JP2008050362A - Monoclonal antibody, cell strain and method for measuring n1, n12-diacetylspermine - Google Patents

Monoclonal antibody, cell strain and method for measuring n1, n12-diacetylspermine Download PDF

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JP2008050362A
JP2008050362A JP2007246794A JP2007246794A JP2008050362A JP 2008050362 A JP2008050362 A JP 2008050362A JP 2007246794 A JP2007246794 A JP 2007246794A JP 2007246794 A JP2007246794 A JP 2007246794A JP 2008050362 A JP2008050362 A JP 2008050362A
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spm
diacetylspermine
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JP4664340B2 (en
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Kunio Fujiwara
邦雄 藤原
Tsunehiro Kitagawa
常広 北川
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Nippon Kayaku Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring N<SP>1</SP>, N<SP>12</SP>-diacetylsperimine expected as a tumor marker. <P>SOLUTION: A method for measuring N<SP>1</SP>, N<SP>12</SP>-diacetylsperimine in a specimen, comprising utilizing the immunization reaction of immobilized or labeled N<SP>1</SP>, N<SP>12</SP>-diacetylspermine or immobilized or labeled N<SP>1</SP>-acetylspermine with an anti-N<SP>1</SP>, N<SP>12</SP>-diacetylsperimine monoclonal antibody, is characterized by using an anti-N<SP>1</SP>, N<SP>12</SP>-diacetylsperimine monoclonal antibody which has a N<SP>1</SP>, N<SP>12</SP>-diacetylsperimine immunization reaction-inhibiting activity of 20 times or more that of N<SP>1</SP>-acetylsperimine in measurement conditions. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、抗N,N12−ジアセチルスペルミンモノクローナル抗体、抗N,N12−ジアセチルスペルミンモノクローナル抗体産生細胞株および該抗体を用いるN,N12−ジアセチルスペルミンの測定法に関するものである。
The present invention relates to an anti-N 1 , N 12 -diacetylspermine monoclonal antibody, an anti-N 1 , N 12 -diacetylspermine monoclonal antibody-producing cell line, and a method for measuring N 1 , N 12 -diacetylspermine using the antibody. .

ポリアミンは、プトレッシン(Put)、カダベリン(Cad)、スペルミジン(Spd)、スペルミン(Spm)及びそれら誘導体等の総称で、生体内に広く分布する生理活性物質である。癌細胞を含む増殖の盛んな細胞でかなりの量が合成され、これらの細胞に局在し、細胞の増殖を促進する因子として作用することから注目されている(Annu.Rev.Biochem.,53,p749−790(1984))。   Polyamine is a general term for putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and derivatives thereof, and is a physiologically active substance widely distributed in the living body. A considerable amount is synthesized in proliferating cells, including cancer cells, and is attracting attention because it is localized in these cells and acts as a factor that promotes cell growth (Annu. Rev. Biochem., 53). , P 749-790 (1984)).

1971年、Russell等は、癌患者の尿中にポリアミンが増加することを見出し(Cancer Res.,31,p1555(1971))、尿中ポリアミンは、現在、腫瘍マーカーとして用いられている。しかしながら、これまでの「ポリアミン全体」を測定することの意義に対して、限界も見えてきた(Prog.Drug Res.,39,p9−33(1992))。   In 1971, Russell et al. Found that polyamines increased in the urine of cancer patients (Cancer Res., 31, p1555 (1971)), and urinary polyamines are currently used as tumor markers. However, there has been a limit to the significance of measuring “the whole polyamine” so far (Prog. Drug Res., 39, p9-33 (1992)).

ポリアミンは、そのまま遊離型として存在する他、アセチル化されたり、結合型としても存在している。Abdel−Monem等は、尿中に排泄されるポリアミンは、主としてモノアセチル体のN−アセチルスペルミジン(N−Ac−Spd)、アセチルプトレッシン(Ac−Put)、N−アセチルスペルミジン(N−Ac−Spd)として存在しており、これら成分の増加や成分比の変動がさらによい腫瘍マーカーとなることを報告している(J.Pharm.Sci.,67,p1671−1673(1978),Cancer Res.,42,p2097−2098(1982))が、これには異論もある(J.Biochem.,118,p1211−1215(1995))。 The polyamine exists as a free form as it is, and is also acetylated or present as a bound form. The like Abdel-Monem, polyamines excreted in the urine mainly N 1 mono acetyl form - acetyl spermidine (N 1 -Ac-Spd), acetyl putrescine (Ac-Put), N 8 - acetyl spermidine ( N 8 -Ac-Spd), and it has been reported that an increase in these components and a change in the component ratio are even better tumor markers (J. Pharm. Sci., 67, p1671-1673 (1978). ), Cancer Res., 42, p2097-2098 (1982)), but this is also controversial (J. Biochem., 118, p1211-1215 (1995)).

最近、平松等は、尿中にポリアミンのジアセチル体のN,N−ジアセチルスペルミジン(N,N−2Ac−Spd)、N,N12−ジアセチルスペルミン(N,N12−2Ac−Spm)が存在することを見出し(J.Biochem.,117,p107−112(1995))、これらが、泌尿器系の腫瘍で著しく増加し、従来のポリアミンやポリアミンのモノアセチル体の変動を凌いでいたことから、新しい腫瘍マーカーとして期待されている(J.Cancer Res.Clin.Oncol.,121,p317−319(1995))。 Recently, Hitoshi Hiramatsu, N 1 of diacetyl of polyamines in urine, N 8 - diacetyl spermidine (N 1, N 8 -2Ac- Spd), N 1, N 12 - Diacetylspermine (N 1, N 12 -2Ac -Spm) (J. Biochem., 117, p107-112 (1995)), which are markedly increased in urological tumors, surpassing the variations of conventional polyamines and polyamine monoacetyls Therefore, it is expected as a new tumor marker (J. Cancer Res. Clin. Oncol., 121, p317-319 (1995)).

従来、これらジアセチル体のN,N−2Ac−Spd、N,N12−2Ac−Spmの測定は、キャピラリー・ガスクロマトグラフィ法(Clin.Chem.,32,p1930−1937(1986))や液体クロマトグラフィ法(J.Biochem.,117,p107−112(1995))で行われてきた。
生化学 1996年第68巻第7号第1211頁 4−P−1075
Conventionally, N 1 , N 8 -2Ac-Spd, N 1 , N 12 -2Ac-Spm of these diacetyl compounds have been measured by capillary gas chromatography (Clin. Chem., 32, p 1930-1937 (1986)) or It has been carried out by a liquid chromatography method (J. Biochem., 117, p107-112 (1995)).
Biochemistry 1996, Vol. 68, No. 7, p. 1211 4-P-1075

しかしながら、ジアセチル体のN,N12−2Ac−Spmを測定するキャピラリー・ガスクロマトグラフィ法や液体クロマトグラフィ法では、煩雑な検体の前処理操作や、装置のメンテナンス技術を必要とするなど、かなりの熟練が要求される。また、煩雑さのため、測定には時間がかかり、臨床的な測定法としては、とても受け入れられるものではない。
そこで、臨床的に使用し得る簡便な測定法の開発を目指し、抗体を用いる免疫的な測定法が検討された。藤原等は、Spmのアルブミン複合体を免疫してN,N12−2Ac−Spmに反応するモノクローナル抗体を作成したが、モノアセチル体のN−Ac−SpdとN−アセチルスペルミン(N−Ac−Spm)とも強く反応し、結果的には、これら3成分の中で最も尿中での存在量の多い、N−Ac−Spdを反映する測定法しかできなかった(J.Biochem.,118,p1211−1215(1995))。従って、N,N12−2Ac−Spmを測定できる免疫測定法は、今だに完成していない。
However, the capillary gas chromatography method and liquid chromatography method for measuring N 1 , N 12 -2Ac-Spm of the diacetyl compound require considerable pretreatment operations for specimens and maintenance techniques for the apparatus, and thus require considerable skill. Is required. Moreover, due to the complexity, the measurement takes time and is not very acceptable as a clinical measurement method.
Therefore, with the aim of developing a simple measurement method that can be used clinically, an immunoassay method using an antibody was examined. Fujiwara et al. Prepared monoclonal antibodies that react with N 1 , N 12 -2Ac-Spm by immunizing Spm albumin complex, but monoacetyl N 1 -Ac-Spd and N 1 -acetylspermine (N 1- Ac-Spm), and as a result, only the measurement method reflecting N 1 -Ac-Spd, which has the largest abundance in urine among these three components, could be carried out (J. Biochem., 118, p1211-1215 (1995)). Therefore, an immunoassay that can measure N 1 , N 12 -2Ac-Spm has not yet been completed.

本発明者らは、前記したような問題点を解決すべく鋭意研究を重ねた結果、N−Ac−Spmのアルブミン複合体で免疫し、実質的に尿中N,N12−2Ac−Spmの測定に使用し得るモノクローナル抗体を作成し、その抗体を用いる測定法を見いだして本発明に至ったものである。 As a result of intensive studies to solve the problems as described above, the present inventors immunized with an N 1 -Ac-Spm albumin complex, and substantially urinary N 1 , N 12 -2Ac- A monoclonal antibody that can be used for the measurement of Spm was prepared, and a measurement method using the antibody was found to arrive at the present invention.

即ち、本発明は、
(1)固相化若しくは標識化N,N12−ジアセチルスペルミン(N,N12−2Ac−Spm)又は固相化若しくは標識化N−アセチルスペルミン(N−Ac−Spm)と抗N,N12−ジアセチルスペルミンモノクローナル抗体の免疫反応を利用した検体中のN,N12−ジアセチルスペルミンの測定系を組んだ場合に、N,N12−ジアセチルスペルミンによる該免疫反応の阻害活性がN−アセチルスペルミジン(N−Ac−Spd)による該免疫反応の阻害活性の20倍以上、好ましくは30倍以上、より好ましくは40倍以上となる測定条件を選択することが可能になる抗N,N12−ジアセチルスペルミンモノクローナル抗体、
(2)固相化若しくは標識化N,N12−ジアセチルスペルミン又は固相化若しくは標識化N−アセチルスペルミンとの免疫反応が50%阻害されるN,N12−ジアセチルスペルミンの濃度が20μM以下、好ましくは15μM以下、より好ましくは1μM以下となる測定条件を選択することが可能になる上記(1)記載のモノクローナル抗体、
(3)検体が尿検体である上記(1)又は(2)記載のモノクローナル抗体、
(4)モノクローナル抗体0520,4914又は8624、
(5)上記(1)、(2)、(3)又は(4)記載のモノクローナル抗体を産生する細胞株、
(6)固相化若しくは標識化N,N12−ジアセチルスペルミン又は固相化若しくは標識化N−アセチルスペルミンと抗N,N12−ジアセチルスペルミンモノクローナル抗体の免疫反応を利用して検体中のN,N12−ジアセチルスペルミンを測定する際に、その測定条件におけるN,N12−ジアセチルスペルミンによる該免疫反応の阻害活性がN−アセチルスペルミジンによる該免疫反応の阻害活性の20倍以上、好ましくは30倍以上、より好ましくは40倍以上となる抗N,N12−ジアセチルスペルミンモノクローナル抗体を用いることを特徴とするN,N12−ジアセチルスペルミンの測定法、
(7)抗N,N12−ジアセチルスペルミンモノクローナル抗体が、測定条件において固相化若しくは標識化N,N12−ジアセチルスペルミン又は固相化若しくは標識化N−アセチルスペルミンとの免疫反応が50%阻害するN,N12−ジアセチルスペルミンの濃度が20μM以下、好ましくは15μM以下、より好ましくは1μM以下となる抗N,N12−ジアセチルスペルミンモノクローナル抗体である上記(6)記載の測定法、
(8)抗N,N12−ジアセチルスペルミンモノクローナル抗体がモノクローナル抗体0520,4914又は8624である上記(7)記載の測定法、
(9)検体が尿検体である上記(6)、(7)又は(8)記載の測定法、
に関する。
That is, the present invention
(1) immobilized or labeled N 1, N 12 - Diacetylspermine (N 1, N 12 -2Ac- Spm) or immobilized or labeled N 1 - and spermine (N 1 -Ac-Spm) anti Inhibition of the immune reaction by N 1 , N 12 -diacetylspermine when a measurement system for N 1 , N 12 -diacetylspermine in a sample utilizing the immune reaction of N 1 , N 12 -diacetylspermine monoclonal antibody is assembled. activity N 1 - acetyl spermidine (N 1 -Ac-Spd) by more than 20 times the inhibitory activity of the immune response, preferably 30 times or more, more preferably to be capable of selecting the measuring conditions to be 40 times or more An anti-N 1 , N 12 -diacetylspermine monoclonal antibody,
(2) The concentration of N 1 , N 12 -diacetylspermine at which 50% inhibition of the immunoreaction with solid-phased or labeled N 1 , N 12 -diacetylspermine or solid-phased or labeled N 1 -acetylspermine is The monoclonal antibody according to the above (1), which makes it possible to select measurement conditions of 20 μM or less, preferably 15 μM or less, more preferably 1 μM or less,
(3) The monoclonal antibody according to (1) or (2) above, wherein the specimen is a urine specimen,
(4) monoclonal antibody 0520, 4914 or 8624,
(5) A cell line producing the monoclonal antibody according to (1), (2), (3) or (4) above,
(6) Solid phase or labeled N 1 , N 12 -diacetylspermine or solid phase or labeled N 1 -acetyl spermine and anti-N 1 , N 12 -diacetylspermine monoclonal antibody in the sample of N 1, N 12 - in measuring the diacetylspermine, N 1, N 12 in the measurement conditions - the inhibitory activity of the immune response by diacetylspermine is N 1 - 20 times the inhibitory activity of the immune response by acetyl spermidine Or more, preferably 30 times or more, more preferably 40 times or more anti-N 1 , N 12 -diacetylspermine monoclonal antibody, a method for measuring N 1 , N 12 -diacetylspermine,
(7) The anti-N 1 , N 12 -diacetylspermine monoclonal antibody has an immunoreaction with immobilized or labeled N 1 , N 12 -diacetylspermine or immobilized or labeled N 1 -acetylspermine under measurement conditions. N 1, N 12 50% inhibition - concentration of diacetylspermine is 20μM or less, preferably 15μM or less, the anti-N 1 and more preferably less than or equal to 1 [mu] M, N 12 - (6) above is diacetylspermine monoclonal antibody measurement according Law,
(8) The measuring method according to the above (7), wherein the anti-N 1 , N 12 -diacetylspermine monoclonal antibody is monoclonal antibody 0520, 4914 or 8624,
(9) The measurement method according to (6), (7) or (8) above, wherein the sample is a urine sample,
About.

本発明のN,N12−ジアセチルスペルミンと反応するモノクローナル抗体は、尿等の検体中のN,N12−ジアセチルスペルミンを測定することができ、癌の診断に有用である。 The monoclonal antibody that reacts with N 1 , N 12 -diacetylspermine of the present invention can measure N 1 , N 12 -diacetylspermine in a sample such as urine and is useful for diagnosis of cancer.

以下、本発明を詳細に説明する。
固相化N,N12−2Ac−Spm又は固相化N−Ac−Spmとしては、N,N12−2Ac−Spm又はN−Ac−Spmを、スペーサーを介して、不溶性物質の表面上に繋ぎ止めたものが挙げられる。
スペーサーの結合位置は、N,N12−2Ac−Spmの場合はその末端であってもその両末端の間のいずれの位置であってもよいが、N−Ac−Spmの場合はその末端アミノ基に結合させるのが望ましい。スペーサーの種類と導入方法については多くの方法が知られているが、これらのいずれであっても良い。例えば、N,N12−2Ac−Spmのいずれかの位置に、末端に反応基を有するスペーサーを導入し(N,N12−2Ac−Spmのアセチル基からスペーサーを誘導する場合は、N−Ac−SpmのN12−アミノ基に導入したアシル基がスペーサーとなる)、この反応基を介して蛋白質や合成高分子などに結合し、生成したN,N12−2Ac−Spmと蛋白質や合成高分子の複合体を、免疫反応の場となる固相担体上に吸着させる方法、予め化学的に活性化されたスペーサーを持つ固相担体上に、N,N12−2Ac−Spmそのもの、もしくは、N−Ac−Spmの末端アミノ基を反応させる方法などがあるが、これらに限定されるものではない。N,N12−2Ac−Spm又はN−Ac−Spmのいずれの位置に、どのようなスペーサーを導入するかは、用いる抗体の性質によって適宜選択すればよい。
Hereinafter, the present invention will be described in detail.
The immobilized N 1, N 12 -2Ac-Spm or immobilized N 1 -Ac-Spm, the N 1, N 12 -2Ac-Spm or N 1 -Ac-Spm, via a spacer, insoluble material What was tied on the surface of the.
In the case of N 1 , N 12 -2Ac-Spm, the binding position of the spacer may be either the end or between the both ends, but in the case of N 1 -Ac-Spm It is desirable to bind to the terminal amino group. Many methods are known for the type of spacer and the introduction method, and any of these may be used. For example, at any position of the N 1, N 12 -2Ac-Spm , may induce introducing a spacer having a reactive group at its terminal (N 1, N 12 spacer acetyl group -2Ac-Spm is, N The acyl group introduced into the N 12 -amino group of 1- Ac-Spm serves as a spacer), and N 1 , N 12 -2Ac-Spm produced by binding to a protein or a synthetic polymer via this reactive group A method of adsorbing a complex of a protein or a synthetic polymer on a solid phase carrier that serves as an immune reaction field, N 1 , N 12 -2Ac- on a solid phase carrier having a chemically activated spacer in advance There is a method of reacting Spm itself or the terminal amino group of N 1 -Ac-Spm, but it is not limited thereto. What spacer should be introduced at any position of N 1 , N 12 -2Ac-Spm or N 1 -Ac-Spm may be appropriately selected depending on the properties of the antibody used.

スペーサーとしては、例えばグルタルアルデヒド(GA)を用いた場合はホルミルブチリル鎖が、N−(4−マレイミドブチルオキシ)コハク酸イミド(GMBS)を用いた場合はマレイミドブチリル鎖が、無水コハク酸を用いた場合は、カルボキシプロピオニル鎖等が挙げられるが、公知のものはいずれも使用できる。
又、蛋白質や合成高分子としては、例えば、アルブミンやポリリジン等が挙げられるが、これらに限定されるものでなはい。固相担体としては、例えば、96穴等のマイクロタイタープレート、ポリスチレンビーズ、各種ラテックス粒子、ニトロセルロース膜等が挙げられるが、これらに限定されるものではない。
For example, when glutaraldehyde (GA) is used as a spacer, formylbutyryl chain is used. When N- (4-maleimidobutyloxy) succinimide (GMBS) is used, maleimidobutyryl chain is used as succinic anhydride. In this case, a carboxypropionyl chain and the like can be mentioned, but any known one can be used.
Examples of proteins and synthetic polymers include albumin and polylysine, but are not limited thereto. Examples of the solid phase carrier include, but are not limited to, a microtiter plate having 96 holes, polystyrene beads, various latex particles, a nitrocellulose film, and the like.

また、標識化N,N12−2Ac−Spm又は標識化N−Ac−Spmとしては、放射性同位元素を導入してN,N12−2Ac−Spm又はN−Ac−Spmそのものを標識したり、N,N12−2Ac−Spm又はN−Ac−Spmに、放射性元素の入った化合物、ユーロピウム等の遅延蛍光性のある元素を保持できる化合物、ルテニウム等の電気化学発光の触媒となる元素を保持できる化合物、蛍光物質等を結合させて標識したり、N,N12−2Ac−Spm又はN−Ac−Spmに、前記のようなスペーサーを介して酵素標識したり、N,N12−2Ac−Spm又はN−Ac−Spmをビオチンで標識し、免疫反応後にアビジンの酵素標識体と反応させ、ビオチン−アビジン複合体として間接的に検出できるようにしたもの等が挙げられる。 Further, as labeled N 1 , N 12 -2Ac-Spm or labeled N 1 -Ac-Spm, N 1 , N 12 -2 Ac-Spm or N 1 -Ac-Spm itself is introduced by introducing a radioisotope. A compound capable of labeling, N 1 , N 12 -2Ac-Spm or N 1 -Ac-Spm containing a radioactive element, a compound capable of retaining a delayed fluorescent element such as europium, or electrochemiluminescence such as ruthenium A compound capable of holding an element serving as a catalyst, a fluorescent substance or the like is bound and labeled, or N 1 , N 12 -2Ac-Spm or N 1 -Ac-Spm is enzyme-labeled via a spacer as described above , labeled N 1, N 12 -2Ac-Spm or N 1 -Ac-Spm with biotin is reacted with avidin enzyme label after the immunological reaction, biotin - avidin complex and Such as those to be able to indirectly detect the like Te.

本発明のモノクローナル抗体は、クローン化されたイムノグロブリン抗体であれば何であってもよく、抗体の由来する動物種、イムノグロブリンのタイプやサブタイプ、抗体の産生方法は問わない。また、抗体を断片化して免疫反応部位を残したもの、それら断片の修飾物、抗体そのものの修飾物、2種類の抗体を結合させたキメラ抗体等も包含する。本発明のモノクローナル抗体は、抗体産生株の組織培養法や、抗体のアミノ酸配列から予想されるDNAを用いて、遺伝子工学を用いる製造法等によって製造することができる。   The monoclonal antibody of the present invention may be any cloned immunoglobulin antibody, regardless of the animal species from which the antibody is derived, the type or subtype of the immunoglobulin, and the method for producing the antibody. Also included are those obtained by fragmenting an antibody to leave an immune reaction site, a modified product of these fragments, a modified product of the antibody itself, a chimeric antibody in which two types of antibodies are bound, and the like. The monoclonal antibody of the present invention can be produced by a tissue culture method of an antibody-producing strain, a production method using genetic engineering, etc. using DNA predicted from the amino acid sequence of the antibody.

本発明のモノクローナル抗体を産生する抗体産生株は、公知の方法に準じた方法、即ち、マウス、ラット等の動物を免疫原で免疫し、次いで免疫した動物のB細胞とミエローマ細胞を融合し、得られたハイブリドーマの中から本発明のモノクローナル抗体を産生する細胞株を選択することにより得ることができる。   The antibody-producing strain producing the monoclonal antibody of the present invention is a method according to a known method, that is, immunizing an animal such as a mouse or rat with an immunogen, and then fusing the B cell and myeloma cell of the immunized animal, It can be obtained by selecting a cell line that produces the monoclonal antibody of the present invention from the obtained hybridomas.

免疫原には、ハプテン(N,N12−2Ac−Spm又はN−Ac−Spm)とキャリア物質(蛋白質又は合成高分子)の複合体が使用でき、これは、固相化N,N12−2Ac−Spm又は固相化N−Ac−Spmの作成法で述べた方法と同様にして製造することができる。
本発明のモノクローナル抗体としては、例えばモノクローナル抗体0520,4914及び8624が挙げられ、これらはそれぞれACSPM−1(工業技術院生命工学工業技術研究所寄託番号、FERM P−16297)、ACSPM−2(工業技術院生命工学工業技術研究所寄託番号、FERM P−16298)及びACSPM−3(工業技術院生命工学工業技術研究所寄託番号、FERM P−16299)と名付けられた細胞株を培養することにより得ることができる。
The immunogen hapten can complex the use of (N 1, N 12 -2Ac- Spm or N 1 -Ac-Spm) and carrier material (protein or synthetic polymer), which is immobilized N 1, It can be produced in the same manner as described in the method for producing N 12 -2Ac-Spm or solid-phased N 1 -Ac-Spm.
Monoclonal antibodies of the present invention include, for example, monoclonal antibodies 0520, 4914, and 8624, which are respectively ACSPM-1 (Institute of Biotechnology, Industrial Technology Research Institute, FERM P-16297), ACSPM-2 (Industry It is obtained by culturing cell lines designated as Biotechnology Institute of Technology, FERM P-16298) and ACSPM-3 (Accession of Biotechnology Institute of Technology, FERM P-16299). be able to.

本発明の測定法は、ハプテンの免疫測定法として知らている方法のいずれの方法によっても行なうことができ、特に制限されない。例えば、結合阻害法の場合、例えば、実験例2のように、N,N12−2Ac−Spmによる抗体と固相化ハプテンとの免疫反応の阻害を、固相化ハプテンに結合した抗体量の減少として、色々な手段で検出する方法、固相担体としてラテックスを用いる例としては、ハプテンを固相化したラテックス試薬の抗体による凝集反応をN,N12−2Ac−Spmによる阻害として検出する方法、逆に、抗体を固相化したラテックス試薬を用い、多エピトープ化したポリハプテンの添加によるラテックス凝集反応を、N,N12−2Ac−Spmによって阻害させる方法等がある。また、競合法の場合、固相化した抗体に対して、N,N12−2Ac−Spmと標識化N,N12−2Ac−Spm又は標識化N−Ac−Spmとを競合反応させ、BF分離後に、固相化した抗体に結合した標識化N,N12−2Ac−Spm又は標識化N−Ac−Spmの標識を、それぞれの方法で適宜に検出すればよい。 The measurement method of the present invention can be carried out by any of the methods known as hapten immunoassays, and is not particularly limited. For example, in the case of the binding inhibition method, for example, as in Experimental Example 2, the inhibition of the immune reaction between the antibody and the immobilized hapten by N 1 , N 12 -2Ac-Spm is caused by the amount of antibody bound to the immobilized hapten. As an example of using a latex as a solid phase carrier, as an example of using latex as a solid phase carrier, an agglutination reaction caused by an antibody of a latex reagent in which a hapten is immobilized is detected as inhibition by N 1 , N 12 -2Ac-Spm. On the other hand, there is a method in which a latex agglutination reaction by adding a polyhapten having a multi-epitope is inhibited by N 1 , N 12 -2Ac-Spm using a latex reagent in which an antibody is immobilized. In the case of the competition method, N 1 , N 12 -2Ac-Spm and labeled N 1 , N 12 -2Ac-Spm or labeled N 1 -Ac-Spm are competitively reacted with the immobilized antibody. After the BF separation, the labeled N 1 , N 12 -2Ac-Spm or labeled N 1 -Ac-Spm bound to the immobilized antibody may be appropriately detected by each method.

本発明の測定法を実施する測定条件は特に限定されないが、従来知られている免疫反応を利用した測定条件が使用できる。例えば、検体と試薬(固相化若しくは標識化N,N12−2Ac−Spm又は固相化若しくは標識化N−Ac−Spm及び抗N,N12−2Ac−Spmモノクローナル抗体)を混合して行なう免疫反応は通常0〜45℃、好ましくは10〜40℃で行なう。本発明の測定を実施するにあたり、抗N,N12−2Ac−Spmモノクローナル抗体は必要により固相化又は標識化して使用する。 The measurement conditions for carrying out the measurement method of the present invention are not particularly limited, but conventionally known measurement conditions using an immune reaction can be used. For example, mixing the sample and reagent (immobilized or labeled N 1, N 12 -2Ac-Spm or immobilized or labeled N 1 -Ac-Spm and anti N 1, N 12 -2Ac-Spm monoclonal antibody) The immune reaction carried out is usually carried out at 0 to 45 ° C, preferably 10 to 40 ° C. Carrying out the measurement of the present invention, anti-N 1, N 12 -2Ac-Spm monoclonal antibody used in immobilized or labeled necessary.

本発明の測定法を実施するにあたり、固相化若しくは標識化N,N12−2Ac−Spm又は固相化若しくは標識化N−Ac−Spmとしては、抗N,N12−2Ac−Spmモノクローナル抗体を作成する際に用いた免疫原を作成する際に使用したスペーサーの種類、N,N12−2Ac−Spm若しくはN−Ac−Spmへの結合位置又は化学結合方法のひとつ以上を変えて作成したものが望ましい。 In carrying out the measurement method of the present invention, solid-phased or labeled N 1 , N 12 -2Ac-Spm or solid-phased or labeled N 1 -Ac-Spm is anti-N 1 , N 12 -2Ac- One or more types of spacers used in preparing the immunogen used in preparing the Spm monoclonal antibody, the binding position to N 1 , N 12 -2Ac-Spm or N 1 -Ac-Spm, or the chemical binding method What was created by changing

なお、本発明において、固相化若しくは標識化N,N12−2Ac−Spm又は固相化若しくは標識化N−Ac−Spmと抗N,N12−2Ac−Spmモノクローナル抗体との免疫反応が50%阻害されるN,N12−2Ac−Spmの濃度は、測定条件下で両者を反応させて得られるシグナルを半減させるN,N12−2Ac−Spmの濃度を求めることにより得ることができ、これは、反応系の検出感度の尺度となり得る。 In the present invention, immunization between solid-phased or labeled N 1 , N 12 -2Ac-Spm or solid-phased or labeled N 1 -Ac-Spm and anti-N 1 , N 12 -2Ac-Spm monoclonal antibody The concentration of N 1 , N 12 -2Ac-Spm at which the reaction is inhibited by 50% is obtained by determining the concentration of N 1 , N 12 -2Ac-Spm that halves the signal obtained by reacting both under the measurement conditions. Which can be a measure of the detection sensitivity of the reaction system.

本発明において、検体としては尿、血清、血漿等各種のものが使用できるが、特に尿が好ましい。   In the present invention, various samples such as urine, serum and plasma can be used as the specimen, and urine is particularly preferable.

以下に、実験例及び実施例により本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。   Hereinafter, the present invention will be described more specifically with reference to experimental examples and examples, but the present invention is not limited thereto.

実験例1 抗体のスクリーニング法
96穴マイクロタイタープレート(ヌンク社製 Nunc Immunoplate II)の各ウェルに、抗原としてN−アセチルスペルミンのヒト血清アルブミン複合体(N−Ac−Spm−GMBS−HSA)15μg/mLを含む10mMトリス・塩酸緩衝液(pH8.5)100μLを入れ、40℃で20分間放置してコーティングした。次に、コーティング液を捨てて0.1%のツウィーン20を含む10mMリン酸で緩衝化された生食液(PBST)で洗浄後、スキムミルク1%を含む50mMトリス・塩酸で緩衝化された生食液(pH7.3)200μLでブロッキング処理した。
ブロッキング液を捨ててPBSTで洗浄後、各ウェルにハイブリドーマの培養上清60μLと、BSA20mg/mLを含む10mMリン酸で緩衝化された生食液20μLを加え、4℃で一晩反応させた。ウェルを、PBSTで洗浄後、PBSTで2000倍に希釈した西洋わさびペルオキシダーゼ(HRP)標識−ヤギ抗マウスIgG溶液(カッペル社)50μLを加え、37℃、40分間反応させた。
結合した酵素の活性は、PBSTで洗浄した各ウェルに、o−フェニレンジアミン0.5mg/mLと過酸化水素0.012%を含む0.1Mクエン酸リン酸緩衝液(pH5.3)100μLを加え、室温下に9分間発色反応させ、ELISAプレートリーダー(SLT−Lab Instruments社)を用い、492nmにおける吸光度の増加として測定した。
To each well of the experimental example 1 antibody screening method 96 well microtiter plates (Nunc Nunc Immunoplate II), N 1 as an antigen - spermine human serum albumin conjugate (N 1 -Ac-Spm-GMBS -HSA) 100 μL of 10 mM Tris / HCl buffer (pH 8.5) containing 15 μg / mL was added, and the coating was allowed to stand at 40 ° C. for 20 minutes. Next, after discarding the coating solution and washing with a 10 mM phosphate buffered saline solution (PBST) containing 0.1% Tween 20, the saline solution buffered with 50 mM Tris-HCl containing 1% skim milk. (PH 7.3) Blocking treatment was performed with 200 μL.
After discarding the blocking solution and washing with PBST, 60 μL of the hybridoma culture supernatant and 20 μL of a saline solution buffered with 10 mM phosphoric acid containing 20 mg / mL of BSA were added to each well and reacted at 4 ° C. overnight. The wells were washed with PBST, and then added with 50 μL of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG solution (Kappel) diluted 2000 times with PBST and reacted at 37 ° C. for 40 minutes.
The activity of the bound enzyme was determined by adding 100 μL of 0.1 M citrate phosphate buffer (pH 5.3) containing 0.5 mg / mL o-phenylenediamine and 0.012% hydrogen peroxide to each well washed with PBST. In addition, the color reaction was allowed to proceed at room temperature for 9 minutes, and the increase in absorbance at 492 nm was measured using an ELISA plate reader (SLT-Lab Instruments).

実験例2 ELISA結合阻害法
[N,N12−ジアセチルスペルミンのヒト血清アルブミン複合体(N,N12−2Ac−Spm−GA−HSA)の調製]
6.5mgのN,N12−ジアセチルスペルミン(N,N12−2Ac−Spm)を含む1M酢酸ナトリウム溶液0.5mLに、0.083Mグルタルアルデヒド溶液1mLを加えて攪拌し、30秒間放置した。さらに、10.5mgのヒト血清アルブミン(HSA)を含む1M酢酸ナトリウム溶液0.5mLを加えて攪拌し、室温で30分間反応させた。さらに、この反応液に水素化ホウ素ナトリウム5mgを加えて室温下に10分間反応させた後、10mM酢酸ナトリウム溶液300mLで、4回液を交換しながら、3時間45分透析した。
[N−アセチルスペルミジンのヒト血清アルブミン複合体(N−Ac−Spd−GA−HSA)の調製]
3mgのN−アセチルスペルミジン(N−Ac−Spd)を含む1M酢酸ナトリウム溶液0.5mLに、0.021Mグルタルアルデヒド溶液1mLを加えて攪拌し、30秒間放置した。さらに、6mgのHSAを含む1M酢酸ナトリウム溶液0.5mLを加えて攪拌し、室温で30分間反応させた。さらに、この反応液に水素化ホウ素ナトリウム2.5mgを加えて室温下に10分間反応させた後、10mM酢酸ナトリウム溶液300mLで、4回液を交換しながら、3時間透析した。
Experimental Example 2 ELISA binding inhibition method [N 1, N 12 - human serum albumin conjugate of Diacetylspermine of (N 1, N 12 -2Ac- Spm-GA-HSA) prepared]
Add 1 mL of 0.083 M glutaraldehyde solution to 0.5 mL of 1 M sodium acetate solution containing 6.5 mg of N 1 , N 12 -diacetylspermine (N 1 , N 12 -2Ac-Spm), and stir for 30 seconds. did. Further, 0.5 mL of 1 M sodium acetate solution containing 10.5 mg of human serum albumin (HSA) was added and stirred, and reacted at room temperature for 30 minutes. Further, 5 mg of sodium borohydride was added to this reaction solution and reacted at room temperature for 10 minutes, and then dialyzed with 300 mL of 10 mM sodium acetate solution for 3 hours and 45 minutes while changing the solution four times.
[N 1 - Preparation of human serum albumin complex of acetyl spermidine (N 1 -Ac-Spd-GA -HSA)]
1 mL of a 0.021 M glutaraldehyde solution was added to 0.5 mL of 1 M sodium acetate solution containing 3 mg of N 1 -acetylspermidine (N 1 -Ac-Spd), and the mixture was allowed to stand for 30 seconds. Furthermore, 0.5 mL of 1 M sodium acetate solution containing 6 mg of HSA was added and stirred, and reacted at room temperature for 30 minutes. Furthermore, 2.5 mg of sodium borohydride was added to this reaction solution and reacted at room temperature for 10 minutes, and then dialyzed with 300 mL of 10 mM sodium acetate solution for 4 hours while changing the solution four times.

[測定法]
96穴マイクロタイタープレート(ヌンク社製 Nunc Immunoplate II)の各ウェルに、抗原としてN−アセチルスペルミンのヒト血清アルブミン複合体(N−Ac−Spm−GMBS−HSA)、N−アセチルスペルミジンのヒト血清アルブミン複合体(N−Ac−Spd−GA−HSA)、又は、N,N12−ジアセチルスペルミンのヒト血清アルブミン複合体(N,N12Ac−Spm−GA−HSA)を15μg/mL含む10mMトリス・塩酸緩衝液(pH8.5)200μLを入れ、40℃で20分間放置してコーティングした。次に、コーティング液を捨ててPBSTで洗浄後、スキムミルク1%を含む50mMトリス・塩酸緩衝液(pH7.3)200μLでブロッキング処理した。
ブロッキング液を捨ててPBSTで洗浄後、各ウェルに、ハイブリドーマ3株のそれぞれの培養上清をPBSTで100倍希釈した液50μLと、測定対象物のスペルミン(Spm)、N−アセチルスペルミジン(N−Ac−Spd)、N−アセチルスペルミン(N−Ac−Spm)、N,N12−ジアセチルスペルミン(N,N12−2Ac−Spm)等を、PBSTで各種濃度に希釈した液50μLを加え、37℃で1.5時間反応させた。ウェルを、PBSTで洗浄後、PBSTで2000倍に希釈した西洋わさびペルオキシダーゼ標識−ヤギ抗マウスIgG溶液50μLを加え、37℃、40分間反応させた。
結合した酵素の活性は、PBSTで洗浄した各ウェルに、o−フェニレンジアミン0.5mg/mLと過酸化水素0.012%を含む0.1Mクエン酸リン酸緩衝液(pH5.3)100μLを加え、室温下に5分間発色反応させ、ELISAプレートリーダー(SLT−Lab Instruments社)を用い、492nmにおける吸光度の増加として測定した。
[Measurement method]
To each well of a 96-well microtiter plates (Nunc Nunc Immunoplate II), N 1 as an antigen - human serum albumin conjugate of spermine (N 1 -Ac-Spm-GMBS -HSA), N 1 - acetyl spermidine human serum albumin conjugate (N 1 -Ac-Spd-GA -HSA), or, N 1, N 12 - diacetylspermine human serum albumin conjugate (N 1, N 12 - 2 Ac-Spm-GA-HSA) 200 μL of 10 mM Tris / HCl buffer (pH 8.5) containing 15 μg / mL of the solution was allowed to stand at 40 ° C. for 20 minutes for coating. Next, the coating solution was discarded and washed with PBST, followed by blocking treatment with 200 μL of 50 mM Tris / HCl buffer solution (pH 7.3) containing 1% skim milk.
After discarding the blocking solution and washing with PBST, 50 μL of each culture supernatant of the hybridoma 3 strain diluted 100-fold with PBST, spermine (Spm), N 1 -acetylspermidine (N 1 -Ac-Spd), N 1 - spermine (N 1 -Ac-Spm), N 1, N 12 - diacetylspermine (N 1, N 12 -2Ac- Spm) or the like, and diluted to various concentrations with PBST 50 μL of the solution was added and reacted at 37 ° C. for 1.5 hours. After washing the wells with PBST, 50 μL of horseradish peroxidase-labeled goat anti-mouse IgG solution diluted 2000 times with PBST was added and reacted at 37 ° C. for 40 minutes.
The activity of the bound enzyme was determined by adding 100 μL of 0.1 M citrate phosphate buffer (pH 5.3) containing 0.5 mg / mL o-phenylenediamine and 0.012% hydrogen peroxide to each well washed with PBST. In addition, the color reaction was allowed to proceed at room temperature for 5 minutes, and the increase in absorbance at 492 nm was measured using an ELISA plate reader (SLT-Lab Instruments).

実施例1 N,N12−ジアセチルスペルミンに対するモノクローナル抗体の作成[N−アセチルスペルミンのBSA複合体(抗原)の調製]
50mgのS−アセチルメルカプトコハク酸無水物(AMS)を含むテトラヒドロフラン溶液1mLを、200mgのウシ血清アルブミン(BSA)を含む0.1Mリン酸緩衝液(pH7.0)5mLに加えて攪拌し、1N水酸化ナトリウム溶液でpHを7.0に保ちながら、室温下に1時間反応させた。この反応液を、5mMリン酸緩衝液(pH6.8)で膨潤化したセファデックスG−75のカラム(2cm×100cm)にかけ、5mMリン酸緩衝液(pH6.8)で溶出した。誘導体化されたBSA画分を集めて凍結乾燥し、AMS化されたBSA(AMS−BSA)180mgを得た。BSAへのSH基の導入数は17±0.5であった。
次に、10.4mgのN−アセチルスペルミン・3塩酸塩を含む0.1Mリン酸緩衝液(pH6.9)1mLに、1mgのN−(4−マレイミドブチルオキシ)コハク酸イミド(GMBS)を含むテトラヒドロフラン溶液0.5mLを加え、pHを7付近に保ちながら、室温で100分間反応させ、GMBS化N−アセチルスペルミン溶液を調製した。
Example 1 N 1, N 12 - [ - Preparation of BSA conjugates of spermine (antigen) N 1] create a monoclonal antibody against Diacetylspermine
1 mL of a tetrahydrofuran solution containing 50 mg of S-acetylmercaptosuccinic anhydride (AMS) was added to 5 mL of 0.1 M phosphate buffer (pH 7.0) containing 200 mg of bovine serum albumin (BSA), and stirred. While maintaining the pH at 7.0 with a sodium hydroxide solution, the mixture was reacted at room temperature for 1 hour. This reaction solution was applied to a Sephadex G-75 column (2 cm × 100 cm) swollen with 5 mM phosphate buffer (pH 6.8) and eluted with 5 mM phosphate buffer (pH 6.8). The derivatized BSA fractions were collected and lyophilized to obtain 180 mg of AMS-modified BSA (AMS-BSA). The number of SH groups introduced into BSA was 17 ± 0.5.
Next, 1 mg of N- (4-maleimidobutyloxy) succinimide (GMBS) was added to 1 mL of 0.1M phosphate buffer (pH 6.9) containing 10.4 mg of N 1 -acetylspermine · 3 hydrochloride. A tetrahydrofuran solution containing 0.5 mL was added, and the mixture was allowed to react at room temperature for 100 minutes while maintaining the pH at around 7, to prepare a GMBS N 1 -acetylspermine solution.

一方、17.3mgのAMS−BSAを含む0.1Mリン酸緩衝液(pH6.9)0.2mLに、0.5Mヒドロキシルアミン溶液(pH7.0)50μLを加えて室温下に10分間放置後、さらに、0.1Mリン酸緩衝液(pH6.9)2mLを加えた。この溶液と、先に調製したGMBS化N−アセチルスペルミン溶液を混合してボルテックスミキサー攪拌後、10mMトリス・塩酸緩衝液(pH7.5)で平衡化したセファデックスG−100のカラム(2.2cm×43cm)にかけ、同緩衝液で溶出した。280nmにおける蛋白質吸収画分を集め、N−アセチルスペルミンのBSA複合体(N−Ac−Spm−GMBS−BSA)を得た。
また、同様の方法で、ヒト血清アルブミン(HSA)に対応するN−アセチルスペルミンのHSA複合体(N−Ac−Spm−GMBS−HSA)も調製した。
On the other hand, after adding 50 μL of 0.5 M hydroxylamine solution (pH 7.0) to 0.2 mL of 0.1 M phosphate buffer (pH 6.9) containing 17.3 mg of AMS-BSA, the mixture was allowed to stand at room temperature for 10 minutes. Furthermore, 2 mL of 0.1 M phosphate buffer (pH 6.9) was added. This solution was mixed with the previously prepared GMBS N 1 -acetylspermine solution, stirred with a vortex mixer, and equilibrated with 10 mM Tris-HCl buffer (pH 7.5) (2. 2 cm × 43 cm) and eluted with the same buffer. Collected protein fraction absorbed at 280 nm, N 1 - was obtained BSA conjugates of spermine and (N 1 -Ac-Spm-GMBS -BSA).
Further, in the same way, N 1 corresponds to the human serum albumin (HSA) - HSA complex of spermine (N 1 -Ac-Spm-GMBS -HSA) were also prepared.

[免疫]
雌性BALB/cマウスに、コンプリート・フロイント・アジュバントで乳化したN−Ac−Spm−GMBS−BSA抗原100μgを腹腔内投与した。さらに、100μgのN−Ac−Spm−GMBS−BSA抗原を10mMリン酸で緩衝化された生食液(PBS)で希釈したもので、2週間毎に、4回追加免疫を行った。
[細胞融合]
5回目の最終感作を行ってから4日後に、マウスの脾細胞を取り出し、これとミエローマ細胞P3/NS−1/1Ag4−1とを、40%ポリエチレングリコール1500(ベーリンガー社)存在下に、Shulmanらの方法に従って細胞融合した。次いで、融合細胞は、96穴培養プレート(コーニング社)を用いて、ウェル当たり105個の細胞密度で、HAT培地中で培養した。細胞融合後10〜20日目に、960ウェル中708ウェル(74%)に、細胞の増殖が認められた。
[Immunity]
Female BALB / c mice were intraperitoneally administered 100 μg of N 1 -Ac-Spm-GMBS-BSA antigen emulsified with complete Freund's adjuvant. Further, 100 μg of N 1 -Ac-Spm-GMBS-BSA antigen was diluted with a saline solution (PBS) buffered with 10 mM phosphate, and boosted 4 times every 2 weeks.
[Cell fusion]
Four days after the fifth final sensitization, the mouse spleen cells were removed and myeloma cells P3 / NS-1 / 1Ag4-1 were added in the presence of 40% polyethylene glycol 1500 (Boehringer). Cell fusion was performed according to the method of Sulman et al. The fused cells were then cultured in HAT medium using a 96-well culture plate (Corning) at a density of 105 cells per well. From 10 to 20 days after cell fusion, cell proliferation was observed in 708 wells (74%) of 960 wells.

[細胞の選択]
各ウェルの培養上清中の抗体価を、実験例1の「抗体のスクリーニング法」で検索し、免疫反応陽性の細胞3個を得た。これらを限界希釈法でクローン化し、継続的に抗体を産生する細胞、ACSPM−1、ACSPM−2、ACSPM−3の3株を樹立した。さらに、これら3株の抗原部位への反応性を確認するため、抗原作成時に副生する可能性のあるGMBA−HSA(AMS−HSAにスペーサーのGMBAを導入したもの)も調製し、実験例1の抗原N−Ac−Spm−GMBS−HSAの代わりに、GMBA−HSA、AMS−HSA(HSAにSH基を導入したもの)やキャリア蛋白そのもののHSAをコーティングしたプレートも調製し、倍々希釈したACSPM−1株、ACSPM−2株、ACSPM−3株の培養上清との反応性を確認した。結果を、それぞれ図1〜図3に示した。いずれの培養上清も、N−Ac−Spm−GMBS−HSAにのみ反応し、GMBA−HSA、AMS−HSAやHSAとは、全く反応しなかった。
[クローン細胞のサブタイプ]
各クローン細胞が産生する免疫グロブリンのサブタイプは、マウスモノクローナルSub−isotyping キット(Zymed社コードNo.97−6550)を用いて決定した。その結果、0520抗体(ACSPM−1株)と4914抗体(ACSPM−2株)はIgG、8624抗体(ACSPM−3株)がIgG2bであった。
[Select cells]
The antibody titer in the culture supernatant of each well was searched using the “antibody screening method” in Experimental Example 1 to obtain 3 immune-positive cells. These were cloned by the limiting dilution method, and three strains of cells that continuously produce antibodies, ACSPM-1, ACSPM-2, and ACSPM-3 were established. Furthermore, in order to confirm the reactivity of these three strains to the antigenic site, GMBA-HSA (in which a spacer GMBA was introduced into AMS-HSA) that may be by-produced at the time of antigen preparation was also prepared. Instead of the antigen N 1 -Ac-Spm-GMBS-HSA, a plate coated with GMBA-HSA, AMS-HSA (in which SH group was introduced into SHA) or HSA of the carrier protein itself was also prepared and diluted twice. The reactivity with the culture supernatant of ACSPM-1 strain, ACSPM-2 strain, and ACSPM-3 strain was confirmed. The results are shown in FIGS. All the culture supernatants reacted only with N 1 -Ac-Spm-GMBS-HSA, and did not react with GMBA-HSA, AMS-HSA or HSA at all.
[Clone cell subtype]
The subtype of immunoglobulin produced by each clonal cell was determined using a mouse monoclonal Sub-isotyping kit (Zymed code No. 97-6550). As a result, the 0520 antibody (ACSPM-1 strain) and the 4914 antibody (ACSPM-2 strain) were IgG 1 , and the 8624 antibody (ACSPM-3 strain) was IgG 2b .

実施例2 モノクローナル抗体の特異性
作成したモノクローナル抗体の特異性は、抗体の固相化抗原への結合を、測定対象物質がどの程度阻害するかを検出する系、「ELISA結合阻害法」で評価した。即ち、実験例2の方法に従い、3つの抗原、N−Ac−Spm−GMBS−HSA、N−Ac−Spd−GA−HSA、N,N12−2Ac−Spm−GA−HSAを、それぞれ固相化したプレートを用いて、各濃度のポリアミン類が、抗体と固相化抗原との反応を、どの程度阻害するかを評価した。免疫原に相当するN−Ac−Spm−GMBS−HSAを固相化した系では、検討したポリアミン類の何れによっても全く阻害されず、測定系としては不適切であった。一方、N−Ac−Spd−GA−HSA、又は、N,N12−2Ac−Spm−GA−HSAを固相化した系では、検討した各成分、Spm、N−Ac−Spd、N−Ac−Spm、N,N12−2Ac−Spmによって、図4〜9に示した結合阻害曲線(検量線)が得られた。0520、4914、8624のいずれの抗体も、N−Ac−SpmとN,N12−2Ac−Spmに強く、N−Ac−Spdと極めて弱く反応(阻害)し、Spmとは全く反応(阻害)しなかった。図から、これらの抗体を用いてポリアミン類を測定した場合の測定感度を50%結合阻害濃度(EC50値)として求め、結果を表1に示した。
Example 2 Specificity of Monoclonal Antibody The specificity of the prepared monoclonal antibody is evaluated by “ELISA binding inhibition method”, a system for detecting how much the measurement target substance inhibits the binding of the antibody to the immobilized antigen. did. That is, according to the method of Experimental Example 3, three antigens, N 1 -Ac-Spm-GMBS-HSA, N 1 -Ac-Spd-GA-HSA, N 1 , N 12 -2Ac-Spm-GA-HSA, Using the respective solid-phased plates, it was evaluated how much each concentration of polyamines inhibits the reaction between the antibody and the solid-phased antigen. In the system in which N 1 -Ac-Spm-GMBS-HSA corresponding to the immunogen was immobilized, it was not inhibited at all by any of the examined polyamines and was inappropriate as a measurement system. On the other hand, in the system in which N 1 -Ac-Spd-GA-HSA or N 1 , N 12 -2Ac-Spm-GA-HSA is solid-phased, each component examined, Spm, N 1 -Ac-Spd, The binding inhibition curves (calibration curves) shown in FIGS. 4 to 9 were obtained by N 1 -Ac-Spm and N 1 , N 12 -2Ac-Spm. Any of the antibodies 0520, 4914, and 8624 is strong against N 1 -Ac-Spm and N 1 , N 12 -2Ac-Spm, reacts very weakly with N 1 -Ac-Spd (inhibits), and does not react with Spm at all. Did not (inhibit). From the figure, the measurement sensitivity when polyamines were measured using these antibodies was determined as 50% binding inhibition concentration (EC50 value), and the results are shown in Table 1.

なお、図4はN,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での0520抗体の特異性を、図5は、N,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での4914抗体の特異性を、図6は、N,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での8624抗体の特異性を、図7は、N−Ac−Spd−GA−HSA固相化ELISA結合阻害法での0520抗体の特異性を、図8は、N−Ac−Spd−GA−HSA固相化ELISA結合阻害法での4914抗体の特異性を、図9は、N−Ac−Spd−GA−HSA固相化ELISA結合阻害法での8624抗体の特異性を示したものである。 4 shows the specificity of the 0520 antibody in the N 1 , N 12 -2Ac-Spm-GA-HSA solid-phase ELISA binding inhibition method, and FIG. 5 shows the N 1 , N 12 -2Ac-Spm-GA- FIG. 6 shows the specificity of the antibody 4914 in the N 1 , N 12 -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method, and the specificity of the 4914 antibody in the HSA immobilized ELISA binding inhibition method. FIG. 7 shows the specificity of the 0520 antibody in the N 1 -Ac-Spd-GA-HSA immobilized ELISA binding inhibition method, and FIG. 8 shows the inhibition of the N 1 -Ac-Spd-GA-HSA immobilized ELISA binding. FIG. 9 shows the specificity of the 8624 antibody in the N 1 -Ac-Spd-GA-HSA solid-phase ELISA binding inhibition method.

0520、4914、8624のいずれの抗体も、N−Ac−SpmとN,N12−2Ac−Spmに特異的であった。平松ら(J.Biochem.,117,p107−112(1995))やG.A.van den Bergら(Clin.Chem.,32(10),p1930−1937(1986))によると、尿中のN−Ac−Spm濃度は、N,N12−2Ac−Spmより著しく低いことから、この測定法で、実質的に尿中のN,N12−2Ac−Spm濃度を反映する測定値を得ることができる。 All antibodies 0520, 4914, and 8624 were specific for N 1 -Ac-Spm and N 1 , N 12 -2Ac-Spm. Hiramatsu et al. (J. Biochem., 117, p107-112 (1995)) and G. A. According to van den Berg et al. (Clin. Chem., 32 (10), p1930-1937 (1986)), the concentration of N 1 -Ac-Spm in urine is significantly lower than that of N 1 , N 12 -2Ac-Spm. From this, it is possible to obtain a measurement value that substantially reflects the concentration of N 1 , N 12 -2Ac-Spm in urine by this measurement method.

Figure 2008050362
Figure 2008050362

実施例3
96穴マイクロタイタープレート(ヌンク社製 Nunc Immunoplate)の各wellに抗原としてN,N12−2Ac−Spm−GA−HSAを16.1μg/mL含む10mMトリス・塩酸緩衝液(pH8.5)150μLを入れ、40℃で30分間放置してコーティングした。次にPBST300μLで洗浄後、スキムミルク1%を含む50mMトリス・塩酸緩衝液(pH7.2)300μLでブロッキング処理した。PBSTで洗浄後、各wellにACSPM−1の培養上清をPBSTで25倍希釈した液50μLと、測定対象物のPut(プトレッシン)、Ac−Put、Orn(オルニチン)、Cad(カダベリン)、Spd、N−Ac−Spd、N−Ac−Spd、N,N−2Ac−Spd、Spm、N−Ac−Spm、N,N12−2Ac−SpmをPBSTで各種濃度に希釈した液50μLを加え、4℃で一晩反応させた。各ウェルをPBSTで洗浄後、PBSTで2000倍に希釈したヤギ抗マウスIgG(H&L)−ビオチン(AMERICAN QUALEX社)100μLを加え、室温で1.5時間反応し、PBSTで洗浄後、PBSTで3000倍希釈したHRP−ストレプトアビジン(フナコシ社)100μLを加え、室温で30分反応させた。結合した酵素の活性は、PBSTで洗浄した各wellに、o−フェニレンジアミン0.5mg/mLと過酸化水素0.012%を含む0.1Mクエン酸・リン酸緩衝液(pH5.3)100μLを加え、室温下に6分間反応させ、ELISAプレートリーダー(SLT−Lab Instruments社)を用い、492nmにおける吸光度の増加として測定した。結果を図10に示した。図10から明らかなようにこの反応系によれば、N,N12−2Ac−Spmに対する特異性が向上していることが判る。
Example 3
150 μL of 10 mM Tris / hydrochloric acid buffer (pH 8.5) containing 16.1 μg / mL of N 1 , N 12 -2Ac-Spm-GA-HSA as an antigen in each well of a 96-well microtiter plate (Nunc Immunoplate manufactured by Nunk) And allowed to stand at 40 ° C. for 30 minutes for coating. Next, after washing with 300 μL of PBST, blocking treatment was performed with 300 μL of 50 mM Tris-HCl buffer (pH 7.2) containing 1% skim milk. After washing with PBST, 50 μL of the ACSPM-1 culture supernatant diluted 25 times with PBST in each well, and Put (putrescine), Ac-Put, Orn (ornithine), Cad (cadaverine), Spd N 1 -Ac-Spd, N 8 -Ac-Spd, N 1 , N 8 -2 Ac-Spd, Spm, N 1 -Ac-Spm, N 1 , N 12 -2Ac-Spm diluted with PBST to various concentrations 50 μL of the solution was added and reacted at 4 ° C. overnight. After washing each well with PBST, 100 μL of goat anti-mouse IgG (H & L) -biotin (AMERICA QUALEX) diluted 2000 times with PBST was added, reacted at room temperature for 1.5 hours, washed with PBST, and then 3000 times with PBST. 100 [mu] L of HRP-streptavidin (Funakoshi) diluted 1-fold was added and reacted at room temperature for 30 minutes. The activity of the bound enzyme is as follows. Each well washed with PBST is 100 μL of 0.1 M citrate / phosphate buffer (pH 5.3) containing 0.5 mg / mL o-phenylenediamine and 0.012% hydrogen peroxide. The mixture was allowed to react at room temperature for 6 minutes, and the increase in absorbance at 492 nm was measured using an ELISA plate reader (SLT-Lab Instruments). The results are shown in FIG. As can be seen from FIG. 10, according to this reaction system, the specificity to N 1 , N 12 -2Ac-Spm is improved.

実施例4 最適化されたELISA結合阻害法でのモノクローナル抗体の特異性
実験例2の測定法を改良し、再度、抗体の特異性と測定感度を確認した。96穴マイクロタイタープレート(ヌンク社製 Nunc Immunoplate II)の各ウェルに、抗原としてN−アセチルスペルミンのヒト血清アルブミン複合体(N−Ac−Spm−GA−HSA)、又は、N,N12−ジアセチルスペルミンのヒト血清アルブミン複合体(N,N12−2Ac−Spm−GA−HSA)15μg/mLを含む10mMトリス・塩酸緩衝液(pH8.5)150μLを入れ、40℃で30分間放置してコーティングした。次に、コーティング液を捨ててPBSTで洗浄後、スキムミルク1%を含む50mMトリス・塩酸で緩衝液(pH7.4)300μL加え、37℃で1時間ブロッキング処理した。
Example 4 Specificity of Monoclonal Antibody in Optimized ELISA Binding Inhibition Method The measurement method in Experimental Example 2 was improved, and the specificity and sensitivity of the antibody were confirmed again. To each well of a 96-well microtiter plates (Nunc Nunc Immunoplate II), N 1 as an antigen - human serum albumin conjugate of spermine (N 1 -Ac-Spm-GA -HSA), or, N 1, N 150 μL of 10 mM Tris-HCl buffer (pH 8.5) containing 15 μg / mL of human serum albumin complex of 12 -diacetylspermine (N 1 , N 12 -2Ac-Spm-GA-HSA) was added, and the mixture was kept at 40 ° C. for 30 minutes. Allowed to coat. Next, after discarding the coating solution and washing with PBST, 300 μL of a buffer solution (pH 7.4) was added with 50 mM Tris-HCl containing 1% skim milk, followed by blocking treatment at 37 ° C. for 1 hour.

ブロッキング液を捨ててPBSTで洗浄後、各ウェルに、ハイブリドーマ3株のそれぞれの培養上清液を表2に示した倍率(200〜20,000倍)にPBSTで希釈した液25μLと、測定対象物のスペルミン(Spm)、N,N−ジアセチルスペルミジン(N,N−2Ac−Spd)、N−アセチルスペルミジン(N−Ac−Spd)、N−アセチルスペルミン(N−Ac−Spm)、N,N12−ジアセチルスペルミン(N,N12−2Ac−Spm)をPBSTで各濃度に希釈した液75μLを加え、室温下に3時間反応させた。ウェルをPBSTで洗浄後、PBSTで2000倍に希釈したビオチン標識ヤギ抗マウスIgG抗体溶液50μLを加え、室温下に1時間反応させた。ウェルを再びPBSTで洗浄後、PBSTで3000倍に希釈した西洋わさびペルオキシダーゼ標識ストレプトアビジン溶液50μLを加え、室温下に30分間反応させた。 After discarding the blocking solution and washing with PBST, each well was diluted with PBST at the magnification (200 to 20,000 times) shown in Table 2 for each culture supernatant of the hybridoma 3 strain, and the measurement target objects of spermine (Spm), N 1, N 8 - diacetyl spermidine (N 1, N 8 -2Ac- Spd), N 1 - acetyl spermidine (N 1 -Ac-Spd), N 1 - spermine (N 1 - Ac-Spm), N 1, N 12 - diacetylspermine (N 1, N 12 -2Ac- Spm) a liquid 75μL added diluted to each concentration with PBST, and reacted for 3 hours at room temperature. After the wells were washed with PBST, 50 μL of a biotin-labeled goat anti-mouse IgG antibody solution diluted 2000 times with PBST was added and reacted at room temperature for 1 hour. The wells were washed again with PBST, and then 50 μL of a horseradish peroxidase-labeled streptavidin solution diluted 3000 times with PBST was added and reacted at room temperature for 30 minutes.

結合した酵素の活性は、PBSTで洗浄した各ウェルに、o−フェニレンジアミン0.5mg/mLと過酸化水素0.012%を含む0.1Mクエン酸リン酸緩衝液(pH5.3)100μLを加え、室温下に5分間発色反応させ、ELISAプレートリーダー(SLT−Lab Instruments社)を用い、492nmにおける吸光度の増加として測定した。
結果を表2にまとめた。測定系を改良し、抗体の希釈倍率を上げることにより、N,N12−Ac−Spm−GA−HSAを固相化した系の測定感度と特異性は、大幅に向上した。即ち、N,N12−Ac−Spmの測定感度は、0520抗体が8倍、4914抗体が22倍、8624抗体が70倍に向上し、50%結合阻害濃度で比較した感度は、0.06〜0.2μMになった。また、特異性をN,N12−Ac−Spmによる50%結合阻害活性とN−Ac−Spdによる50%結合阻害活性との比として表現した場合、0520抗体が48倍、4914抗体が117倍、8624抗体が45倍となり、何れも改良前より向上した。
The activity of the bound enzyme was determined by adding 100 μL of 0.1 M citrate phosphate buffer (pH 5.3) containing 0.5 mg / mL o-phenylenediamine and 0.012% hydrogen peroxide to each well washed with PBST. In addition, the color reaction was allowed to proceed at room temperature for 5 minutes, and the increase in absorbance at 492 nm was measured using an ELISA plate reader (SLT-Lab Instruments).
The results are summarized in Table 2. By improving the measurement system and increasing the dilution ratio of the antibody, the measurement sensitivity and specificity of the system in which N 1 , N 12 -Ac-Spm-GA-HSA was immobilized were greatly improved. That is, the measurement sensitivity of N 1 , N 12 -Ac-Spm was improved 8 times for the 0520 antibody, 22 times for the 4914 antibody and 70 times for the 8624 antibody, and the sensitivity compared with the 50% binding inhibition concentration was 0. It became 06-0.2 micromol. In addition, when the specificity is expressed as a ratio of 50% binding inhibitory activity by N 1 , N 12 -Ac-Spm to 50% binding inhibitory activity by N 1 -Ac-Spd, 0520 antibody is 48 times and 4914 antibody is 117 times and 8624 antibody became 45 times, both improved from before improvement.

Figure 2008050362
Figure 2008050362

実施例5 4914モノクローナル抗体の特異性
実施例4で最も測定感度と特異性の高かった4914モノクローナル抗体を用い、実施例3の方法でプトレッシン(Put)、アセチルプトレッシン(Ac−Put)、L−オルニチン(Orn)、カダベリン(Cad)、スペルミジン(Spd)、N−アセチルスペルミジン(N−Ac−Spd)、N−アセチルスペルミジン(N−Ac−Spd)、N,N−ジアセチルスペルミジン(N,N−2Ac−Spd)、スペルミン(Spm)、N−アセチルスペルミン(N−Ac−Spm)やN,N12−ジアセチルスペルミン(N,N12−2Ac−Spm)の希釈系列を測定し、4914モノクローナル抗体の特異性検討した。図11のように、この測定系は、N,N12−2Ac−Spmに特異的で、N−Ac−SpmとはN,N12−2Ac−Spmの24%、N−Ac−Spdとは0.85%、N,N−2Ac−Spdとは0.6%、Spmとは0.1%、その他のポリアミン類とはほとんど反応しなかった。
Example 5 Specificity of 4914 Monoclonal Antibody The 4914 monoclonal antibody having the highest measurement sensitivity and specificity in Example 4 was used, and putrescine (Put), acetylputrescine (Ac-Put), L - ornithine (Orn), cadaverine (Cad), spermidine (Spd), N 8 - acetyl spermidine (N 8 -Ac-Spd), N 1 - acetyl spermidine (N 1 -Ac-Spd), N 1, N 8 - diacetyl spermidine (N 1, N 8 -2Ac- Spd), spermine (Spm), N 1 - spermine (N 1 -Ac-Spm) and N 1, N 12 - diacetylspermine (N 1, N 12 -2Ac- (Spm) dilution series was measured to examine the specificity of the 4914 monoclonal antibody. As shown in FIG. 11, this measurement system is specific to N 1 , N 12 -2Ac-Spm, and N 1 -Ac-Spm is 24% of N 1 , N 12 -2Ac-Spm, N 1 -Ac. 0.85% the -Spd, 0.6% and N 1, N 8 -2Ac-Spd , 0.1% and Spm, and other polyamines hardly react.

実施例6 健常者の尿中N,N12−2Ac−Spmの測定
実施例5の方法で、健常者の尿検体16例(男性8例、女性8例)の希釈系列を測定し、尿中のN,N12−2Ac−Spm濃度を求めた。測定例の一部を図12に示した。男子及び女子8例ずつの平均値±SDは、それぞれ0.34±0.16、0.39±0.14μM/g−クレアチニンで、全体の平均値は0.36μM/g−クレアチニンであった。平松らの報告(J.Biochem.,117,p107−112(1995))によると、尿中におけるN,N12−2Ac−Spm:N−Ac−Spm:N−Ac−Spd:N,N−2Ac−Spdの存在比は、3.2%:1.0%:86.2%:9.6%であることが報告されている。この測定系の特異性からすれば、N,N12−2Ac−Spm以外のポリアミン成分の影響は軽微と考えられ、尿中のN,N12−2Ac−Spmは、ほぼ正確に測定されているものと考えられる。
Example 6 Measurement of N 1 , N 12 -2Ac-Spm in urine of healthy subjects By the method of Example 5, a dilution series of 16 urine specimens of healthy subjects (8 men and 8 women) was measured, and urine The concentration of N 1 , N 12 -2Ac-Spm was determined. A part of the measurement example is shown in FIG. The mean ± SD for each of the 8 boys and girls was 0.34 ± 0.16 and 0.39 ± 0.14 μM / g-creatinine, respectively, and the overall mean was 0.36 μM / g-creatinine. . According to a report by Hiramatsu et al. (J. Biochem., 117, p107-112 (1995)), N 1 , N 12 -2Ac-Spm: N 1 -Ac-Spm: N 1 -Ac-Spd: N in urine 1 , N 8 -2Ac-Spd is reported to be 3.2%: 1.0%: 86.2%: 9.6%. Considering the specificity of this measurement system, the influence of polyamine components other than N 1 , N 12 -2Ac-Spm is considered to be minor, and urinary N 1 , N 12 -2Ac-Spm is measured almost accurately. It is thought that.

ACSPM−1株培養上清液の免疫原への特異性Specificity of ACSPM-1 strain culture supernatant for immunogen ACSPM−2株培養上清液の免疫原への特異性Specificity of ACSPM-2 strain culture supernatant for immunogen ACSPM−3株培養上清液の免疫原への特異性Specificity of ACSPM-3 strain culture supernatant for immunogen ,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での0520抗体の特異性Specificity of 0520 antibody in N 1 , N 12 -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method ,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での4914抗体の特異性Specificity of antibody 4914 in N 1 , N 12 -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method ,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での8624抗体の特異性Specificity of 8624 antibody in N 1 , N 12 -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method −Ac−Spd−GA−HSA固相化ELISA結合阻害法での0520抗体の特異性Specificity of 0520 antibody in N 1 -Ac-Spd-GA-HSA immobilized ELISA binding inhibition method −Ac−Spd−GA−HSA固相化ELISA結合阻害法での4914抗体の特異性4914 antibody specificity in N 1 -Ac-Spd-GA- HSA immobilized ELISA binding inhibition method −Ac−Spd−GA−HSA固相化ELISA結合阻害法での8624抗体の特異性Specificity of the 8624 antibody in the N 1 -Ac-Spd-GA-HSA immobilized ELISA binding inhibition method ,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での0520抗体の特異性Specificity of 0520 antibody in N 1 , N 12 -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method ,N12−2Ac−Spm−GA−HSA固相化ELISA結合阻害法での4914抗体の特異性Specificity of antibody 4914 in N 1 , N 12 -2Ac-Spm-GA-HSA immobilized ELISA binding inhibition method ,N12−2Ac−Spmの検量線及び尿検体中のN,N12−2Ac−Spmの濃度N 1, N 12 concentration of N 1, N 12 -2Ac-Spm calibration curve and urine specimens -2Ac-Spm

Claims (5)

固相化N,N12−ジアセチルスペルミン又は固相化N−アセチルスペルミンと抗N,N12−ジアセチルスペルミンモノクローナル抗体との免疫反応において、N,N12−ジアセチルスペルミンによる該免疫反応の50%阻害活性がN−アセチルスペルミジンによる該免疫反応の50%阻害活性の少なくとも20倍となる抗N,N12−ジアセチルスペルミンモノクロナール抗体。 In the immunoreaction of solid-phased N 1 , N 12 -diacetylspermine or solid-phased N 1 -acetylspermine and an anti-N 1 , N 12 -diacetylspermine monoclonal antibody, the immune reaction by N 1 , N 12 -diacetylspermine 50% inhibitory activity N 1 - anti N 1 comprising at least 20 times the 50% inhibitory activity of the immune response by acetyl spermidine, N 12 - diacetylspermine monoclonal antibodies. 固相化N,N12−ジアセチルスペルミン又は固相化N−アセチルスペルミンと抗N,N12−ジアセチルスペルミンモノクローナル抗体との免疫反応において、該免疫反応を50%阻害するN,N12−ジアセチルスペルミンの濃度が20μM以下である請求項1記載の抗N,N12−ジアセチルスペルミンモノクローナル抗体。 In an immunoreaction of immobilized N 1 , N 12 -diacetylspermine or immobilized N 1 -acetylspermine and an anti-N 1 , N 12 -diacetylspermine monoclonal antibody, N 1 , N that inhibits the immune reaction by 50% 12 - anti-N 1 according to claim 1, wherein the concentration of diacetylspermine is below 20 [mu] M, N 12 - diacetylspermine monoclonal antibodies. モノクローナル抗体が、細胞株ACSPM−1(工業技術院生命工学工業技術研究所寄託番号、FERM P−16297)を培養して得られるモノクローナル抗体0520、細胞株ACSPM−2(工業技術院生命工学工業技術研究所寄託番号、FERM P−16298)を培養して得られるモノクローナル抗体4914、又は、細胞株ACSPM−3(工業技術院生命工学工業技術研究所寄託番号、FERM P−16299)を培養して得られるモノクローナル抗体8624である請求項1又は2に記載の抗N,N12−ジアセチルスペルミンモノクローナル抗体。 Monoclonal antibody 0520 obtained by culturing the cell line ACSPM-1 (Institute of Biotechnology, Industrial Technology Research Institute deposit number, FERM P-16297), cell line ACSPM-2 (Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology) Obtained by culturing the monoclonal antibody 4914 obtained by culturing the laboratory deposit number, FERM P-16298), or the cell line ACSPM-3 (according to the Biotechnology Industrial Technology Laboratory deposit number, FERM P-16299). anti N 1 according to claim 1 or 2 which is a monoclonal antibody 8624 that is, N 12 - diacetylspermine monoclonal antibodies. 請求項1〜3のいずれか一項に記載の抗N,N12−ジアセチルスペルミンモノクローナル抗体を産生する細胞株。 Anti N 1 according to any one of claims 1 to 3, N 12 - cells producing diacetylspermine monoclonal antibody strains. 動物(但し、ヒトを除く)をN,N12−ジアセチルスペルミン又はN−アセチルスペルミンと、キャリア物質の複合体で免疫し、次いで免疫した動物のB細胞とミエローマ細胞を融合し、得られたハイブリドーマから選別された細胞株を培養して得ることを特徴とする抗N,N12−ジアセチルスペルミンモノクロナール抗体の製造法。 Animals (excluding human) N 1 a, N 12 - Diacetylspermine or N 1 - fused and spermine were immunized with the complex of carrier substance and then the B cells with myeloma cells of the immunized animals, obtained anti N 1, N 12, characterized in that obtained by culturing the cell lines selected from hybridomas - preparation of diacetylspermine monoclonal antibodies.
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