JPH09249699A - Antihuman pivka-ii monoclonal antibody, hybridoma capable of producing the same antibody and measuring reagent and measurement using the same antibody - Google Patents

Antihuman pivka-ii monoclonal antibody, hybridoma capable of producing the same antibody and measuring reagent and measurement using the same antibody

Info

Publication number
JPH09249699A
JPH09249699A JP8080496A JP8049696A JPH09249699A JP H09249699 A JPH09249699 A JP H09249699A JP 8080496 A JP8080496 A JP 8080496A JP 8049696 A JP8049696 A JP 8049696A JP H09249699 A JPH09249699 A JP H09249699A
Authority
JP
Japan
Prior art keywords
pivka
antibody
monoclonal antibody
peptide
human
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
Application number
JP8080496A
Other languages
Japanese (ja)
Inventor
Yoshiaki Uchida
好昭 内田
Yoshihiro Kurano
義裕 倉野
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.)
Fujirebio Inc
Original Assignee
Fujirebio Inc
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 Fujirebio Inc filed Critical Fujirebio Inc
Priority to JP8080496A priority Critical patent/JPH09249699A/en
Publication of JPH09249699A publication Critical patent/JPH09249699A/en
Pending legal-status Critical Current

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Abstract

PROBLEM TO BE SOLVED: To produce a monoclonal antibody and a reagent for carrying out the measurement of a human protein induced by vitamin K absence-II (PIVKA- II) in a specimen. SOLUTION: A lymphocyte obtained by immunizing a mammal with an immunogen comprising a peptide represented by the formula is fused to a cell of a myeloma of a mammal to prepare a hybridoma. An antihuman PIVKA-II monoclonal antibody is obtained therefrom. The resultant antibody is used to produce a measuring reagent to carry out the immunoassay of the human PIVKA-III contained in a specimen.

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【発明の属する技術分野】本発明は、ヒトPIVKA−
II(Protein induced by vitamin K absense-II )の
測定に用いられるモノクローナル抗体を製造するための
ペプチド、該抗体を産生するハイブリドーマ、抗ヒトP
IVKA−IIモノクローナル抗体、該抗体からなる免
疫測定試薬及びその測定方法に関する。
TECHNICAL FIELD The present invention relates to human PIVKA-
II (Protein induced by vitamin Kabsense-II) peptide for producing a monoclonal antibody, a hybridoma producing the antibody, and anti-human P
The present invention relates to an IVKA-II monoclonal antibody, an immunoassay reagent comprising the antibody, and a method for measuring the same.

【0002】[0002]

【従来の技術】ヒトPIVKA−IIは、血液凝固に関
連するプロトロンビンに類似した構造を有する糖蛋白質
である。プロトロンビンは、蛋白質のN末端の近傍に1
0個のγ−カルボキシグルタミン酸(Gla) 残基を有し、
そのアミノ酸配列は Ala Asn Thr Phe Leu Gla Gla Val Arg Lys Gly Asn Leu Gla Arg Gla Cys Val 1 10 Gla Gla Thr Cys Ser Thr Gla Gla Ala Phe Gla Ala Leu Gla Ser ・・・・ 20 30 で示される。このプロトロンビンが生体内で産生される
際に、ビタミンKの欠乏、肝機能不全、ビタミンK拮抗
剤の投与、肝細胞障害等に起因して、プロトロンビン中
の10個のγ−カルボキシグルタミン酸(Gla) の一部あ
るいは全部のカルボキル化が不完全なグルタミン酸(Gl
u) 残基を有する糖蛋白質が血液中に見出されることが
知られている。この蛋白質は異常プロトロンビン即ちヒ
トPIVKA−IIと呼ばれている。近年、肝細胞癌患
者において、血漿中にヒトPIVKA−IIが高率で発
現することが報告され、肝細胞癌のマーカー、診断のモ
ニターに利用されるようになった。
Human PIVKA-II is a glycoprotein having a structure similar to prothrombin, which is related to blood coagulation. Prothrombin is 1 near the N-terminal of the protein.
Having 0 γ-carboxyglutamic acid (Gla) residues,
The amino acid sequence is shown by Ala Asn Thr Phe Leu Gla Gla Val Arg Lys Gly Asn Leu Gla Arg Gla Cys Val 1 10 Gla Gla Thr Cys Ser Thr Gla Gla Ala Phe Gla Ala Leu Gla Ser. When this prothrombin is produced in vivo, 10 γ-carboxyglutamic acids (Gla) in prothrombin due to deficiency of vitamin K, liver dysfunction, administration of vitamin K antagonist, hepatocyte disorder, etc. Incompletely or partially carboxylated glutamic acid (Gl
u) Glycoproteins with residues are known to be found in blood. This protein is called abnormal prothrombin or human PIVKA-II. In recent years, it has been reported that human PIVKA-II is highly expressed in plasma in patients with hepatocellular carcinoma, and it has come to be used as a marker for hepatocellular carcinoma and a monitor for diagnosis.

【0003】従来ヒトPIVKA−IIを測定するため
に、ポリクローナル抗体による競合RIA法が行われて
いたが、更に選択的な測定を実施するためにヒトPIV
KA−IIに対するモノクローナル抗体が作成された。
この抗ヒトPIVKA−II抗体は、ワーファリン服用
患者血漿からプロトロンビンを除去後、正常プロトロン
ビン及びPIVKA−IIの両者に対する共通部分のモ
ノクローナル抗体を用いたアフィニティーカラムで精製
したヒトPIVKA−IIを得、これを免疫原として用
い、哺乳動物に免疫後得られた脾臓細胞と腫瘍細胞との
ハイブリドーマを作成し取得していた。さらにこのモノ
クローナル抗体を固相に結合させ、二抗体サンドイッチ
法を利用した酵素免疫測定試薬が製造されていた(特公
平5−43357号参照)。
Conventionally, a competitive RIA method using a polyclonal antibody has been performed to measure human PIVKA-II, but human PIV has been used to perform more selective measurement.
A monoclonal antibody against KA-II was made.
This anti-human PIVKA-II antibody was obtained by removing prothrombin from the plasma of a patient taking warfarin, and then obtaining human PIVKA-II purified by an affinity column using a monoclonal antibody of a common portion against both normal prothrombin and PIVKA-II. It was used as an immunogen, and a hybridoma of spleen cells and tumor cells obtained after immunization of a mammal was prepared and obtained. Further, this monoclonal antibody was bound to a solid phase to produce an enzyme immunoassay reagent using the two-antibody sandwich method (see Japanese Patent Publication No. 5-43357).

【0004】[0004]

【発明が解決しようとする課題】従来ヒトPIVKA−
IIと反応するモノクローナル抗体を製造するには、ヒ
ト血漿中に含まれる精製したヒトPIVKA−IIを免
疫原として用いているが、このヒトPIVKA−IIが
混合物であるため精製は難しく、取得した精製抗原を用
いモノクローナル抗体を製造することは容易ではなかっ
た。さらにヒトPIVKA−IIペプチドは、構成アミ
ノ酸のγ−カルボキシグルタミン酸が不安定なために容
易には合成することができない等の問題点があった。ま
た、肝細胞癌患者の早期診断にはヒトPIVKA−II
の高感度測定が望まれている。しかしながら従来法で得
たモノクローナル抗体から製造した測定試薬では、検体
中に含まれる低濃度のヒトPIVKA−IIを測定する
のは非常に難しかった。そこで測定試薬にはヒトPIV
KA−IIとの反応性が高く、ヒトトロンビンとの交差
反応を起こさない新たなモノクローナル抗体が求められ
ていた。
[Problems to be Solved by the Invention] Conventional human PIVKA-
In order to produce a monoclonal antibody that reacts with II, purified human PIVKA-II contained in human plasma is used as an immunogen, but since this human PIVKA-II is a mixture, purification is difficult, and thus the obtained purified It has not been easy to produce a monoclonal antibody using an antigen. Furthermore, the human PIVKA-II peptide has a problem that it cannot be easily synthesized because the constituent amino acid γ-carboxyglutamic acid is unstable. In addition, human PIVKA-II is used for early diagnosis of hepatocellular carcinoma patients.
Highly sensitive measurement of is desired. However, it was very difficult to measure low-concentration human PIVKA-II contained in a sample with a measurement reagent produced from a monoclonal antibody obtained by a conventional method. Therefore, human PIV is used as the measurement reagent.
There is a need for new monoclonal antibodies that are highly reactive with KA-II and that do not cross-react with human thrombin.

【0005】[0005]

【課題を解決するための手段】本発明者らは鋭意研究の
結果、ヒトプロトロンビンのN末端のアミノ酸のγ−カ
ルボキシグルタミン酸(Gla) が全てグルタミン酸(Glu)
に変換された式 Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala (I) で表されるペプチドを見出し、このペプチドから製造さ
れた免疫原を哺乳動物に免疫しハイブリドーマを作成
し、次いでモノクローナル抗体を得て本発明を完成する
に至った。
[Means for Solving the Problems] As a result of intensive studies, the present inventors have found that all of the N-terminal amino acids γ-carboxyglutamic acid (Gla) of human prothrombin are glutamic acid (Glu).
A peptide represented by the formula Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala (I) was found, and an immunogen produced from this peptide was immunized to a mammal. The present invention was completed by preparing a hybridoma and then obtaining a monoclonal antibody.

【0006】本発明の前記式(I)で表されるペプチド
は、20個のアミノ酸残基で構成される。この前記式
(I)で表されるペプチドは、前記ヒトプロトロンビン
のアミノ酸配列のうちN末端の11番目から30番目の
アミノ酸残基において、γ−カルボキシグルタミン酸
(Gla )がグルタミン酸(Glu )に置換した化合物であ
る。このペプチドは、公知ペプチド合成の方法に従い製
造することができる。ペプチド合成の方法としては、例
えばα−アミノ基をt−ブトキシカルボニル(Boc)
基、側鎖官能基をベンジルアルコール系保護基で保護す
るBoc法、α−アミノ基を9−フルオレニルメトキシ
カルボニル(Fmoc)基、側鎖官能基をt−ブチルア
ルコール系保護基で保護するFmoc法等を挙げること
ができる。これらのペプチド合成法は、液相法又は固相
法で実施することができる。液相法は、前記保護アミノ
酸を順次縮合反応と脱保護反応とを溶液中で繰り返し目
的とするペプチドを製造することができる。また固相法
は、担体として架橋したポリスチレン樹脂、ポリアクリ
ルアミドで被覆したシリカ担体等の不溶性の担体上で保
護アミノ酸の縮合反応、N−α−アミノ保護基の脱保護
反応等を繰り返すことにより実施することができる。さ
らに固相法は、自動合成機を用いて行うことが効率よく
目的のペプチドを製造するためには好ましい。前記合成
法で製造されたペプチドは、保護基をフッ化水素、トリ
フルオロ酢酸(TFA)、トリフルオロメタンスルホン
酸(TFMSA)/TFA、トリメチルシリルトリフル
オロメタンスルホン酸(TMSOTf)/TFA、トリ
メチルシリルブロミド(TMSBr)/TFA、テトラ
フルオロホウ酸(HBF4 )/TFA等を用いて除去
し、精製して目的のペプチドを製造することができる。
The peptide represented by the above formula (I) of the present invention is composed of 20 amino acid residues. In the peptide represented by the above formula (I), γ-carboxyglutamic acid (Gla) is replaced with glutamic acid (Glu) at the N-terminal 11th to 30th amino acid residues in the amino acid sequence of human prothrombin. It is a compound. This peptide can be produced by a known peptide synthesis method. As a method for peptide synthesis, for example, an α-amino group is added to t-butoxycarbonyl (Boc).
Group, side chain functional group is protected by benzyl alcohol-based protecting group, Boc method, α-amino group is protected by 9-fluorenylmethoxycarbonyl (Fmoc) group, side chain functional group is protected by t-butyl alcohol-based protecting group The Fmoc method and the like can be mentioned. These peptide synthesis methods can be performed by a liquid phase method or a solid phase method. In the liquid phase method, the desired peptide can be produced by repeating a condensation reaction and a deprotection reaction of the protected amino acid in order in a solution. The solid phase method is carried out by repeating the condensation reaction of the protected amino acid and the deprotection reaction of the N-α-amino protecting group on an insoluble carrier such as a crosslinked polystyrene resin or a silica carrier coated with polyacrylamide. can do. Furthermore, the solid phase method is preferably performed using an automatic synthesizer in order to efficiently produce the target peptide. The peptide produced by the above synthetic method has hydrogen fluoride, trifluoroacetic acid (TFA), trifluoromethanesulfonic acid (TFMSA) / TFA, trimethylsilyltrifluoromethanesulfonic acid (TMSOTf) / TFA, and trimethylsilylbromide (TMSBr) as protective groups. / TFA, tetrafluoroboric acid (HBF 4 ) / TFA and the like can be removed and purified to produce the desired peptide.

【0007】このようにして製造された前記式(I)で
表されるペプチドは、ペプチド鎖中のCysが還元型で
形成されているが、前記式(I)で表されるペプチド中
の2個のCys残基のチオール基を酸化し、ジスルフィ
ド結合で分子内架橋した環状のペプチド構造を有する酸
化型の式
In the peptide represented by the above formula (I) produced in this manner, Cys in the peptide chain is formed in a reduced form, but 2 of the peptides represented by the above formula (I) are formed. Oxidation formula having a cyclic peptide structure in which the thiol group of each Cys residue is oxidized and intramolecularly bridged by a disulfide bond

【化2】 で表されるペプチドを製造することができる。前記式
(II)で表されるペプチドは、PIVKA−IIの三
次元構造と類似するため、抗PIVKA−IIモノクロ
ーナル抗体を製造するための免疫原として用いるために
は好ましい。
Embedded image The peptide represented by can be produced. The peptide represented by the formula (II) is preferable for use as an immunogen for producing an anti-PIVKA-II monoclonal antibody because it has a three-dimensional structure similar to that of PIVKA-II.

【0008】次いで前記式(II)で表されるペプチド
は免疫原として哺乳動物に投与しリンパ球細胞を得、ヒ
トPIVKA−IIを認識するモノクローナル抗体を産
生するハイブリドーマを製造することができる。この前
記式(II)で表されるペプチドを免疫原として用いる
ためには、ペプチドのN末端又はC末端のそれぞれに1
0個までのアミノ酸残基を結合させてペプチド誘導体を
製造して用いることもできる。またこれらのペプチドは
前記式(II)で表されるペプチド又はその誘導体を単
独に哺乳動物に投与し免疫することもできるが、当業者
には周知の方法により各種キャリアーとなる蛋白質との
複合体を製造し、この複合体を投与することがさらに効
率よく抗体産生リンパ球を取り出すためには好ましい。
キャリアー蛋白質としては例えばKLH(Keyhole Limp
et Hemocyanin )、BSA(ウシ血清アルブミン)、M
SA(マウス血清アルブミン)等を用いるとができる。
この複合体を製造するには、例えば前記式(II)で表
されるペプチドとキャリアー蛋白質との官能基を縮合し
化学結合させる方法、架橋剤を用い両者を化学結合させ
る方法等を用いることができる。
Then, the peptide represented by the above formula (II) can be administered as an immunogen to a mammal to obtain lymphocytes, and a hybridoma producing a monoclonal antibody that recognizes human PIVKA-II can be produced. In order to use the peptide represented by the above formula (II) as an immunogen, 1 is added to each of the N-terminal and C-terminal of the peptide.
It is also possible to produce and use a peptide derivative by linking up to 0 amino acid residues. These peptides can also be administered to a mammal by immunizing a mammal with the peptide represented by the above formula (II) or a derivative thereof alone, and a complex with a protein serving as various carriers by a method well known to those skilled in the art. It is preferable to produce and to administer this complex in order to more efficiently remove antibody-producing lymphocytes.
Examples of carrier proteins include KLH (Keyhole Limp
et Hemocyanin), BSA (bovine serum albumin), M
SA (mouse serum albumin) or the like can be used.
In order to produce this complex, for example, a method of condensing the functional group of the peptide represented by the above formula (II) and the carrier protein to chemically bond them, a method of chemically bonding both with a crosslinking agent, and the like are used. it can.

【0009】さらに、前記した免疫原となるペプチドを
免疫しモノクローナル抗体を産生するハイブリドーマを
製造することができる。この製造方法は、まずハイブリ
ドーマを作成するため抗原を免疫した哺乳動物のリンパ
球と、これと融合させる哺乳動物のミエローマ細胞(骨
髄腫細胞)を用意する。この前記リンパ球を採取するに
は、例えば前記前記式(II)で表されるペプチドとキ
ャリアー蛋白質とを結合させた複合体からなる抗原を作
成し、この抗原をマウス、ラット等の哺乳動物に免疫す
る。
Furthermore, a hybridoma which immunizes with the above-mentioned peptide serving as an immunogen and produces a monoclonal antibody can be produced. In this production method, first, lymphocytes of a mammal immunized with an antigen to prepare a hybridoma and mammalian myeloma cells (myeloma cells) to be fused with the lymphocytes are prepared. To collect the lymphocytes, for example, an antigen composed of a complex in which the peptide represented by the formula (II) and a carrier protein are bound is prepared, and the antigen is transferred to mammals such as mice and rats. Immunize.

【0010】この免疫法は、従来の抗血清を取得する方
法に準じ、抗原の使用量、投与部位、アジュバンド等を
使用し実施することができる。この方法として例えばマ
ウスを用いる場合、マウス一匹あたり一回につき0.0
01mg〜1mg、好ましくは0.01mg〜0.1m
gの前記複合体を初回はアジュバンド(例えばフロイン
トの完全アジュバンド)とよく混合して、皮下、腹腔内
等に投与し、2週間以上経過後、再びアジュバンド(例
えばフロイントの不完全アジュバンド)をよく混合し
て、皮下、腹腔内等に投与する。さらに二週間経過後、
前記複合体を静脈内、皮下、腹腔内等に投与して充分免
疫する。このように免疫されたマウスを好ましくは最終
免疫から2〜4日後に殺し、リンパ球を採取する。リン
パ球の調整には、脾臓、リンパ節、末梢血等が用いられ
る。得られたリンパ球は培養液中に懸濁した状態で保存
される。
This immunization method can be carried out according to the conventional method for obtaining an antiserum, using the amount of the antigen used, the administration site, the adjuvant and the like. For example, when using a mouse as this method, 0.0
01 mg to 1 mg, preferably 0.01 mg to 0.1 m
g of the complex is first mixed well with an adjuvant (eg, Freund's complete adjuvant) and administered subcutaneously, intraperitoneally, etc., and after 2 weeks or more, an adjuvant (eg, Freund's incomplete adjuvant) is again administered. ) Is mixed well and administered subcutaneously, intraperitoneally, etc. After another two weeks,
The above complex is administered intravenously, subcutaneously, intraperitoneally, etc. for sufficient immunization. The mice thus immunized are preferably killed 2 to 4 days after the final immunization and lymphocytes collected. For the preparation of lymphocytes, spleen, lymph nodes, peripheral blood, etc. are used. The obtained lymphocytes are stored in a state of being suspended in the culture medium.

【0011】一方ミエローマ細胞は、前記免疫に用いた
動物と同じ種由来のものを使用することが好ましい。さ
らにそのミエローマは薬剤抵抗性の変異株であることが
好ましく、さらに未融合のミエローマ細胞がハイブリド
ーマ選択培地で生育しないものが好ましい。ミエローマ
細胞としては、例えば市販のマウスミエローマP3・X
63・Ag8・6・5・3(X63・6・5・3);P
3・X63・Ag8・U1(P3U1);ラットミエロ
ーマ 210・RCY3・Ag1・2・3等を用いるこ
とができる。
On the other hand, it is preferable to use myeloma cells derived from the same species as the animal used for the immunization. Further, the myeloma is preferably a drug-resistant mutant strain, and further, one in which unfused myeloma cells do not grow in a hybridoma selection medium is preferable. Examples of myeloma cells include commercially available mouse myeloma P3.X.
63 / Ag8 / 6/5/3 (X63 / 6/5/3); P
3 * X63 * Ag8 * U1 (P3U1); Rat myeloma 210 * RCY3 * Ag1 * 2 * 3 etc. can be used.

【0012】このミエローマ細胞を血清、好ましくは牛
胎児血清を含有するダルベッコ変法イーグル最少培地
(DMEM)、RPMI1640培地等の培地中で培養
する。次にDMEM、RPMI1640等の培地に上記
で得たリンパ球及びミエローマ細胞を各々懸濁し、混合
する。この時の混合比は任意に選択できるが、好ましく
はリンパ球:ミエローマ細胞が細胞数で1:1〜20:
1、好ましくは2:1〜5:1の比率で用いることがで
きる。混合した細胞は、融合促進剤を用いて融合を行
う。融合方法としては、例えばImmunological Methods;
Vol.2, 1981, Academic Press に従い行うことができ
る。融合促進剤としては、種々の高分子化合物、ウイル
ス等を用いることがきる。この融合促進剤として、例え
ばポリエチレングリコール(PEG),センダイウイル
スを挙げることができる。PEGは平均分子量400〜
20,000のものを使用することができるが、1,0
00〜7,500のものを用いることが好ましい。融合
促進剤の使用濃度は、40〜60vol%である。
The myeloma cells are cultured in a medium containing serum, preferably fetal bovine serum, such as Dulbecco's modified Eagle's minimal medium (DMEM) and RPMI1640 medium. Next, the lymphocytes and myeloma cells obtained above are suspended and mixed in a medium such as DMEM or RPMI1640. The mixing ratio at this time can be arbitrarily selected, but preferably the lymphocyte: myeloma cell number is 1: 1 to 20 :.
It can be used in a ratio of 1, preferably 2: 1 to 5: 1. The mixed cells are fused using a fusion promoter. As the fusion method, for example, Immunological Methods;
It can be done according to Vol.2, 1981, Academic Press. As the fusion promoter, various polymer compounds, viruses and the like can be used. Examples of this fusion promoter include polyethylene glycol (PEG) and Sendai virus. PEG has an average molecular weight of 400-
20,000 can be used, but 1,0
It is preferable to use the one of 00 to 7,500. The concentration of the fusion promoter used is 40 to 60% by volume.

【0013】融合させた細胞は、洗浄して融合促進剤を
除去し、5〜15vol%の血清を含むDMEM又はR
PMI1640培地に懸濁し、96穴培養皿等に0.1
〜1×106 /穴の割合で分注する。さらに、各穴に選
択培地(例えば、HAT培地)を加え、適宜選択培地を
交換することによりハイブリドーマを選択することがで
きる。
The fused cells are washed to remove the fusion promoter, and DMEM or R containing 5 to 15 vol% serum is added.
Suspend in PMI1640 medium and add 0.1 to 96-well culture dish.
Dispense ~ 1 x 10 6 / hole. Furthermore, a hybridoma can be selected by adding a selective medium (for example, HAT medium) to each well and exchanging the selective medium appropriately.

【0014】次にヒトPIVKA−IIに対する抗体を
産生するハイブリドーマを検索、選別する。その方法に
はELISA法を用いることができる。精製PIVKA
−IIをELISAプレートに吸着させ、これにハイブ
リドーマ上清を加え反応を行った後、洗浄し市販の免疫
動物免疫グロブリンに対する標識抗体(例えば西洋ワサ
ビパーオキシダーゼ(HRP)標識抗体あるいはヨウ素
125標識抗体)を添加する。その結果ハイブリドーマ
上清中にヒトPIVKA−IIに対する抗体が存在する
場合には、その抗体が固相ヒトPIVKA−IIに結合
し、さらに免疫グロブリンに対する標識抗体がこれに結
合して、標識によるシグナルが得られる。一方、上清中
にヒトPIVKA−IIに対する抗体が存在しない場合
には、固相の複合体には何も結合せず従ってシグナルも
得られない。このように、標識によるシグナルの有無を
手がかりとしてハイブリドーマの選択を行うことができ
る。
Next, a hybridoma producing an antibody against human PIVKA-II is searched and selected. The ELISA method can be used for the method. Purified PIVKA
-II is adsorbed on an ELISA plate, a hybridoma supernatant is added thereto, and the reaction is carried out, followed by washing and labeling antibody against commercially available immunized animal immunoglobulin (for example, horseradish peroxidase (HRP) -labeled antibody or iodine 125-labeled antibody) Is added. As a result, when an antibody against human PIVKA-II is present in the hybridoma supernatant, the antibody binds to solid-phase human PIVKA-II, and a labeled antibody against immunoglobulin binds to it, resulting in a signal from the label. can get. On the other hand, when the antibody against human PIVKA-II is not present in the supernatant, nothing is bound to the solid-phase complex and therefore no signal is obtained. In this way, hybridomas can be selected based on the presence or absence of a signal by the label.

【0015】このハイブリドーマ細胞株は、通常用いら
れる培地で増殖可能である。例えば牛胎児血清を5〜2
0%含有するRPMI1640又はDMEMを培地とし
て用い、37℃炭酸ガス濃度5vol%含有空気下でよ
く増殖する。また、ハイブリドーマ細胞株はミエローマ
の持つ増殖性を有するので、生体内(例えば同系の動
物、ヌードマウス等)で増殖し、ヒトPIVKA−II
に対する抗体を産生することができる。
This hybridoma cell line can grow in a medium which is usually used. For example, 5 to 2 fetal bovine serum
Using RPMI1640 or DMEM containing 0% as a medium, it grows well in the air containing carbon dioxide gas concentration of 5 vol% at 37 ° C. In addition, since the hybridoma cell line has the proliferative property of myeloma, it proliferates in vivo (for example, an animal of the same strain, a nude mouse, etc.) and human PIVKA-II.
Antibodies can be produced.

【0016】このようにして得られた抗体は、必要に応
じ精製して使用することができる。精製には例えば硫安
分画、イオン交換クロマトグラフィー、プロテインAを
固定したアフィニティークロマトグラフィー等、通常の
蛋白質を精製する手段を用いることができる。
The antibody thus obtained can be purified before use, if necessary. For purification, ordinary means for purifying proteins such as ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography with protein A immobilized thereon can be used.

【0017】前記方法により製造したモノクローナル抗
体を用いてPIVKA−IIの測定試薬を製造すること
ができる。試薬を製造するには、まず間接凝集免疫測
定、標識免疫測定等の免疫測定用固相に抗体を結合させ
製造することができる。固相としては、例えばゼラチ
ン、アラビアゴム及びメタリン酸塩を不溶化して得られ
るゼラチン粒子、羊、山羊、馬、牛、家兎等の哺乳動物
赤血球、ラテックス、カオリン等の凝集免疫測定用粒
子、プラスチック試験管、マイクロタイタープレート、
ガラスビーズ、プラスチックビーズ、メンブレン等の標
識免疫測定用固相を挙げることができる。
The PIVKA-II assay reagent can be produced using the monoclonal antibody produced by the above method. In order to produce the reagent, first, the antibody can be bound to a solid phase for immunoassay such as indirect agglutination immunoassay, labeled immunoassay and the like for production. As the solid phase, for example, gelatin, gelatin particles obtained by insolubilizing gum arabic and metaphosphate, sheep red blood cells such as goats, horses, cows and rabbits, latex, and particles for agglutination immunoassay such as kaolin, Plastic test tube, microtiter plate,
Examples include solid phase for labeled immunoassay such as glass beads, plastic beads, and membranes.

【0018】固相とモノクローナル抗体との結合には、
公知の共有結合又は非共有結合を作る方法を利用して製
造することができる。結合の方法には、例えばグルタル
アルデヒド法、過ヨウ素酸法、マレイミド法、ピリジル
・ジスルフィド法、各種架橋剤を用いる方法等を挙げる
ことができる(例えば「蛋白質核酸酵素」別冊31号、
37〜45頁(1985年)参照)。共有結合による方
法では、モノクローナル抗体に存在する官能基を利用で
きるほか、抗体に例えばチオール基、アミノ基、カルボ
キシル基、水酸基等の基を導入した後、前記結合方法に
従い反応を行うことができる。また非共有結合による方
法としては物理吸着法等を挙げるとができる。結合に用
いるモノクローナル抗体は、前記抗体を酵素処理して製
造した抗体のFab、F(ab’)2 等のフラグメント
であってもよい。
For binding the solid phase to the monoclonal antibody,
It can be produced by using a known method for producing a covalent bond or a non-covalent bond. Examples of the binding method include the glutaraldehyde method, the periodate method, the maleimide method, the pyridyl disulfide method, and methods using various crosslinking agents (for example, “Protein Nucleic Acid Enzyme” Supplement No. 31,
37-45 (1985)). In the method using a covalent bond, a functional group existing in the monoclonal antibody can be used, or after introducing a group such as a thiol group, an amino group, a carboxyl group, and a hydroxyl group into the antibody, the reaction can be performed according to the above-mentioned bonding method. In addition, examples of the method using non-covalent bond include physical adsorption method. The monoclonal antibody used for binding may be a fragment of the antibody such as Fab and F (ab ′) 2 produced by enzymatically treating the antibody.

【0019】前記ヒトPIVKA−IIの免疫測定試薬
は、検体と反応させて検体中のヒトPIVKA−IIを
測定することができる。前記間接凝集試薬では、検体と
反応させその凝集像から検体中のヒトPIVKA−II
の測定を行うことができる。また標識免疫測定試薬は、
標識した抗ヒトPIVKA−II抗体又は抗プロトロン
ビン抗体等と組み合わせたサンドイッチ法、標識ヒトP
IVKA−IIを用いた競合法等の周知の方法に従い測
定を行うことができる。この標識物としては、免疫測定
に用いられる例えば酵素、放射性同位元素、蛍光物質、
発光物質、着色粒子、コロイド粒子等を挙げるとができ
る。標識免疫測定法では、反応後固相に結合した前記標
識物を直接的又は間接的に検出し測定を行うことができ
る。検出には標識物によりそれぞれ対応する目視による
方法の他、シンチレーションカウンター、比色計、蛍光
光度計、フォトンカウンター、感光フィルム等の測定装
置を用い標識物の測定を行うことができる。標識物が酵
素の場合には発光基質、蛍光基質、発色基質等を加えて
反応液に生ずる発光、蛍光、発色等を目視又は前記測定
装置を用いて測定を行うことができる。
The human PIVKA-II immunoassay reagent can be reacted with a sample to measure human PIVKA-II in the sample. The indirect agglutination reagent is reacted with the sample, and from the agglutination image, human PIVKA-II in the sample is detected.
Can be measured. The labeled immunoassay reagent is
Sandwich method in combination with labeled anti-human PIVKA-II antibody or anti-prothrombin antibody, labeled human P
The measurement can be performed according to a well-known method such as a competitive method using IVKA-II. As the labeled substance, for example, an enzyme used for immunoassay, a radioisotope, a fluorescent substance,
Examples include luminescent substances, colored particles, colloidal particles, and the like. In the labeled immunoassay, the labeled substance bound to the solid phase can be directly or indirectly detected and measured after the reaction. For the detection, in addition to a visual method corresponding to each labeled substance, the labeled substance can be measured using a measuring device such as a scintillation counter, a colorimeter, a fluorometer, a photon counter, or a photosensitive film. When the labeling substance is an enzyme, a luminescent substrate, a fluorescent substrate, a color-developing substrate, etc. are added, and the luminescence, fluorescence, color development, etc. generated in the reaction solution can be measured visually or by using the above-mentioned measuring device.

【0020】本発明は、測定検体に制限はなく例えば血
清、血漿、全血、尿、リンパ液等の各種体液中のPIV
KA−IIの測定に適用することができる。
In the present invention, there is no limitation on the sample to be measured, and for example, PIV in various body fluids such as serum, plasma, whole blood, urine and lymph.
It can be applied to the measurement of KA-II.

【0021】[0021]

【実施例】以下、本発明を実施例によりさらに詳細に説
明する。
The present invention will be described in more detail with reference to the following examples.

【0022】実施例1 ペプチド(前記式(I)で表さ
れるペプチド)の製造 アプライドバイオシステムズ431Aペプチド合成機
(パーキンエルマー社製)を用いて合成を行った。アミ
ノ酸はグリシンを除いて、すべてL体を用いた。各アミ
ノ酸のα−アミノ基はFmoc(9−フルオレニルメト
キシカルボニル)基で保護し、アミノ酸側鎖官能基の保
護には以下の保護基を使用した。Cysのβ−スルフヒ
ドリル基とAsnのβ−カルボキサミド基はTrt(ト
リフェニルメチル)基、Gluのγ−カルボキシル基、
Ser、Thrのβ−水酸基及びTyrのフェノール性
水酸基はすべてt−Bu(t−ブチル)基、Argのグ
アニジノ基はPMC基(2,2,5,7,8-ペンタメチルクロマ
ン-6- スルホニル)基。
Example 1 Production of peptide (peptide represented by the above formula (I)) Synthesis was carried out using an Applied Biosystems 431A peptide synthesizer (manufactured by Perkin Elmer). Amino acids used were all L-forms except glycine. The α-amino group of each amino acid was protected with an Fmoc (9-fluorenylmethoxycarbonyl) group, and the following protecting groups were used to protect the amino acid side chain functional group. The β-sulfhydryl group of Cys and the β-carboxamide group of Asn are Trt (triphenylmethyl) group, γ-carboxyl group of Glu,
The β-hydroxyl group of Ser and Thr and the phenolic hydroxyl group of Tyr are all t-Bu (t-butyl) group, and the guanidino group of Arg is PMC group (2,2,5,7,8-pentamethylchroman-6-sulfonyl). ) Group.

【0023】ペプチド合成を開始するための固相担体
は、HMPレジン(パーキンエルマー社)を用い最初に
Fmoc−Alaを431合成機に添付されている合成
プログラムであるLoading Cycleにより導
入した。ペプチド鎖の延長は、431A合成機の標準的
な方法であるFmoc/HOBt/NMP法で自動的に
行った。反応を0.25mmolのスケールで開始し、
1070mg(収率86%)の保護基を含むペプチド担
体を得た。
As a solid phase carrier for initiating peptide synthesis, HMP resin (Perkin Elmer) was used, and Fmoc-Ala was first introduced by a loading program, which is a synthesis program attached to the 431 synthesizer. The peptide chain extension was automatically performed by the Fmoc / HOBt / NMP method, which is a standard method of the 431A synthesizer. Start the reaction on a 0.25 mmol scale,
1070 mg (yield 86%) of a peptide carrier containing a protecting group was obtained.

【0024】上記担体を添加物を含むトリフルオロ酢酸
溶液(トリフルオロ酢酸10ml,水0.5ml,フェ
ノール0.4g,チオアニソール0.5ml,エタンジ
チオール0.25ml)中室温で90分間処理して、遊
離のペプチド鎖を取り出し、ジエチルエーテルで沈殿さ
せ、ろ取した。さらにこの沈殿を添加剤を含むトリフル
オロ酢酸溶液(トリフルオロ酢酸10ml,チオアニソ
ール0.5ml,エタンジチオール0.25ml)中で
氷冷下40分処理し、ジエチルエーテルで沈殿させ、ろ
取し、水に懸濁後凍結乾燥して前記式(I)で表される
粗ペプチド390mg(収率68%)を得た。
The above carrier was treated in a trifluoroacetic acid solution containing additives (10 ml of trifluoroacetic acid, 0.5 ml of water, 0.4 g of phenol, 0.5 ml of thioanisole, 0.25 ml of ethanedithiol) at room temperature for 90 minutes. The free peptide chain was taken out, precipitated with diethyl ether, and collected by filtration. Further, this precipitate was treated with an additive-containing trifluoroacetic acid solution (trifluoroacetic acid 10 ml, thioanisole 0.5 ml, ethanedithiol 0.25 ml) under ice cooling for 40 minutes, precipitated with diethyl ether, and collected by filtration. After suspending in water and freeze-drying, 390 mg (yield 68%) of the crude peptide represented by the formula (I) was obtained.

【0025】前記式(I)で表されるペプチドに含まれ
る2つのCys残基がスルフヒドリル(SH)型の還元
型であることを確認するため以下の実験を行った。得ら
れた前記式(I)で表される粗ペプチドを1MTris
−HCl緩衝液(pH8.9)に溶解し、逆相カラムを
用いた高速液体クロマトグラフィで分析した。溶解直後
の分析(図1,a)では、主に保持時間が14.5分、
16.9分、19.9分にピークが見られたが。2時間
後(図1,b)には16.9分のピークはほぼ消失し、
主に14.5分、19.9分のピークが見られた。この
溶液に還元剤であるジチオスレイトール(DTT)を過
剰量加えたものを分析したところ(図1,c)14.5
分、19.9分のピークはなくなり、(DTTの還元
型、酸化型のピークを除いて)16.9分のピークだけ
となった。
The following experiment was carried out to confirm that the two Cys residues contained in the peptide represented by the above formula (I) are sulfhydryl (SH) type reduced forms. The obtained crude peptide represented by the formula (I) was added to 1 MTris
-Dissolved in HCl buffer (pH 8.9) and analyzed by high performance liquid chromatography using a reverse phase column. In the analysis immediately after dissolution (Fig. 1, a), the retention time was mainly 14.5 minutes,
Peaks were seen at 16.9 and 19.9 minutes. After 2 hours (Fig. 1, b), the peak at 16.9 minutes almost disappeared,
Peaks at 14.5 minutes and 19.9 minutes were mainly observed. When an excess amount of dithiothreitol (DTT) as a reducing agent was added to this solution, it was analyzed (Fig. 1, c).
The peak for 1 minute and 19.9 minutes disappeared, and only the peak for 16.9 minutes (excluding the reduced and oxidized peaks of DTT).

【0026】これらの結果から、14.5分のピークは
Cys残基側鎖がジスルフィド結合した酸化型の一量
体、16.9分のピークは還元型、19.9分のピーク
は酸化型の二量体であることが明らかとなった。従って
溶解前の粗ペプチドは還元型を主に含むと考えられる。
From these results, the peak at 14.5 minutes is an oxidized monomer in which the side chain of the Cys residue is disulfide-bonded, the peak at 16.9 minutes is reduced, and the peak at 19.9 minutes is oxidized. It was revealed to be a dimer of. Therefore, it is considered that the crude peptide before dissolution mainly contains the reduced form.

【0027】実施例2 前記式(II)で表されるペプ
チド(E7)の製造 前記実施例1で製造した式(I)で表されるペプチド5
3.2mgを0.2MTris-HCl 緩衝液(pH8.9) 50m
lに攪拌しながら加え溶解した。この溶液を室温で激し
く攪拌し、空気酸化を行った。高速液体クロマトグラフ
ィで反応を追跡し、3時間後に反応液を酢酸で中和し
た。この溶液をMCI GEL CHP20P(三菱化
学)カラム(12ml)に通してペプチドを吸着させ、
30%アセトニトリルを含む0.1%アンモニア水で溶
出し、パウリ反応陽性の部分を集めて凍結乾燥した。4
5.35mg(収率82%)の前記式(II)で表され
る粗ペプチドを得た。さらにこのペプチドを逆相高速液
体クロマトグラフィ(カラム:Asahipack ODP-90 21.5m
ml.D. × 300mmL.; 溶媒:0.1%トリフルオロ酢酸/
水と0.1%トリフルオロ酢酸/アセトニトリルの10
〜60%直線濃度勾配)で精製し、溶出画分を減圧下濃
縮後、凍結乾燥し前記式(II)で表されるペプチド
(以下E7という。)12.6mg(収率28%)を得
た。
Example 2 Production of Peptide (E7) Represented by Formula (II) The peptide 5 represented by Formula (I) produced in Example 1 above
3.2 mg of 0.2 M Tris-HCl buffer solution (pH 8.9) 50 m
It was added to 1 with stirring and dissolved. This solution was vigorously stirred at room temperature and air-oxidized. The reaction was followed by high performance liquid chromatography, and after 3 hours, the reaction solution was neutralized with acetic acid. This solution was passed through an MCI GEL CHP20P (Mitsubishi Chemical) column (12 ml) to adsorb the peptide,
Elution was performed with 0.1% ammonia water containing 30% acetonitrile, and the Pauli reaction positive portions were collected and lyophilized. Four
5.35 mg (yield 82%) of the crude peptide represented by the above formula (II) was obtained. Furthermore, this peptide was applied to reversed-phase high performance liquid chromatography (column: Asahipack ODP-90 21.5m
ml.D. × 300mmL .; Solvent: 0.1% trifluoroacetic acid /
10 with water and 0.1% trifluoroacetic acid / acetonitrile
˜60% linear concentration gradient), and the eluate fraction is concentrated under reduced pressure and freeze-dried to obtain 12.6 mg (yield 28%) of the peptide represented by the formula (II) (hereinafter referred to as E7). It was

【0028】E7ペプチドを加水分解しアミノ酸分析を
行った。加水分解は、ペプチド1.03mgを試験管に
とり、濃塩酸−トリフルオロ酢酸(2:1)を加え、減
圧下脱気、封管し、166℃のヒートブロック中で25
分間行った(A. Tsugita, J.-J. Scheffler, Eur. J. B
iochem., 124, 585 (1982)参照)。アミノ酸分析は、J
LC−300全自動アミノ酸分析計(日本電子製)でニ
ンヒドリン法で行った。その結果を以下に示す。Asp 1.
02(1), Thr 0.70(1), Ser 0.56(1), Glu 6.64(7), Gly
1.00(1), Ala 2.00(2), Cys 1.70(2), Val 0.96(1), Le
u 1.00(1), Tyr 0.95(1), Phe 0.99(1), Arg 0.99(1)
カッコ内は配列からの予想値
The E7 peptide was hydrolyzed and subjected to amino acid analysis. For hydrolysis, take 1.03 mg of the peptide in a test tube, add concentrated hydrochloric acid-trifluoroacetic acid (2: 1), degas under reduced pressure, seal the tube, and heat in a heat block at 166 ° C. for 25 hours.
I went for a minute (A. Tsugita, J.-J. Scheffler, Eur. J. B
iochem., 124, 585 (1982)). Amino acid analysis is J
It was carried out by a ninhydrin method with an LC-300 fully automatic amino acid analyzer (manufactured by JEOL Ltd.). The results are shown below. Asp 1.
02 (1), Thr 0.70 (1), Ser 0.56 (1), Glu 6.64 (7), Gly
1.00 (1), Ala 2.00 (2), Cys 1.70 (2), Val 0.96 (1), Le
u 1.00 (1), Tyr 0.95 (1), Phe 0.99 (1), Arg 0.99 (1)
Expected value from the array in parentheses

【0029】また、ペプチドをProtein Sequencer Pr
ocise 494(パーキンエルマー社製)にかけて
分析を行った。その結果を図2に示す。
In addition, the peptide was labeled with Protein Sequencer Pr.
The analysis was carried out by using ocise 494 (manufactured by Perkin Elmer). The result is shown in FIG.

【0030】実施例3 E7−KLH複合体の作製 KLH(Keyhole Limpet Hemocyanin ;CALBIOC
HEM社製)5mgを400μlの1%のジチオスレイ
トールを含む0.1Mリン酸緩衝液(pH7.5)に溶
解し、同緩衝液100μlに溶解した2−イミノチオラ
ン塩酸塩(SIGMA CHEMICAL社製)1mg
を加え、室温で1時間攪拌した。反応液を1mMのED
TAを含む0.1Mリン酸緩衝液(pH7.0)で平衡
化したPD−10カラム(ファルマシアバイオテク社
製)にかけ、同緩衝液で溶出した。カラム溶出液の初め
の2.5mlは廃棄し、それに続く2.0mlを集め
た。
Example 3 Preparation of E7-KLH Complex KLH (Keyhole Limpet Hemocyanin; CALBIOC)
2-iminothiolane hydrochloride (manufactured by SIGMA CHEMICAL) dissolved in 100 μl of a 0.1 M phosphate buffer (pH 7.5) containing 400 μl of 1% dithiothreitol. 1 mg
Was added and stirred at room temperature for 1 hour. The reaction solution is 1 mM ED
It was applied to a PD-10 column (Pharmacia Biotech) equilibrated with 0.1 M phosphate buffer (pH 7.0) containing TA and eluted with the same buffer. The first 2.5 ml of column eluate was discarded and the subsequent 2.0 ml was collected.

【0031】一方、E7 2.31mgを0.1Mリン
酸緩衝液(pH7.5)800μlに溶解し、DMF
(ジメチルホルムアミド)230μlに溶解したGMB
S0.30mg加え、室温で1時間攪拌した。反応液を
前記KLH溶液と混合し、室温で1時間攪拌した。0.
1Mリン酸緩衝液(pH7.5)100μlを溶解した
マレイミド1mgを加えさらに1時間攪拌した後、反応
液を透析チューブに移し、PBS(リン酸化生理食塩
水)に対し4℃で一夜透析し、E7−KLH複合体溶液
を得た。この溶液の蛋白濃度は、BCA Protein Assa
y Reagent (PIERCE社製)により定量し、810
μg/mlであった。
On the other hand, 2.31 mg of E7 was dissolved in 800 μl of 0.1 M phosphate buffer (pH 7.5) to prepare DMF.
GMB dissolved in 230 μl (dimethylformamide)
S0.30 mg was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was mixed with the KLH solution and stirred at room temperature for 1 hour. 0.
After adding 1 mg of maleimide dissolved in 100 μl of 1M phosphate buffer (pH 7.5) and stirring for 1 hour, the reaction solution was transferred to a dialysis tube and dialyzed against PBS (phosphorylated physiological saline) at 4 ° C. overnight, An E7-KLH complex solution was obtained. The protein concentration of this solution is BCA Protein Assa
810 quantified by y Reagent (PIERCE)
μg / ml.

【0032】実施例4 (1)抗PIVKA−IIモノクローナル抗体の作製 実施例3で作製したE7−KLH複合体をフロイント完
全アジュバンドに充分分散させ、マウス(BALB/
C)腹腔に100μl(約40μg/マウス)で免疫し
た。約1ケ月後同じくE7−KLHをフロイント不完全
アジュバンドに分散させ腹腔に免疫した。抗体価の上昇
の確認後、静注によりE7−KLH複合体を投与し、3
日後脾臓を摘出し、ミエローマ細胞との融合を実施し
た。融合した細胞は、96ウエル カルチャー プレー
トに分注し、炭酸ガスインキュベーターで培養した。培
養上清中の抗ヒトPIVKA−II抗体の有無は、ワー
ファリン投与患者血漿から精製したヒトPIVKA−I
Iを固相化したELISAプレートで調べた。又特異性
に関しては同時にプロトロンビン(シグマ社製)を固相
化したELISAプレートを用いて調べた。これらEL
ISAにて確認された抗PIVKA−II特異抗体産生
ウエルの細胞を限界希釈法にてモノクローン化した。抗
ヒトPIVKA−IIモノクローナル抗体を産生する細
胞は、大量に培養しマウス腹腔に投与し、抗PIVKA
−IIモノクローナル抗体を大量に含む腹水を回収し
た。さらにProteinA−Sepharoseを用
い腹水より抗体を精製し、モノクローナル抗体を得た。
この抗ヒトPIVKA−IIモノクローナル抗体をE7
−222抗体と命名し、E7−222抗体を産生するハ
イブリドーマを平成8年1月9日に通産省工業技術院生
命工学工業研究所特許微生物寄託センターに寄託手続き
を行い、菌寄第15386号(FERM P−1538
6)として受け入れられた。
Example 4 (1) Preparation of anti-PIVKA-II monoclonal antibody The E7-KLH complex prepared in Example 3 was sufficiently dispersed in Freund's complete adjuvant, and the mouse (BALB / BALB /
C) The abdominal cavity was immunized with 100 μl (about 40 μg / mouse). About one month later, E7-KLH was similarly dispersed in Freund's incomplete adjuvant and the abdominal cavity was immunized. After confirming the increase in antibody titer, the E7-KLH complex was administered by intravenous injection to
After a day, the spleen was removed and fused with myeloma cells. The fused cells were dispensed into a 96-well culture plate and cultured in a carbon dioxide incubator. The presence or absence of the anti-human PIVKA-II antibody in the culture supernatant depends on whether human PIVKA-I purified from the plasma of a patient receiving warfarin.
I was examined on an ELISA plate immobilized with solid phase. The specificity was simultaneously examined using an ELISA plate on which prothrombin (manufactured by Sigma) was immobilized. These EL
The cells in the wells producing the anti-PIVKA-II specific antibody confirmed by ISA were monocloned by the limiting dilution method. The cells producing the anti-human PIVKA-II monoclonal antibody were cultured in a large amount and administered intraperitoneally to the mouse to obtain anti-PIVKA-II.
Ascites fluid containing a large amount of the -II monoclonal antibody was collected. Further, the antibody was purified from ascites using Protein A-Sepharose to obtain a monoclonal antibody.
This anti-human PIVKA-II monoclonal antibody was labeled with E7.
-222 antibody, and the hybridoma producing the E7-222 antibody was deposited at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, on January 9, 1996, and the bacterial strain No. 15386 (FERM P-1538
6) was accepted as

【0033】(2)固相化抗原に対するE7−222抗
体の反応性 Nunc社製96ウエルELISAプレートに精製PI
VKA−II、プロトロンビンをそれぞれ2μg/ml
の濃度に75μl/ウエルで4℃一夜放置し、コートし
た。プレートは1%スキムミルクPBSを各ウエルに1
50μl入れ、37℃、5時間ブロッキングした。次に
プレートを0.05%Tween(登録商標)20を含
むPBSで3回洗浄した後、1%BSA含有Tris緩
衝液pH7.4に5μg/mlに溶かしたE7−222
抗体とコントロールとしてF1−3抗体(抗プロトロン
ビンモノクローナル抗体)75μl/ウエルで加え37
℃、1時間反応させた。反応後、0.05%Tween
20含有PBSで3回洗浄し、ペルオキシダーゼ標識抗
マウス抗体(DACO社製)を加え、37℃1時間反応
させた。反応後0.05%Tween20含有PBSで
4回洗浄し、基質ABTSを加え405nmの吸光度を
測定した。結果を図3に示す。E7−222抗体は固相
化ヒトPIVKA−IIには反応するが、プロトロンビ
ンとは実質的に反応せずヒトPIVKA−IIに特異的
である。
(2) Reactivity of E7-222 antibody to immobilized antigen Purified PI was added to 96-well ELISA plate manufactured by Nunc.
VKA-II, prothrombin 2 μg / ml each
Was coated at 75 μl / well at 4 ° C. overnight. Plate 1% skim milk PBS in each well
50 μl was added and blocking was performed at 37 ° C. for 5 hours. The plates were then washed 3 times with PBS containing 0.05% Tween® 20 and then E7-222 dissolved in Tris buffer pH 7.4 containing 1% BSA at 5 μg / ml.
As antibody and control, F1-3 antibody (anti-prothrombin monoclonal antibody) was added at 75 μl / well 37
The reaction was carried out at ℃ for 1 hour. After the reaction, 0.05% Tween
The cells were washed 3 times with 20-containing PBS, a peroxidase-labeled anti-mouse antibody (manufactured by DACO) was added, and the mixture was reacted at 37 ° C for 1 hour. After the reaction, the plate was washed 4 times with PBS containing 0.05% Tween 20, the substrate ABTS was added, and the absorbance at 405 nm was measured. The results are shown in FIG. The E7-222 antibody reacts with immobilized human PIVKA-II but does not substantially react with prothrombin and is specific to human PIVKA-II.

【0034】実施例5 固相化E7−222抗体を用い
たELISAによる検体中のヒトPIVKA−IIの測
定 精製E7−222抗体をPBSに10μg/mlに溶か
し、Nunc社製96ウエルELISAプレートに75
μl/ウエルで加え、4℃一夜放置し、抗体をコートし
た。プレートは1%スキムミルクPBSを各ウエルに1
50μl入れ、37℃5時間ブロッキングした。0.0
5%Tween20含有PBSで洗浄した後、10%の
Calf Serumを含んだ0.1%Tris緩衝液
(pH7.4)35μlと血清35μlをウエルに加
え、37℃1時間反応させた。反応後プレートを0.0
5%Tween20を含むPBSで3回洗浄し、アルカ
リホスファターゼ標識F1−3抗体(抗プロトロンビン
モノクローナル抗体)を75μl/ウエルで加え、37
℃1時間反応させた。プレートは同様に0.05%Tw
een20含有PBSで3回洗浄後、蒸留水で1回洗浄
し、基質パラニトロフェニルホスフェート(PNPP)
を加え、37℃1時間反応後405nmの吸光度を測定
した。陽性検体10例、陰性検体5例について、本発明
の測定試薬と従来のPIVKA−II測定試薬(エイテ
ストモノP−II;エ−ザイ社製)で測定を行い、その
結果を図4に示す。
Example 5 Measurement of human PIVKA-II in a sample by ELISA using solid-phased E7-222 antibody Purified E7-222 antibody was dissolved in PBS at 10 μg / ml and put on a Nunc 96-well ELISA plate.
μl / well, and left overnight at 4 ° C. to coat the antibody. Plate 1% skim milk PBS in each well
50 μl was added and blocking was performed at 37 ° C. for 5 hours. 0.0
After washing with PBS containing 5% Tween 20, 35 μl of 0.1% Tris buffer (pH 7.4) containing 10% Calf Serum and 35 μl of serum were added to the wells and reacted at 37 ° C. for 1 hour. After reaction, plate 0.0
The plate was washed 3 times with PBS containing 5% Tween 20, and alkaline phosphatase-labeled F1-3 antibody (anti-prothrombin monoclonal antibody) was added at 75 μl / well.
The reaction was performed at 1 ° C for 1 hour. The plate is also 0.05% Tw
After washing 3 times with PBS containing een20, washing once with distilled water, and substrate para-nitrophenyl phosphate (PNPP)
Was added, and after reacting at 37 ° C. for 1 hour, the absorbance at 405 nm was measured. Ten positive samples and five negative samples were measured with the measurement reagent of the present invention and the conventional PIVKA-II measurement reagent (Etest Mono P-II; manufactured by Eisai Co., Ltd.), and the results are shown in FIG. .

【0035】[0035]

【発明の効果】本発明は、前記式(II)で表されるペ
プチドからなる免疫原を用いることにより、検体中のヒ
トPIVKA−IIと特異的で高い反応性を持つ抗ヒト
PIVKA−IIモノクローナル抗体が得られた。該抗
体から製造された免疫測定試薬は、検体中のヒトプロト
ロンビン等の影響を受けず、低濃度のヒトPIVKA−
IIを感度よく測定することができる。
INDUSTRIAL APPLICABILITY The present invention uses an immunogen consisting of the peptide represented by the above formula (II) to obtain an anti-human PIVKA-II monoclonal antibody having high specificity and high reactivity with human PIVKA-II in a sample. Antibodies were obtained. The immunoassay reagent produced from the antibody is not affected by human prothrombin or the like in the sample and has a low concentration of human PIVKA-.
II can be measured with high sensitivity.

【0036】[0036]

【配列表】[Sequence list]

配列番号:1 配列の長さ:20 配列の型:アミノ酸 トポロジー:直鎖状 配列の種類:ペプチド 配列 Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu 1 5 10 15 Ala Phe Glu Ala 20 SEQ ID NO: 1 Sequence length: 20 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu 1 5 10 15 Ala Phe Glu Ala 20

【図面の簡単な説明】[Brief description of drawings]

【図1】前記式(I)で表されるペプチド及び前記式
(II)で表されるペプチドの高速液体クロマトグラフ
ィでの分析結果を示す図である。
FIG. 1 is a diagram showing the results of high-performance liquid chromatography analysis of the peptide represented by the formula (I) and the peptide represented by the formula (II).

【図2】前記式(II)ペプチドのアミノ酸配列分析結
果を示す図である。
FIG. 2 shows the results of amino acid sequence analysis of the peptide of formula (II).

【図3】PIVKA−II又はプロトロンビンへのE7
−222抗体の反応性を示すELISA法の測定結果を
示す図である。
FIG. 3. E7 to PIVKA-II or prothrombin.
It is a figure which shows the measurement result of the ELISA method which shows the reactivity of the -222 antibody.

【図4】従来のPIVKA−II測定試薬(エイテスト
モノP−II;エーザイ社製)と本発明の測定試薬との
ELISA法での測定結果を示す図である。
FIG. 4 is a diagram showing measurement results by an ELISA method of a conventional PIVKA-II measurement reagent (Etest Mono P-II; manufactured by Eisai Co., Ltd.) and the measurement reagent of the present invention.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 FI 技術表示箇所 C12P 21/08 G01N 33/53 D G01N 33/53 33/566 33/577 33/566 C12N 5/00 B 33/577 9282−4B 15/00 C //(C12P 21/08 C12R 1:91) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Office reference number FI technical display location C12P 21/08 G01N 33/53 D G01N 33/53 33/566 33/577 33/566 C12N 5 / 00 B 33/577 9282-4B 15/00 C // (C12P 21/08 C12R 1:91)

Claims (8)

【特許請求の範囲】[Claims] 【請求項1】 式 Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala で表されるペプチド。1. A peptide represented by the formula Gly Asn Leu Glu Arg Glu Cys Val Glu Glu Thr Cys Ser Tyr Glu Glu Ala Phe Glu Ala. 【請求項2】 式 【化1】 で表されるペプチド。2. The formula: A peptide represented by 【請求項3】 請求項2記載のペプチドからなる免疫原
を哺乳動物に免疫して得られたリンパ球と哺乳動物のミ
エローマ細胞との融合によって得られるハイブリドー
マ。
3. A hybridoma obtained by fusing a lymphocyte obtained by immunizing a mammal with an immunogen comprising the peptide according to claim 2 and a myeloma cell of the mammal.
【請求項4】 請求項2記載のペプチドからなる免疫原
を免疫した哺乳動物より取得したリンパ球と哺乳動物の
ミエローマ細胞との融合によって作成されたハイブリド
ーマを培養することにより得られるヒトPIVKA−I
Iと反応するモノクローナル抗体。
4. Human PIVKA-I obtained by culturing a hybridoma prepared by fusing lymphocytes obtained from a mammal immunized with an immunogen comprising the peptide of claim 2 and mammalian myeloma cells.
A monoclonal antibody that reacts with I.
【請求項5】 ヒトPIVKA−IIとは反応するが、
ヒトトロンビンとは実質的に反応しない請求項4記載の
モノクローナル抗体。
5. Reacting with human PIVKA-II,
The monoclonal antibody according to claim 4, which does not substantially react with human thrombin.
【請求項6】 E7−222(FERM P−1538
6)細胞が産生する請求項4又は5記載のモノクローナ
ル抗体。
6. E7-222 (FERM P-1538)
6) The monoclonal antibody according to claim 4 or 5, which is produced by cells.
【請求項7】 請求項4ないし6記載のモノクローナル
抗体からなるヒトPIVKA−IIの免疫測定試薬。
7. An immunoassay reagent for human PIVKA-II comprising the monoclonal antibody according to claim 4.
【請求項8】 請求項7記載の測定試薬を用いてなるヒ
トPIVKA−IIの免疫測定方法。
8. An immunoassay method for human PIVKA-II, which comprises using the assay reagent according to claim 7.
JP8080496A 1996-03-11 1996-03-11 Antihuman pivka-ii monoclonal antibody, hybridoma capable of producing the same antibody and measuring reagent and measurement using the same antibody Pending JPH09249699A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP8080496A JPH09249699A (en) 1996-03-11 1996-03-11 Antihuman pivka-ii monoclonal antibody, hybridoma capable of producing the same antibody and measuring reagent and measurement using the same antibody

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP8080496A JPH09249699A (en) 1996-03-11 1996-03-11 Antihuman pivka-ii monoclonal antibody, hybridoma capable of producing the same antibody and measuring reagent and measurement using the same antibody

Publications (1)

Publication Number Publication Date
JPH09249699A true JPH09249699A (en) 1997-09-22

Family

ID=13719923

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Country Status (1)

Country Link
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001044810A1 (en) * 1999-12-14 2001-06-21 Sanko Junyaku Co., Ltd. Method for immunologically assyaing pivka-ii
WO2010104815A1 (en) * 2009-03-10 2010-09-16 Abbott Laboratories Antibodies binding to pivka-ii amino acids 13-27
WO2012161288A1 (en) * 2011-05-20 2012-11-29 Abbott Japan Co. Ltd. Immunoassay methods and reagents for decreasing nonspecific binding
WO2013008900A1 (en) * 2011-07-08 2013-01-17 Abbott Japan Co. Ltd. Methods and kits for decreasing interferences from leukocytes in specific binding assays
JP2013541936A (en) * 2010-07-26 2013-11-21 アボット・ラボラトリーズ Antibodies for PIVKA-II and uses thereof
WO2014024853A1 (en) 2012-08-09 2014-02-13 富士レビオ株式会社 Pivka-ii measurement method, measurement reagent, and measurement kit
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6893831B1 (en) 1999-12-14 2005-05-17 Sanko Junyaku Co., Ltd. Immunoassay of PIVKA-II
WO2001044810A1 (en) * 1999-12-14 2001-06-21 Sanko Junyaku Co., Ltd. Method for immunologically assyaing pivka-ii
WO2010104815A1 (en) * 2009-03-10 2010-09-16 Abbott Laboratories Antibodies binding to pivka-ii amino acids 13-27
JP2012520075A (en) * 2009-03-10 2012-09-06 アボット・ラボラトリーズ Antibodies that bind to PIVKA-II amino acids 13-27
US8283162B2 (en) 2009-03-10 2012-10-09 Abbott Laboratories Antibodies relating to PIVKAII and uses thereof
EP3059249A1 (en) * 2009-03-10 2016-08-24 Abbott Laboratories Antibodies binding to pivka-ii amino acids 13-27
JP2013541936A (en) * 2010-07-26 2013-11-21 アボット・ラボラトリーズ Antibodies for PIVKA-II and uses thereof
JP2014515476A (en) * 2011-05-20 2014-06-30 アボットジャパン株式会社 Immunoassay methods and reagents for reducing non-specific binding
CN103620409A (en) * 2011-05-20 2014-03-05 阿波特日本有限公司 Immunoassay methods and reagents for decreasing nonspecific binding
WO2012161288A1 (en) * 2011-05-20 2012-11-29 Abbott Japan Co. Ltd. Immunoassay methods and reagents for decreasing nonspecific binding
US10107826B2 (en) 2011-05-20 2018-10-23 Abbott Japan Co. Ltd. Immunoassay methods and reagents for decreasing nonspecific binding
EP2717054A1 (en) * 2011-05-23 2014-04-09 Sekisui Medical Co., Ltd. Method for inhibiting non-specific reaction in pivka-ii measurement reagent
KR20140057495A (en) * 2011-05-23 2014-05-13 세키스이 메디칼 가부시키가이샤 Method for inhibiting non-specific reaction in pivka-ii measurement reagent
EP2717054A4 (en) * 2011-05-23 2014-12-31 Sekisui Medical Co Ltd Method for inhibiting non-specific reaction in pivka-ii measurement reagent
US9952230B2 (en) 2011-05-23 2018-04-24 Sekisui Medical Co., Ltd. Method of inhibiting nonspecific reaction in PIVKA-II assay reagent
WO2013008900A1 (en) * 2011-07-08 2013-01-17 Abbott Japan Co. Ltd. Methods and kits for decreasing interferences from leukocytes in specific binding assays
WO2014024853A1 (en) 2012-08-09 2014-02-13 富士レビオ株式会社 Pivka-ii measurement method, measurement reagent, and measurement kit

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