JPH0283337A - Diagnostic agent for cancer and recovery of tumor marker using the same agent - Google Patents
Diagnostic agent for cancer and recovery of tumor marker using the same agentInfo
- Publication number
- JPH0283337A JPH0283337A JP63233314A JP23331488A JPH0283337A JP H0283337 A JPH0283337 A JP H0283337A JP 63233314 A JP63233314 A JP 63233314A JP 23331488 A JP23331488 A JP 23331488A JP H0283337 A JPH0283337 A JP H0283337A
- Authority
- JP
- Japan
- Prior art keywords
- cancer
- immobilized
- lectin
- tumor markers
- fraction
- 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.)
- Granted
Links
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 52
- 201000011510 cancer Diseases 0.000 title claims abstract description 21
- 239000000439 tumor marker Substances 0.000 title claims abstract description 18
- 239000000032 diagnostic agent Substances 0.000 title claims abstract description 14
- 229940039227 diagnostic agent Drugs 0.000 title claims abstract description 14
- 238000011084 recovery Methods 0.000 title description 4
- 239000003795 chemical substances by application Substances 0.000 title 1
- 239000002523 lectin Substances 0.000 claims abstract description 28
- 108090001090 Lectins Proteins 0.000 claims abstract description 27
- 102000004856 Lectins Human genes 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 17
- 125000005630 sialyl group Chemical group 0.000 claims abstract description 6
- 239000004480 active ingredient Substances 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 2
- 239000011324 bead Substances 0.000 abstract description 12
- 206010061902 Pancreatic neoplasm Diseases 0.000 abstract description 6
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 abstract description 6
- 201000002528 pancreatic cancer Diseases 0.000 abstract description 6
- 208000008443 pancreatic carcinoma Diseases 0.000 abstract description 6
- 238000005406 washing Methods 0.000 abstract description 5
- 229920000936 Agarose Polymers 0.000 abstract description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 abstract description 3
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 abstract description 3
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 abstract description 3
- 206010033128 Ovarian cancer Diseases 0.000 abstract description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 abstract description 3
- 229920002684 Sepharose Polymers 0.000 abstract description 3
- 210000001124 body fluid Anatomy 0.000 abstract description 2
- 239000010839 body fluid Substances 0.000 abstract description 2
- 229920002678 cellulose Polymers 0.000 abstract description 2
- 239000001913 cellulose Substances 0.000 abstract description 2
- 239000003550 marker Substances 0.000 abstract description 2
- 229920002401 polyacrylamide Polymers 0.000 abstract description 2
- 208000009956 adenocarcinoma Diseases 0.000 abstract 2
- 239000007788 liquid Substances 0.000 abstract 2
- 210000001035 gastrointestinal tract Anatomy 0.000 abstract 1
- 230000002685 pulmonary effect Effects 0.000 abstract 1
- 238000010828 elution Methods 0.000 description 7
- 239000000499 gel Substances 0.000 description 6
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 4
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 125000002446 fucosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)[C@@H](O1)C)* 0.000 description 3
- 230000003100 immobilizing effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 2
- 102000004641 Fetal Proteins Human genes 0.000 description 2
- 108010003471 Fetal Proteins Proteins 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 201000005249 lung adenocarcinoma Diseases 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 108010041181 Aleuria aurantia lectin Proteins 0.000 description 1
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 1
- 206010004593 Bile duct cancer Diseases 0.000 description 1
- 101100191768 Caenorhabditis elegans pbs-4 gene Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 238000011891 EIA kit Methods 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 101100383698 Secale cereale rscc gene Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 208000026900 bile duct neoplasm Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 208000024558 digestive system cancer Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 201000010231 gastrointestinal system cancer Diseases 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000011240 wet gel Substances 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
【発明の詳細な説明】
［産業上の利用分野］
この発明は、癌性フコシル化糖鎖を有する腫瘍マーカー
を生じる癌のための癌診断薬及び上記腫瘍マーカーの回
収方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a cancer diagnostic agent for cancer that produces a tumor marker having cancerous fucosylated sugar chains, and a method for recovering the tumor marker.
［従来の技術］
腫瘍マーカーは腫瘍なスクリーニングする、腫瘍の診断
をする、腫瘍の実態をモニターする等に応用され、大き
な成果をあげている。特に膵癌のマーカーＣＡ１９−９
、卵巣癌のマーカーＣＡ１２５等の癌関連抗原、α−フ
ェトプロティン（ＡＦＰ）、癌胎児性抗原（ＣＥＡ）
、塩基性フェトプロティン等の胎児性タンパク質等は特
異性の優れたモノクローナル抗体、ポリクローナル抗体
の開発があり、血清診断キットとしても簡便かつ有効な
診断法として成功をおさめている。[Prior Art] Tumor markers have been applied to tumor screening, tumor diagnosis, and monitoring of the actual state of tumors, and have achieved great results. Especially pancreatic cancer marker CA19-9
, cancer-related antigens such as ovarian cancer marker CA125, α-fetoprotein (AFP), carcinoembryonic antigen (CEA)
Monoclonal and polyclonal antibodies with excellent specificity have been developed for fetal proteins such as basic fetoprotein, and serum diagnostic kits have been successful as simple and effective diagnostic methods.
これらの腫瘍マーカーについては、血中抗原測定用のＲ
ＩＡキット、次いでＥＩＡキットが開発され、広く普及
している。膵癌、胆管系の癌で最も優れた血中マーカー
と言われるＣＡ１９−９ではこれらの癌の約８０％に高
い血中抗原濃度が検出されるが、胃癌３８．６％、大腸
癌５９．１％、肺腺癌２８．６％、肝細胞癌３２．３％
等と癌組織に広く分布しており、低いとはいえ健常人で
も検出される。For these tumor markers, R
IA kits and then EIA kits were developed and are now widely used. CA19-9, which is said to be the best blood marker for pancreatic cancer and bile duct cancer, has a high blood antigen concentration detected in approximately 80% of these cancers, but 38.6% of gastric cancer and 59.1% of colon cancer. %, lung adenocarcinoma 28.6%, hepatocellular carcinoma 32.3%
It is widely distributed in cancer tissues, and can be detected even in healthy people, although at low levels.
従って、偽陰性、偽陽性の出現を低くする手段が必要で
ある。Therefore, a means is needed to reduce the occurrence of false negatives and false positives.
また、これらのＩＩ！Ｉマーカーを検出するためのモノ
クローナル抗体は、それらの腫瘍マーカを抗原として用
いて作製されるので、もし、腫瘍マーカーを簡便に回収
することができる方法があれば、癌の診断薬として用い
られるモノクローナル抗体の作製にとって有利である。Also, these II! Monoclonal antibodies for detecting I markers are produced using these tumor markers as antigens, so if there was a method that could easily collect tumor markers, it would be possible to develop monoclonal antibodies that can be used as cancer diagnostics. It is advantageous for the production of antibodies.
［発明が解決しようとする問題点］
従って、この発明の目的は、腫瘍マーカーを検出するこ
とによっていくつかの種類の癌の診断に用いることがで
きる新規な癌診断薬を提供することである。[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a novel cancer diagnostic agent that can be used for diagnosing several types of cancer by detecting tumor markers.
さらにまた、この発明の目的は、いくつかの種類の腫瘍
マーカーの簡便な回収方法を提供することである。Furthermore, it is an object of the present invention to provide a simple method for collecting several types of tumor markers.
［問題点を解決するための手段］
本願発明者らは、鋭意研究の結果、癌性フコシル化糖鎖
を有する腫瘍マーカーが固定化レクチンに吸着される性
質を何することを見出し、この発明を完成した。[Means for Solving the Problems] As a result of intensive research, the inventors of the present application discovered that the property of a tumor marker having a cancerous fucosylated sugar chain being adsorbed to an immobilized lectin, and developed the present invention. completed.
すなわち、この発明は、固定化ＡＡＬレクチンを有効成
分とする、癌性フコシル化糖鎖を有する腫瘍マーカーを
生じる癌のための診断薬を提供する。That is, the present invention provides a diagnostic agent for cancer that produces a tumor marker having cancerous fucosylated sugar chains, which contains immobilized AAL lectin as an active ingredient.
さらにまた、この発明は、癌性フコシル化糖鎖を有する
腫瘍マーカーを含む流体を固定化ＡＡＬレクチンに作用
させ、固定化ＡＡＬレクチンに吸着された腫瘍マーカー
を回収することから成る、上記腫瘍マーカーの回収方法
を提供する。Furthermore, the present invention provides a method for treating the tumor marker, which comprises applying a fluid containing a tumor marker having cancerous fucosylated sugar chains to the immobilized AAL lectin and recovering the tumor marker adsorbed to the immobilized AAL lectin. Provide collection methods.
［発明の効果］
この発明により、癌性フコシル化糖鎖を有する腫瘍マー
カーを生じる癌に対する新規な診断薬が提供された。こ
の発明の癌診断薬は、免疫反応により腫瘍マーカーと結
合するのではなく、全く別の原理により結合するので、
腫瘍マーカーに対する従来のモノクローナル抗体と併用
することにより、従来の癌診断における偽陽性及び偽陰
性が生じる確率を低下させることができる。[Effects of the Invention] The present invention provides a novel diagnostic agent for cancer that produces a tumor marker having cancerous fucosylated sugar chains. The cancer diagnostic agent of this invention does not bind to tumor markers through an immune reaction, but by a completely different principle.
By using it in combination with conventional monoclonal antibodies against tumor markers, the probability of false positives and false negatives in conventional cancer diagnosis can be reduced.
さらにまた、この発明により、癌性フコシル化糖鎖を有
する腫瘍マーカーの新規な回収方法が提供された。この
発明の方法によると、−段階のアフィニティクロマトグ
ラフィー操作によりフコシル化糖鎖を担っている腫瘍マ
ーカーを簡便に回収、精製することができ、しかも、フ
コシル化糖鎖を担う種々の腫瘍マーカーに適用できるの
で汎用性に優れている。また、固定化ＡＡＬレクチンは
何回でも繰り返し使用することができる。さらに、ＡＡ
Ｌレクチンは界面活性剤の存在下でも活性を有するので
例えば可滴化膜タンパク質の回収にも適用することがで
きる。Furthermore, the present invention provides a novel method for collecting tumor markers having cancerous fucosylated sugar chains. According to the method of the present invention, tumor markers carrying fucosylated sugar chains can be easily recovered and purified by the -step affinity chromatography operation, and moreover, it can be applied to various tumor markers carrying fucosylated sugar chains. It is highly versatile because it can be used. Moreover, the immobilized AAL lectin can be used repeatedly any number of times. Furthermore, A.A.
Since L-lectin has activity even in the presence of a surfactant, it can be applied, for example, to the recovery of dropletized membrane proteins.
［発明の詳細な説明］
上述したように、この発明の癌診断薬は、固定化された
ＡＡＬレクチンを（Ａｌｅｕｒｉａ ａｕｒａｎｔｉａ
ｌｅｃｔｉｎ、 Ｎ、 Ｋｏｃｈｉｂｅ ａｎｄ
Ｋ、 Ｆｕｒｕｋａｗａ。[Detailed Description of the Invention] As described above, the cancer diagnostic agent of the present invention contains immobilized AAL lectin (Aleuria aurantia
lectin, N., Kochibe and
K, Furukawa.
”Ｂｉｏｃｈｅｍｉｓｔｒｙ”、 １９巻ｐＰ、 ２
８４１−２８４６ ｆ１９８０＋＋ を有効成分と
する。ＡＡＬレクチン自身は公知であり、その調製方法
及び性質も公知であって上記文献に記載されている。ま
た、ＡＡＬレクチンの固定化方法も種々の公知の方法を
採用することができ、例えばＹａｍａｓｈｉｔａ、 Ｋ
ｏｃｈｉｂｅ、 ０ｈｋｕｒａ、 Ｕｅｄａａｎｄ Ｋ
ｏｂａｔａ、 ”Ｊ、 Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅ
ｍｉｓｔｒｙ 、 ｖｏｌ。"Biochemistry", vol. 19 pP, 2
841-2846 f1980++ is the active ingredient. AAL lectin itself is known, and its preparation method and properties are also known and described in the above-mentioned literature. Furthermore, various known methods can be used for immobilizing AAL lectin, such as Yamashita, K.
Ochibe, 0hkura, Uedaand K
obata, ”J, Biological Che
mistry, vol.
２６０、 ｐｐ、 ４６８８−４６９３ （１９８５１
に記載されている。260, pp. 4688-4693 (19851
It is described in.
好ましい担体としてはセファロースビーズ、アガロース
ビーズ、セルロースビーズ、ポリアクリルアミドビーズ
等を挙げることかでき、固相化するレクチンの濃度は通
常２０ Ｂ／ｍｌないしｌ ｍｇ／ｍｌ、好ましくは約
３ ｍｇ／ｍ１程度である。さらにまた、担体がアガロ
ースゲル（例えばセファロース４Ｂ、６Ｂ（ファルマシ
ア社裂）、バイオゲルＡ０．５ｍ、Ａ１．５ｍ （登録
商標）をＣＮＢｒで活性化してカプリングする（例えば
Ｍａｒｃｈ ｅｔ ａｌ、、 Ａｎａｌ。Preferred carriers include sepharose beads, agarose beads, cellulose beads, polyacrylamide beads, etc., and the concentration of lectin to be immobilized is usually about 20 B/ml to 1 mg/ml, preferably about 3 mg/ml. It is. Furthermore, the carrier can be activated with CNBr and coupled to an agarose gel (eg, Sepharose 4B, 6B (Pharmacia), Biogel A0.5m, A1.5m (registered trademark)) (eg, March et al., Anal.
Ｂｉｏｃｈｅｍ、 ６０．１４９−５２．１９７４）こ
ともできる、また、この方法をＣＮＢｒ−活性化ゲル（
ファルマシア社製）に適用して固定化することもできる
。Biochem, 60.149-52.1974), and this method can also be applied to CNBr-activated gels (
(manufactured by Pharmacia) for immobilization.
さらにまた、活性化スペーサーを導入したゲル（例えば
アフィゲル１０．１５（バイオラド社製））にＡＡＬレ
クチンを作用させて固定化することもできる。さらに、
ホルミル基を導入したゲル（例えば、）オルミルアガロ
ース
ミルトヨパール（東ソー株式会社製））にＡＡＬレクチ
ンを作用させてカプリングし、これをＮａＣＮＢＨｓで
還元する方法も用いることができる。Furthermore, AAL lectin can be immobilized by acting on a gel into which an activated spacer has been introduced (for example, Affigel 10.15 (manufactured by Bio-Rad)). moreover,
A method can also be used in which AAL lectin is applied to a gel into which a formyl group has been introduced (for example, ormylated agarose miltoyopearl (manufactured by Tosoh Corporation)) and coupled thereto, and then this is reduced with NaCNBHs.
さらに、他のタンパク質（例えばゼラチン）を上記いず
れかの方法により固定化した後、ＡＡＬレクチンをグル
タルアルデヒドによって上記タンパク質と架橋する方法
も用いることができる。上記いずれの方法により固定化
する場合でも、ｌＯｍＭ程度のし一フコースを加えてフ
コシル化糖鎖がＡＡＬレクチンに結合する結合部位を保
護することが好ましい。Furthermore, a method can also be used in which, after immobilizing another protein (eg, gelatin) by any of the above methods, AAL lectin is crosslinked with the above protein using glutaraldehyde. When immobilizing by any of the above methods, it is preferable to add about 10mM of monofucose to protect the binding site where the fucosylated sugar chain binds to AAL lectin.
この発明の診断薬は、特に限定されないが、カラムに充
填して用いることが操作上簡便で好ましい。この場合、
カラムに充填する同相化ＡＡＬレクチンの量（担体を含
む）は特に限定されないが約１ｍｌで十分である。Although the diagnostic agent of the present invention is not particularly limited, it is preferable to use it by filling it in a column because it is easy to use. in this case,
The amount of homophasized AAL lectin packed into the column (including the carrier) is not particularly limited, but about 1 ml is sufficient.
この発明の診断薬により診断することができる癌は、癌
によりフコシル化糖鎖を有する腫瘍マーカーを生じさせ
るものである．この発明の診断薬に吸着される、癌性フ
コシル化糖鎖を有する腫瘍マーカーの例としては、膵癌
に対するＣＡ１９−９、消化器系ガンに対するＣＥＡ、
膵癌に対するＤＩ−ＰＡＮ−２、肺腺癌等に対するシア
リル・ルイス・ｘ−ｉ（文献：「化学と生物」２６巻、
４号、 ２２０−２３４ 、 １９８８） 、腺癌に対
するＬｅＩ′．その他卵巣癌に対するＣＡ１２５を挙げ
ることができる。Cancers that can be diagnosed with the diagnostic agent of the present invention are cancers that produce tumor markers having fucosylated sugar chains. Examples of tumor markers having cancerous fucosylated sugar chains that are adsorbed to the diagnostic agent of this invention include CA19-9 for pancreatic cancer, CEA for digestive system cancer,
DI-PAN-2 for pancreatic cancer, Sialyl Lewis x-i for lung adenocarcinoma, etc. (References: "Chemistry and Biology" vol. 26,
4, 220-234, 1988), LeI'. Other examples include CA125 for ovarian cancer.
この発明の診断薬を用いて癌の診断を行なう場合には、
患者から得た体液、例えば血清を、固定化ＡＡＬレクチ
ン含有カラムにかけ、ＰＢＳのような緩衝液で洗浄して
洗浄画分を集め、次いで例えば約２０ｍＭ程度のフコー
スを含む緩衝液で溶出させて溶出画分を集め、洗浄画分
及び溶出画分中に存在する腫瘍マーカーを定量して比較
する。When diagnosing cancer using the diagnostic agent of this invention,
A body fluid obtained from a patient, such as serum, is applied to a column containing immobilized AAL lectin, washed with a buffer such as PBS to collect the washed fraction, and then eluted with a buffer containing, for example, about 20 mM fucose. Fractions are collected and the tumor markers present in the wash and elution fractions are quantified and compared.
溶出画分に腫瘍マーカーの多くが存在している場合には
、その患者が癌に罹患している確率が高いので，癌の診
断を行なうことができる。なお、腫瘍マーカーの定量は
、モノクローナル抗体を用いたＥＴＡのような周知の免
疫分析法により行なうことができ、そのためのキットも
種々市販されている。If many tumor markers are present in the elution fraction, there is a high probability that the patient is suffering from cancer, and cancer can be diagnosed. The tumor marker can be quantified by a well-known immunoassay method such as ETA using a monoclonal antibody, and various kits for this purpose are commercially available.
また、同様な方法により、溶出画分中に腫瘍マーカーを
溶出させた後、それを回収することができる。溶出画分
中の腫瘍マーカーの回収、精製は、例えば溶出液を透析
チューブに入れ、５０〜１００倍量のＰＢＳに対して半
日毎に４回ＰＢＳを取り替えて透析を行なうことにより
腫瘍マーカーの回収を行なうことができる。Furthermore, by a similar method, a tumor marker can be eluted into the elution fraction and then recovered. Recovery and purification of tumor markers in the elution fraction can be carried out, for example, by putting the eluate into a dialysis tube and performing dialysis against 50 to 100 times the volume of PBS, changing the PBS 4 times every half day. can be done.
なお、ＡＡＬ固定化ゲルは、フコース含有緩衝液で溶出
した後、ＰＢＳで洗浄すれば再生されるので何回でも使
用できる．また、ゲルの保存には０．０２％ＮａＮｚを
含むＰＢＳを用いることができる。The AAL-immobilized gel can be regenerated by washing with PBS after elution with a fucose-containing buffer, so it can be used any number of times. Furthermore, PBS containing 0.02% NaNz can be used to preserve the gel.
［実施例１
以下、本発明を実施例に基づきより具体的に説明する．
なお、本発明は下記実施例に限定されるものではない。[Example 1] Hereinafter, the present invention will be explained in more detail based on an example.
Note that the present invention is not limited to the following examples.
ＡＡＬレクチンカラムの 製
活性化スペーサー（中性のｌＯ原子のアーム、を導入し
たバイオラド社製Ａｆｆｉｇｅｌ １０に以下のように
してＡＡＬレクチンを導入し、４．６ ｍｇ／ｍｌウェ
ットゲルのＡＡＬが導入°された固定化ＡＡＬレクチン
を調製した．すなわち、アフィゲル１０を４℃にし、ゲ
ルを均一にして１ｍｌを測り取り、イソプロパツールを
浸したポリプロピレン性エコツカラムに入れた．インプ
ロパツールを流し出しだ後１０ｍｌの１０ｍＭ酢酸ナト
リウム（ｐＨ４．５ ）で洗浄した．洗浄後すぐ２倍量
のカップリングバッファーで洗浄し、ＡＡＬ５ｍｇを加
え混合した．４℃で一夜撹拌しながら反応させ、０．１
ｍｌのＩＭエタノールアミンＨＣＩ ｆｐＨ８）を加
え一時間反応させた。カラムから反応液を溶出させ．Ａ
ＡＬ含量を測定したところ９０％が結合していた．その
後、カップリングバッフｙｌｏｍｌ、２０ｍｌの２０ｍ
Ｍフコース、０．旧Ｍ ＰＢＳｆｐＨ７．２１ １０
ｍｌで順次洗浄し、固定化ＡＡＬレクチンとした。これ
を０．１３ ｃｌｌ（直径）に７ ｃｍのカラムに充填
し、固定化ＡＡＬカラムを作製した。AAL lectin was introduced into the AAL lectin column's Affigel 10 manufactured by Bio-Rad, into which an activated spacer (neutral lO atom arm) was introduced, and 4.6 mg/ml wet gel of AAL was introduced. The immobilized AAL lectin was prepared by heating Affigel 10 to 4°C, homogenizing the gel, measuring out 1 ml, and placing it in a polypropylene ecotu column soaked with isopropatool. After pouring out the impropatool. Washed with 10 ml of 10 mM sodium acetate (pH 4.5). Immediately after washing, washed with twice the amount of coupling buffer, added 5 mg of AAL and mixed. Reacted overnight at 4°C with stirring, and 0.1
ml of IM ethanolamine HCI fpH8) was added and reacted for one hour. Elute the reaction solution from the column. A
When the AL content was measured, 90% was bound. Then coupling buff yl, 20ml of 20m
M-fucose, 0. Old M PBSfpH7.21 10
ml was sequentially washed to obtain immobilized AAL lectin. This was packed into a 7 cm column at 0.13 cll (diameter) to produce an immobilized AAL column.
腫瘍マーカーの回収
上記のようにして調製した固定化ＡＡＬカラムに、ＣＡ
１９−９ （ｒエルグ・ＣＡ１９−９・キット」ミドＪ
十字社製）、シアリルＬｅｖｉｓ Ｘ−ｉ （Ｓ Ｌ
Ｘ「オーツカ」、天場製薬社製）、ＣＥＡ（ｒＣＥＡ
・リアビーズ」、ダイナボット社製） 、 ＤＩ−ＰＡ
Ｎ−２（ｒデタミナＤＵＰＡＮ２Ｊ 、共和メデイック
ス社製）、 Ｌｅｌ′（ｒＬｅｖｉｓリアビーズ」、第
一ラジオアイソトープ社製）、５ＣＣ（ｒｓｃｃ・リア
ビーズ」、ダイナボット社製） 、 ＣＡ１２５（ｒセ
ントコアＣＡ１２５ ＲＩＡキット」セントコア社製）
又はａ−フェトプロティン（「α−フェト・リアビーズ
」、ダイナボット社製）の各腫瘍マーカーの標品を含む
溶液及び癌患者からの血清１ ｍｌを室温下で流通さ
せ、アフイニティクロマトグラフィーを行なった。洗浄
液としては０．Ｏ１閘ＰＢＳ １ｐＨ７，０）を用い、
溶出液とじてはこのＰＢＳに２０ｍＭのＩ、−フコース
を添加したものを用いた。Recovery of tumor markers The CA
19-9 (rerg・CA19-9・kit” Mido J
), Sialyl Levis X-i (S L
X “Otsuka”, manufactured by Tenba Pharmaceutical Co., Ltd.), CEA (rCEA
・Rear Beads”, manufactured by Dynabot), DI-PA
N-2 (rDetermina DUPAN2J, manufactured by Kyowa Medix Co., Ltd.), Lel' (rLevis Rear Beads, manufactured by Daiichi Radioisotope Co., Ltd.), 5CC (rScc Rear Beads, manufactured by Dynabot), CA125 (r Centocor CA125 RIA Kit) Manufactured by Centocor)
Alternatively, a solution containing preparations of each tumor marker of a-fetoprotein ("α-feto-rea beads", manufactured by Dynabot) and 1 ml of serum from a cancer patient were circulated at room temperature, and affinity chromatography was performed. I did it. 0.0 as a cleaning solution. Using O1 PBS 1pH 7,0),
This PBS to which 20 mM I,-fucose was added was used as the eluate.
洗浄画分及び溶出画分をそれぞれ回収し、蒸留水に対し
て一夜透析しく分子量カットオフ２０００１ 、凍結乾
燥し、上記ＰＢ５１ｍｌに溶解して、各腫瘍マーカーに
ついての市販の診断キットを用いて腫瘍マーカーを定量
した。結果を下記表に示す。The wash fraction and elution fraction were each collected, dialyzed overnight against distilled water, lyophilized, and dissolved in 51 ml of the above PB. was quantified. The results are shown in the table below.
上記表より明らかなように、癌性フコシル糖鎖を担持す
る腫瘍マーカーであるＣＡ１９−９、シアリルＬｅｗｉ
ｓ Ｘ−ｉ 、 ＣＥ Ａ 、 ＤＯ−ＰＡＮ−２、Ｌ
ｅｂ、 ＣＡ１２５については活性の大部分が溶出画分
に回収され、一方、フコシル糖鎖を有さないＳＣＣ及び
ａ−フェトプロテインについてはほとんどが洗浄画分に
回収された。これにより、固定化ＡＡＬレクチンを用い
て癌性フコシル糖鎖を担持する腫瘍マカーの回収及び検
出を行なうことができることが明らかになった。As is clear from the above table, CA19-9, a tumor marker carrying cancerous fucosyl sugar chains, and sialyl Lewis
sX-i, CE A, DO-PAN-2, L
Most of the activity of eb and CA125 was recovered in the elution fraction, while most of the activity of SCC and a-fetoprotein, which do not have fucosyl sugar chains, was recovered in the wash fraction. This revealed that it is possible to recover and detect tumor markers carrying cancerous fucosyl sugar chains using immobilized AAL lectin.
Claims (4)
コシル化糖鎖を有する腫瘍マーカーを生じる癌のための
診断薬。(1) A diagnostic agent for cancer that produces a tumor marker having cancerous fucosylated sugar chains, which contains immobilized AAL lectin as an active ingredient.
ｗｉｓＸ−ｉ、ＣＥＡ、ＤＵ−ＰＡＮ−２、Ｌｅ＾ｂ又
はＣＡ１２５である請求項１記載の診断薬。(2) The above tumor markers are CA19-9, Sialyl Le
The diagnostic agent according to claim 1, which is wisX-i, CEA, DU-PAN-2, Le^b, or CA125.
流体を固定化ＡＡＬレクチンに作用させ、固定化ＡＡＬ
レクチンに吸着された腫瘍マーカーを回収することから
成る、上記腫瘍マーカーの回収方法。(3) A fluid containing a tumor marker having cancerous fucosylated sugar chains is applied to the immobilized AAL lectin, and the immobilized AAL
The above method for collecting tumor markers, which comprises collecting tumor markers adsorbed to lectin.
ｗｉｓＸ−ｉ、ＣＥＡ、ＤＵ−ＰＡＮ−２、Ｌｅ＾ｂ又
はＣＡ１２５である請求項３記載の方法。(4) The above tumor markers are CA19-9, Sialyl Le
4. The method according to claim 3, which is wisX-i, CEA, DU-PAN-2, Le^b or CA125.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63233314A JP2721900B2 (en) | 1988-09-18 | 1988-09-18 | Cancer diagnostic agent and method for recovering tumor marker using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63233314A JP2721900B2 (en) | 1988-09-18 | 1988-09-18 | Cancer diagnostic agent and method for recovering tumor marker using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0283337A true JPH0283337A (en) | 1990-03-23 |
| JP2721900B2 JP2721900B2 (en) | 1998-03-04 |
Family
ID=16953187
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63233314A Expired - Fee Related JP2721900B2 (en) | 1988-09-18 | 1988-09-18 | Cancer diagnostic agent and method for recovering tumor marker using the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2721900B2 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0533834A1 (en) * | 1990-06-15 | 1993-03-31 | Cytel Corporation | Intercellular adhesion mediators |
| US5576305A (en) * | 1990-06-15 | 1996-11-19 | Cytel Corporation | Intercellular adhesion mediators |
| US5753631A (en) * | 1990-06-15 | 1998-05-19 | Cytel Corporation | Intercellular adhesion mediators |
| US6033663A (en) * | 1996-04-10 | 2000-03-07 | Neose Technologies, Inc. | Nucleic acids encoding GDP-Fucose pyrophosphorylase |
| WO2005083440A3 (en) * | 2004-02-19 | 2006-03-16 | Univ Yale | Identification of cancer protein biomarkers using proteomic techniques |
| WO2010010674A1 (en) | 2008-07-22 | 2010-01-28 | 株式会社Ｊ－オイルミルズ | FUCOSE α1→6-SPECIFIC LECTIN |
| WO2011105544A1 (en) * | 2010-02-26 | 2011-09-01 | 地方独立行政法人大阪府立病院機構 | Tumor marker, antibody to same, detection kit for same, and method for detecting same |
-
1988
- 1988-09-18 JP JP63233314A patent/JP2721900B2/en not_active Expired - Fee Related
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0533834A4 (en) * | 1990-06-15 | 1995-04-19 | Cytel Corp | |
| US5576305A (en) * | 1990-06-15 | 1996-11-19 | Cytel Corporation | Intercellular adhesion mediators |
| US5753631A (en) * | 1990-06-15 | 1998-05-19 | Cytel Corporation | Intercellular adhesion mediators |
| EP0533834A1 (en) * | 1990-06-15 | 1993-03-31 | Cytel Corporation | Intercellular adhesion mediators |
| US6033663A (en) * | 1996-04-10 | 2000-03-07 | Neose Technologies, Inc. | Nucleic acids encoding GDP-Fucose pyrophosphorylase |
| US8975379B2 (en) | 2004-02-19 | 2015-03-10 | Yale University | Identification of cancer protein biomarkers using proteomic techniques |
| WO2005083440A3 (en) * | 2004-02-19 | 2006-03-16 | Univ Yale | Identification of cancer protein biomarkers using proteomic techniques |
| US7666583B2 (en) | 2004-02-19 | 2010-02-23 | Yale University | Identification of cancer protein biomarkers using proteomic techniques |
| AU2005217375B2 (en) * | 2004-02-19 | 2011-05-19 | Yale University | Identification of cancer protein biomarkers using proteomic techniques |
| US10168334B2 (en) | 2004-02-19 | 2019-01-01 | Yale University | Identification of cancer protein biomarkers using proteomic techniques |
| AU2005217375C1 (en) * | 2004-02-19 | 2012-06-07 | Yale University | Identification of cancer protein biomarkers using proteomic techniques |
| US9470688B2 (en) | 2004-02-19 | 2016-10-18 | Yale University | Identification of cancer protein biomarkers using proteomic techniques |
| WO2010010674A1 (en) | 2008-07-22 | 2010-01-28 | 株式会社Ｊ－オイルミルズ | FUCOSE α1→6-SPECIFIC LECTIN |
| US8461305B2 (en) | 2008-07-22 | 2013-06-11 | J-Oil Mills, Inc. | L-fucose α1→6 specific lectin |
| WO2011105544A1 (en) * | 2010-02-26 | 2011-09-01 | 地方独立行政法人大阪府立病院機構 | Tumor marker, antibody to same, detection kit for same, and method for detecting same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2721900B2 (en) | 1998-03-04 |
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