JPH08113600A - Antibody against mrp - Google Patents
Antibody against mrpInfo
- Publication number
- JPH08113600A JPH08113600A JP27307594A JP27307594A JPH08113600A JP H08113600 A JPH08113600 A JP H08113600A JP 27307594 A JP27307594 A JP 27307594A JP 27307594 A JP27307594 A JP 27307594A JP H08113600 A JPH08113600 A JP H08113600A
- Authority
- JP
- Japan
- Prior art keywords
- mrp
- antibody
- cells
- protein
- resistant
- 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
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、一部の癌細胞（多剤耐
性細胞）に発現される多剤耐性蛋白質（ｍｕｌｔｉｄｒ
ｕｇ ｒｅｓｉｓｔａｎｃｅ−ａｓｓｏｃｉａｔｅｄ
ｐｒｏｔｅｉｎ：ＭＲＰ）に対する特異的抗体、その製
造及び利用に関するものである。The present invention relates to a multidrug resistance protein (multidr) expressed in some cancer cells (multidrug resistant cells).
ug resistance-associated
The present invention relates to a specific antibody against protein (MRP), its production and use.
【０００２】[0002]
【従来の技術】癌細胞が投与した抗癌剤に対して耐性に
なるのと同時に他の複数の薬剤に対しても耐性になる事
がある。このように多くの薬剤に同時に耐性を獲得した
多剤耐性細胞では、多くの場合、細胞内の抗癌剤の蓄積
が減少していることがわかっている。これらの現象の一
部は、Ｐ−糖蛋白質と言われる膜蛋白質がＡＴＰのエネ
ルギーを利用して細胞内に入った抗癌剤を細胞外へ排出
するポンプの役割を果たしているためであることが知ら
れている（Ｍｏｌｅｃｕｌａｒ Ｍｅｄｉｃｉｎｅ，３
０（６），６８８−６９５（１９９３））。2. Description of the Related Art A cancer cell sometimes becomes resistant to an anticancer drug administered and at the same time, becomes resistant to a plurality of other drugs. It is known that in many cases, in multidrug resistant cells that have acquired resistance to many drugs at the same time, intracellular anticancer drug accumulation is reduced in many cases. It is known that part of these phenomena is that a membrane protein called P-glycoprotein plays a role of a pump that uses the energy of ATP to expel the anticancer drug entering the cell to the outside of the cell. It is (Molecular Medicine, 3
0 (6), 688-695 (1993)).
【０００３】一方、ＭＲＰは、上記Ｐ−糖蛋白質と同様
に多剤耐性細胞から検出された蛋白質であるが、分子量
が１９０ｋＤａであって、Ｐ−糖蛋白質とは全く別異の
蛋白質であることが知られている。しかしながら、その
作用はＰ−糖蛋白質の作用と類似しており、細胞内に入
ってきた抗癌剤を細胞外に排出するポンプの役割を果た
しているものと考えられている。On the other hand, MRP is a protein detected from multidrug resistant cells like the above-mentioned P-glycoprotein, but has a molecular weight of 190 kDa and is a completely different protein from P-glycoprotein. It has been known. However, its action is similar to that of P-glycoprotein, and it is considered to play a role of a pump for discharging the anticancer drug that has entered the cells to the outside of the cells.
【０００４】そして、癌細胞の多剤耐性獲得のメカニズ
ムのうち約３割はＰ−糖蛋白が関与することが知られて
いるが、残りの７割のうちの一部やＰ−糖蛋白の関与し
ている３割の一部にＭＲＰが関与した多剤耐性獲得が考
えられる（Ｍｏｌｅｃｕｌａｒ Ｍｅｄｉｃｉｎｅ，３
０（６），６８０−６８７（１９９３））。It is known that about 30% of the mechanism of acquiring multidrug resistance of cancer cells is involved in P-glycoprotein, but a part of the remaining 70% and P-glycoprotein are involved. Acquisition of multidrug resistance in which MRP is involved in a part of the 30% involved is considered (Molecular Medicine, 3
0 (6), 680-687 (1993)).
【０００５】[0005]
【発明が解決しようとする課題】上記のような多剤耐性
癌に対しては、通常の抗癌剤をいくら投与しても細胞外
へ排出されてしまうため、抗癌剤治療の効果を得ること
はできない。しかも単にそれだけにとどまらず、更に、
抗癌剤副作用のため患者に負担をかける結果となり、多
剤耐性癌か否かの診断が治療上必要とされている。すな
わち本発明の目的は、特定の多剤耐性癌を特異的に検出
するために有用な抗体及びその関連システムを提供する
ことである。With respect to the above-mentioned multidrug-resistant cancers, no matter how much an ordinary anticancer drug is administered, it is excreted out of the cells, so that the effect of the anticancer drug treatment cannot be obtained. And more than just that,
A side effect of an anti-cancer drug causes a burden on a patient, and a diagnosis of whether or not a multi-drug resistant cancer is therapeutically required. That is, an object of the present invention is to provide an antibody useful for specifically detecting a specific multidrug-resistant cancer and a system related thereto.
【０００６】[0006]
【課題を解決するための手段】本発明は、上記目的を達
成するためになされたものであって、この蛋白質の発現
を免疫学的方法によって確認できるようにするため、各
方面から検討した結果、ＭＲＰの一部を抗原とし、これ
を哺乳類に注射することにより、該蛋白質に対する抗体
の作成に成功し、本発明を完成するに至った。Means for Solving the Problems The present invention has been made in order to achieve the above-mentioned object, and in order to make it possible to confirm the expression of this protein by an immunological method, the results of examination from various directions , A part of MRP was used as an antigen, and this was injected into a mammal, whereby an antibody against the protein was successfully prepared and the present invention was completed.
【０００７】本発明を実施するには、免疫原を用いて動
物を免疫する必要がある。免疫原としては、ＭＲＰの一
部を用いる。具体的には、例えば、抗原としては、ＭＲ
Ｐに由来する下記のアミノ酸配列を含むペプタイドを利
用することができる。 Ｇｌｕ−Ｇｌｎ−Ｇｌｕ−Ａｒｇ−Ｐｈｅ−Ｉｌｅ−Ｈｉｓ− Ｇｌｎ−Ｓｅｒ−Ａｓｐ−Ｌｅｕ−Ｌｙｓ−Ｖａｌ−Ａｓｐ− Ｇｌｕ−Ａｓｎ−Ｇｌｎ−ＬｙｓIn order to carry out the present invention, it is necessary to immunize an animal with an immunogen. A part of MRP is used as an immunogen. Specifically, for example, as the antigen, MR
A peptide containing the following amino acid sequence derived from P can be used. Glu-Gln-Glu-Arg-Phe-Ile-His- Gln-Ser-Asp-Leu-Lys-Val-Asp- Glu-Asn-Gln-Lys.
【０００８】すなわち本発明においてペプタイド抗原と
しては、上記したアミノ酸配列それ自体のほか、該配列
を複数結合したもの、該配列に他のペプタイドを結合し
たもの、及び／又は、該配列を含有するペプタイドが適
宜使用され、必要ある場合には該アミノ酸配列の一部も
抗原として使用することができる。That is, in the present invention, the peptide antigen includes, in addition to the above-mentioned amino acid sequence itself, a combination of a plurality of the sequences, a combination of the sequences with another peptide, and / or a peptide containing the sequence. Are appropriately used, and if necessary, part of the amino acid sequence can also be used as an antigen.
【０００９】免疫は常法に従い、例えば前述したペプタ
イドの全てまたは一部を動物に必要回数注射することに
よって行う。注射する動物としては、マウス、ウサギ、
ラット、ヒツジ等を使用することができ、注射する方法
は公知の方法に従う事ができる。そして注射した動物よ
り採血し、抗血清を得る。得られた抗血清より本発明の
抗体を得るには従来知られているいずれの方法でも構わ
ない。例えば、Ｃ−Ａ５００細胞（Ｃ−Ａ５００細胞
は、白人男性の口腔内類表皮癌より得られた細胞株をア
ドリアマイシン、メゼレイン、セファランチンの３種薬
剤存在下で培養し、得られた細胞株であって、Ｐ−糖蛋
白質とは異なる薬剤能動排出ポンプの役割を果たすＭＲ
Ｐを発現していることが確認されている。）を可溶化
し、蛋白質を電気泳動によって分離した後、ＰＶＤＦ膜
に転写し、採取した抗血清で蛋白質のバンドを検出し、
この検出された蛋白質の分子量が１９０ｋＤａであるか
否かで判断することができる。必要であれば、抗体（抗
血清）は、さらにアフィニティー精製をしてもよい。Immunization is carried out according to a conventional method, for example, by injecting an animal with all or part of the above-mentioned peptide as many times as necessary. Animals to be injected include mice, rabbits,
Rats, sheep, etc. can be used, and the injection method can follow known methods. Then, blood is collected from the injected animal to obtain antiserum. To obtain the antibody of the present invention from the obtained antiserum, any conventionally known method may be used. For example, C-A500 cells (C-A500 cells are cell lines obtained by culturing a cell line obtained from epidermoid carcinoma of the oral cavity of a white male in the presence of three drugs, adriamycin, mezerein, and cepharanthin. MR acting as a drug active efflux pump different from P-glycoprotein
It has been confirmed that P is expressed. ) Is solubilized and the protein is separated by electrophoresis, then transferred to a PVDF membrane, and a protein band is detected with the collected antiserum,
It can be judged whether or not the molecular weight of the detected protein is 190 kDa. If desired, the antibody (antiserum) may be further affinity purified.
【００１０】このようにして得たポリクローナル抗体
は、各種の免疫測定法、これらを直接蛍光色素標識する
方法、あらかじめ蛍光色素標識された二次抗体を用いる
方法などによって、フローサイトメトリーを用いた細胞
表面に発現されたＭＲＰの測定、ひいては多剤耐性癌の
検出、その他の用途に用いることができる。The polyclonal antibody thus obtained can be used for cells using flow cytometry by various immunoassays, direct labeling of these with a fluorescent dye, a method of using a secondary antibody labeled with a fluorescent dye in advance, and the like. It can be used for measurement of MRP expressed on the surface, and eventually for detection of multidrug resistant cancer, and other applications.
【００１１】以下、本発明の実施例について述べるが、
本発明はこれらの実施例のみに限定されるものではな
い。Examples of the present invention will be described below.
The invention is not limited to these examples only.
【００１２】[0012]
【実施例１ ポリクローナル抗体の作製】Example 1 Preparation of polyclonal antibody
【００１３】（１）ペプタイドに対する抗体の作製 上記蛋白質のアミノ酸配列の一部をＭＡＰ合成法により
作製した。得られたペプタイド５００μｇをフロイント
の完全アジュバントと共にウサギの背部に注射した。以
後、一週間おきに５００μｇのペプチドをフロイントの
完全アジュバントと混合して同様に合計５回免疫した。
最終免疫後、７日目に全採血を行い、抗血清を分離し
た。(1) Preparation of antibody against peptide A part of the amino acid sequence of the above protein was prepared by the MAP synthesis method. 500 μg of the obtained peptide was injected into the back of a rabbit together with Freund's complete adjuvant. Thereafter, every other week, 500 μg of the peptide was mixed with Freund's complete adjuvant, and a total of 5 immunizations were performed in the same manner.
Seven days after the final immunization, whole blood was collected and antiserum was separated.
【００１４】（２）ペプタイドに対する抗体の精製 この抗血清をプロテインＡを用いたアフィニティークロ
マトグラフィー処理し、ＩｇＧ画分を単離した。即ち、
アフィゲル−プロテインＡ（バイオラッド社製）をカラ
ムに充填し、同社のバインディングバッファーで平衡化
した。採血した血清を同量のバインディングバッファー
で希釈し、カラムに流してプロテインＡにＩｇＧを結合
させた後、同社のエリューションバッファーで溶出さ
せ、ＩｇＧ画分を得た。この精製抗体の純度はＳＤＳ−
ＰＡＧＥ電気泳動にて検定した。(2) Purification of antibody against peptide This antiserum was subjected to affinity chromatography using protein A to isolate the IgG fraction. That is,
Affi-Gel-Protein A (manufactured by Bio-Rad) was packed in a column and equilibrated with the company's binding buffer. The collected blood serum was diluted with the same amount of binding buffer, passed through a column to bind IgG to protein A, and then eluted with the same elution buffer of the same company to obtain an IgG fraction. The purity of this purified antibody is SDS-
It was assayed by PAGE electrophoresis.
【００１５】得られたＩｇＧ画分は必要に応じてペプタ
イドカラムにてアフィニティー精製した。即ち、免疫原
に用いたペプタイドをＣＮＢｒ−ａｃｔｉｖａｔｅｄ−
Ｓｅｐｈａｒｏｓｅ４Ｂ（ファルマシア社製）に結合さ
せ、カラムに充填しＰＢＳにて平衡化する。上記のアフ
ィニティー精製したＩｇＧ画分をＰＢＳにて透析し、カ
ラムに流して抗体を特異的に結合させた後０．２Ｍグリ
シン（ｐＨ２．５）にて溶出させ、ポリクローナル抗体
ＭＲＰ−Ｐを得た。The obtained IgG fraction was affinity purified with a peptide column as needed. That is, the peptide used as the immunogen was prepared from CNBr-activated-
It is bound to Sepharose 4B (Pharmacia), packed in a column and equilibrated with PBS. The above-mentioned affinity-purified IgG fraction was dialyzed against PBS, passed through a column to specifically bind the antibody, and then eluted with 0.2 M glycine (pH 2.5) to obtain a polyclonal antibody MRP-P. .
【００１６】[0016]
【実施例２】下記により、アフィニティー精製ポリクロ
ーナル抗体とＣ−Ａ５００、ＫＢ細胞の可溶化蛋白質と
のイムノブロッティングによる認識抗原分子量の確認を
行った。Example 2 As described below, the molecular weight of the recognized antigen was confirmed by immunoblotting of the affinity-purified polyclonal antibody with the solubilized protein of C-A500 and KB cells.
【００１７】Ｃ−Ａ５００、ＫＢ細胞それぞれ１×１０
7個ずつＰＢＳにて洗浄し、１％ＴｒｉｔｏｎＸ−１０
０、０．２％ＳＤＳ存在下でホモジナイズ後１０００
ｘ ｇ、１０ｍｉｎ遠心分離し、その上清をさらに１０
０００ ｘ ｇ、５０ｍｉｎにて遠心分離し、沈殿を回収
した。沈殿は、溶解後蛋白質を測定し、７．５％のポリ
アクリルアミドゲルにてＳＤＳ−ＰＡＧＥを行なった。
泳動後ＰＶＤＦ膜に転写し、アフィニティー精製ポリク
ローナル抗体を反応させ、Ａｍｅｒｓｈａｍ社のＥＣＬ
ｄｅｔｅｃｔｉｏｎ ｋｉｔにより検出した。1 × 10 each of C-A500 and KB cells
Wash with 7 PBS each, 1% TritonX-10
0, 1000 after homogenization in the presence of 0.2% SDS
Centrifuge at xg for 10 min and add 10 times to the supernatant.
Centrifugation was performed at 000 xg for 50 minutes to collect the precipitate. For the precipitation, the protein was measured after dissolution, and SDS-PAGE was performed on a 7.5% polyacrylamide gel.
After migration, transfer to a PVDF membrane and react with an affinity-purified polyclonal antibody to obtain ECL from Amersham.
It was detected by the detection kit.
【００１８】得られた結果を図１に示した。図中、レー
ン１、２は抗体濃度０．０３４μｇ／ｍｌ、レーン３、
４は抗体濃度０．０１７μｇ／ｍｌを表わし、レーン
５、６はネガティブコントロールである。そして、レー
ン１〜６において、１、３、５はＫＢ細胞を表わし、
２、４、６はＣ−Ａ５００細胞を表わす。図１に示した
ように、Ｃ−Ａ５００細胞のみに１９０ｋＤａのバンド
が検出された。The results obtained are shown in FIG. In the figure, lanes 1 and 2 have an antibody concentration of 0.034 μg / ml, lane 3,
4 represents an antibody concentration of 0.017 μg / ml, and lanes 5 and 6 are negative controls. And, in lanes 1 to 6, 1, 3, 5 represent KB cells,
2, 4, 6 represent C-A500 cells. As shown in FIG. 1, a 190 kDa band was detected only in C-A500 cells.
【００１９】[0019]
【発明の効果】本発明によってＭＲＰを特異的に認識す
るポリクローナル抗体の製造がはじめて可能となった。
本抗体は特異性が高いのでＭＲＰの検出、定量性にすぐ
れ、例えばＭＲＰを発現している細胞の検出により、多
剤耐性癌の早期発見が可能となる。INDUSTRIAL APPLICABILITY The present invention makes it possible for the first time to produce a polyclonal antibody that specifically recognizes MRP.
Since this antibody has high specificity, it is excellent in MRP detection and quantification. For example, the detection of cells expressing MRP enables early detection of multidrug-resistant cancer.
【００２０】その結果、効果のない通常の抗癌剤の使用
がさし控えられ、患者の苦痛や負担が軽減されると同時
に無駄な医療を行う必要がなくなり、したがって本発明
は、積極的に癌の治療に貢献するのとは異なり、これと
は別の貢献をなすものであって、新しいタイプの有効性
を示すものである。As a result, the use of ineffective ordinary anti-cancer agents is restrained, and the pain and burden on the patient are reduced, and at the same time unnecessary medical treatment is eliminated. Unlike contributing to treatment, it is a separate contribution and represents a new type of efficacy.
【図１】Ｃ−Ａ５００細胞、ＫＢ細胞を可溶化後、イム
ノブロッティングで蛋白質をＰＶＤＦ膜に転写し抗体を
反応させた図面である。FIG. 1 is a drawing showing that after solubilizing C-A500 cells and KB cells, a protein was transferred onto a PVDF membrane by immunoblotting and reacted with an antibody.
Claims (6)
識する抗体。1. An antibody that specifically recognizes a multidrug resistance protein (MRP).
抗原として得られる抗体。 Glu-Gln-Glu-Arg-Phe-Ile-His-Gln-Ser-Asp-Leu-Lys-Va
l-Asp-Glu-Asn-Gln-Lys2. An antibody obtained by using a peptide containing the following amino acid sequence as an antigen. Glu-Gln-Glu-Arg-Phe-Ile-His-Gln-Ser-Asp-Leu-Lys-Va
l-Asp-Glu-Asn-Gln-Lys
特徴とする請求項１又は請求項２に記載の抗体。3. The antibody according to claim 1 or 2, wherein the antibody is a polyclonal antibody.
ＲＰを特異的に認識する抗体であることを特徴とする抗
体。4. The antibody according to claim 2 or 3 is M
An antibody which is an antibody that specifically recognizes RP.
載の抗体を用いることを特徴とするＭＲＰの測定方法。5. A method for measuring MRP, which comprises using the antibody according to any one of claims 1 to 4.
載の抗体を含有することを特徴とするＭＲＰの測定用キ
ット。6. A kit for measuring MRP, which comprises the antibody according to any one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27307594A JPH08113600A (en) | 1994-10-13 | 1994-10-13 | Antibody against mrp |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27307594A JPH08113600A (en) | 1994-10-13 | 1994-10-13 | Antibody against mrp |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08113600A true JPH08113600A (en) | 1996-05-07 |
Family
ID=17522802
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP27307594A Pending JPH08113600A (en) | 1994-10-13 | 1994-10-13 | Antibody against mrp |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08113600A (en) |
-
1994
- 1994-10-13 JP JP27307594A patent/JPH08113600A/en active Pending
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