手続ネｎｉ正書（方式）
１ 事件の表示
昭和５８年特許願第１７６７７４号
２ 発明の名称
診断・検査薬
３ 補正をする寵
事件との関係 特許出願人
（５９６）三菱化成工業株式会社
４代理人 〒１００
東京都千代田区丸の内二丁目５番２号
三菱化成工業株式会社内
（ばか１名）
５ ？ａ正命令の日付 昭和５９年１月３１日（発送日
）手続ネ甫正書（自発）
昭和５９年２月２０日
１ 事件の表示 昭和５８年特許願第１７６７７４号２
発明の名称 診断・検査薬
３ 補正をする者
事件との関係 出願人
（５９６） 三菱化成工業株式会社
４代理人
明細書の１発明の詳細な説明」の欄
６ 補正の内容Figure / shows an example of a calibration curve for measuring the AFp concentration in a sample using the test/diagnostic agent according to the present invention. Applicant Mitsubishi Chemical Industries, Ltd. Agent Patent Attorney Hase - (and 7 others)
1 AFP (/Lt97ml) Procedure Neni Original Book (Method) 1 Indication of the case 1982 Patent Application No. 176774 2 Name of the invention Diagnosis/Testing agent 3 Relationship with the case to be amended Patent applicant (596) Mitsubishi Kasei Kogyo Co., Ltd. 4 agents Mitsubishi Kasei Kogyo Co., Ltd., 2-5-2 Marunouchi, Chiyoda-ku, Tokyo 100 (1 idiot) 5? Date of official order: January 31, 1980 (shipment date) Procedural order: February 20, 1980 1 Case description: 1982 Patent Application No. 176774 2
Title of the invention Diagnostic/Testing agent 3 Relationship with the case of the person making the amendment Applicant (596) Mitsubishi Chemical Industries, Ltd. 4 Column 6 of ``Detailed explanation of the invention'' in the attorney's specification 4 Contents of the amendment
Claims (1)
ＦＰ）’ｉ認識するモノクローナル抗体ガンのマーカー
として、α−フェトプロティ(1) α-fetoprotein (A) present on the cell membrane surface
FP)'i monoclonal antibody that recognizes α-fetoprotein as a cancer marker.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17677483A JPS6067860A (en) | 1983-09-24 | 1983-09-24 | Diagnosing/examination agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP17677483A JPS6067860A (en) | 1983-09-24 | 1983-09-24 | Diagnosing/examination agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6067860A true JPS6067860A (en) | 1985-04-18 |
Family
ID=16019590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP17677483A Pending JPS6067860A (en) | 1983-09-24 | 1983-09-24 | Diagnosing/examination agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6067860A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2635267A1 (en) * | 1988-08-09 | 1990-02-16 | Tokuyama Soda Kk | MONOCLONAL ANTIBODIES AND METHOD FOR PRODUCING THE SAME |
-
1983
- 1983-09-24 JP JP17677483A patent/JPS6067860A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2635267A1 (en) * | 1988-08-09 | 1990-02-16 | Tokuyama Soda Kk | MONOCLONAL ANTIBODIES AND METHOD FOR PRODUCING THE SAME |
| GB2222591A (en) * | 1988-08-09 | 1990-03-14 | Tokuyama Soda Kk | Monoclonal antibodies |
| GB2222591B (en) * | 1988-08-09 | 1992-04-15 | Tokuyama Soda Kk | Monoclonal antibodies and process for production of monoclonal antibodies |
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