JP6508996B2 - Method of detecting heart failure by β-ANP - Google Patents
Method of detecting heart failure by β-ANP Download PDFInfo
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- JP6508996B2 JP6508996B2 JP2015060777A JP2015060777A JP6508996B2 JP 6508996 B2 JP6508996 B2 JP 6508996B2 JP 2015060777 A JP2015060777 A JP 2015060777A JP 2015060777 A JP2015060777 A JP 2015060777A JP 6508996 B2 JP6508996 B2 JP 6508996B2
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Description
本発明は心不全の検出方法及びそのキットに関する。 The present invention relates to a method for detecting heart failure and a kit thereof.
心房性ナトリウム利尿ペプチド（ＡＮＰ：ａｔｒｉａｌ ｎａｔｒｉｕｒｅｔｉｃ ｐｅｐｔｉｄｅ）は松尾・寒川らにより１９８４年に報告されたペプチドホルモンで（非特許文献１参照）、心臓で産生され強力な利尿、ナトリウム利尿活性と平滑筋弛緩活性を持つ。ＡＮＰは、ヒトばかりでなく、げっ歯類以外の哺乳類では同一のアミノ酸配列を有し、例えばヒト、ウシ、ブタのＡＮＰは同一である。ＡＮＰには、分子量約３０００のα型、α型の逆平行二量体であるβ型、α型の前駆物質であるγ型の３種類の分子型が確認されている（図１参照）。 Atrial natriuretic peptide (ANP) is a peptide hormone reported in 1984 by Matsuo and Sakawa et al. (See Non-Patent Document 1), which is produced in the heart and has strong diuretic, natriuretic activity and smooth muscle relaxation. It has activity. ANPs have the same amino acid sequence in mammals as well as humans but rodents, for example, human, bovine and porcine ANPs are identical. In ANP, three molecular types of α-type having a molecular weight of about 3000, β-type which is an antiparallel dimer of α-type, and γ-type which is a precursor of α-type are confirmed (see FIG. 1).
ＡＮＰは、心臓、特に心房から分泌されるホルモンで、利尿作用、Ｎａ利尿作用、血管拡張作用、レニン・アンジオテンシン・アルドステロン系や交感神経系の抑制作用など多様な生物活性を有し、体液量、血圧の調節に重要な役割を担っている（非特許文献２，３参照）。
ANP is a hormone secreted from the heart, particularly the atria, and has various biological activities such as diuretic action, natriuretic action, vasodilator action, renin-angiotensin-aldosterone system and sympathetic nervous system-suppressing action, and It plays an important role in the regulation of blood pressure (see non-patent
ＡＮＰは心血行動態的負荷、特に心房内圧の増加が主要な産生、分泌刺激になると考えられ、体液量あるいは心房内圧の増加する各種心疾患、腎疾患において血中濃度の増加が認められることから、これら病態の把握、重症度の指標として極めて有用であり、ルーチンの検査項目として日常臨床に応用されている（非特許文献４〜６参照）。
ANP is considered to be the stimulation of production and secretion mainly due to an increase in cardiovascular hemodynamic load, especially in atrial pressure, and an increase in blood concentration is observed in various heart diseases and renal diseases in which the fluid volume or atrial pressure is increased. It is extremely useful as a grasp of these pathological conditions and as an indicator of severity, and is applied to daily practice as a routine examination item (see Non-Patent
α−ＡＮＰは血中に存在するＡＮＰ類の中心となる分子種であり、２８アミノ酸残基からなり、Ｎ末端から７番目のＣｙｓと２３番目のＣｙｓが分子内でジスルフィド結合し、その間の配列が環状構造をなしている。一方、β−ＡＮＰは、２分子のα−ＡＮＰが分子間でジスルフィド結合した、α−ＡＮＰの逆平行二量体である（特許文献１参照）。γ−ＡＮＰは１２６アミノ酸残基からなり、そのＣ末端にα−ＡＮＰ配列を有する。健常者においては、γ−ＡＮＰは心房中に保存されており、分泌時にα−ＡＮＰとＮ末端ペプチドに切断され、血中ではこの２つの分子で存在する（非特許文献７参照）。 α-ANP is a central molecular species of ANPs present in blood and consists of 28 amino acid residues, and the seventh Cys from the N terminus and the 23rd Cys are intermolecularly disulfide bonded, and the sequence between them Has a ring structure. On the other hand, β-ANP is an antiparallel dimer of α-ANP in which two molecules of α-ANP are intermolecularly disulfide bonded (see Patent Document 1). γ-ANP consists of 126 amino acid residues and has an α-ANP sequence at its C-terminus. In healthy individuals, γ-ANP is conserved in the atrium, is cleaved into α-ANP and an N-terminal peptide upon secretion, and exists in the blood in these two molecules (see Non-patent Document 7).
ＡＮＰの測定は、酵素やラジオアイソトープで標識した抗体と、担体に固定化した固定化抗体とでサンドイッチされる免疫学的測定法が利用されており、特に臨床検査薬として広く医療現場で使用されており（非特許文献９参照）、α型、β型、γ型の３種類の総和が測定されるものである。 The measurement of ANP uses an immunoassay in which an antibody labeled with an enzyme or radioisotope and an immobilized antibody immobilized on a carrier are used, and in particular, it is widely used as a clinical test drug in the medical field. (See Non-Patent Document 9), and the sum of three types of α-type, β-type, and γ-type is measured.
α−ＡＮＰの有用性は多く報告されているが、β−ＡＮＰとγ−ＡＮＰについても重症心不全症例で増加していることが報告されている（非特許文献８参照）。特にβ−ＡＮＰの動向は顕著であり、健常者では確認されない様式であるが、心不全の重篤度に合わせて出現することが確認され、心不全病態との関係が確認されている（非特許文献１０，１１参照）。心不全治療前後におけるＡＮＰの存在様式を検討すると、心不全時は，α−、γ−ＡＮＰの他に、健常人では認めなかったβ−ＡＮＰと思われる中分子型ＡＮＰが出現し、心不全治療による血行動態・臨床症状が改善した後には、ＡＮＰ濃度の低下とともにこのβ−ＡＮＰの割合が減少あるいは消失している。
Although the utility of α-ANP has been widely reported, it is also reported that β-ANP and γ-ANP are also increasing in severe heart failure cases (see Non-patent Document 8). In particular, the trend of β-ANP is remarkable and is a mode that is not confirmed in healthy people, but it has been confirmed to appear according to the severity of heart failure, and its relationship with heart failure pathologic condition has been confirmed (Non-patent
治療により心不全が改善する過程における血中ＡＮＰ濃度と存在様式の推移では、血中ＡＮＰ濃度は治療により漸減し、存在様式は重症心不全時はβ−ＡＮＰがピークを形成していたが、治療によりβ−ＡＮＰは漸減し、血行動態および臨床症状が改善した時点では存在様式はβ−ＡＮＰの割合は著しく減少あるいは消失し、心不全病態とβ−ＡＮＰの関係が示されている（非特許文献１１参照）。しかしながらこれらの報告では具体的にβ−ＡＮＰ値のカットオフ値、参考基準値あるいは総ＡＮＰに対する存在比率などを用いて心不全の病態や治療効果を見ていない。加えてα型、β型、γ型の各分子型の共通領域（即ちα−ＡＮＰ）を用いる抗体と逆相高速液体クロマト（ＲＰ−ＨＰＬＣ）法を用いた測定法により確認しているために、定量性に乏しく操作による変動も大きい。 In the transition of blood ANP concentration and mode of existence in the process of improvement of heart failure by treatment, blood ANP concentration gradually decreased by treatment, and the mode of existence was that β-ANP formed a peak during severe heart failure, but by treatment The β-ANP gradually decreases, and at the time when hemodynamics and clinical symptoms improve, the proportion of β-ANP significantly decreases or disappears, and the relationship between heart failure disease state and β-ANP is shown (Non-patent Document 11) reference). However, in these reports, specifically, the condition of the heart failure and the treatment effect are not seen using the cutoff value of the β-ANP value, the reference standard value or the abundance ratio to the total ANP. In addition, because it is confirmed by the measurement method using the antibody and the reverse phase high performance liquid chromatography (RP-HPLC) method using the common region (that is, α-ANP) of α-type, β-type and γ-type molecular types There is little quantitativeness and the fluctuation due to operation is large.
このように、従来は、β−ＡＮＰの分子型に対し特異的な抗体や測定方法は見出されていなかったために、β−ＡＮＰの総ＡＮＰに対する存在比率から心不全の状態を判断する例はなかった。 Thus, conventionally, no antibody or measurement method specific to the molecular form of β-ANP has been found, so there is no example of judging the state of heart failure from the abundance ratio of β-ANP to total ANP The
β−ＡＮＰは、心不全の重篤度に合わせて出現することが確認され、心不全病態との関係が確認されている。しかし市販されているＡＮＰを測定するサンドイッチ免疫測定法においては、α型、α型の２量体であるβ型、α型の前駆物質とされるγ型の３種類の分子型も測定され、β−ＡＮＰのみを特異的に測定する方法は一般化されていない。 It is confirmed that (beta) -ANP appears according to the severity of heart failure, and the relationship with a heart failure pathology is confirmed. However, in a sandwich immunoassay that measures ANP that is commercially available, three types of molecular forms of α-type and α-type dimers, β-type and α-type precursors, which are regarded as precursors of α-type, are also measured. The method of specifically measuring only β-ANP is not generalized.
β−ＡＮＰに特異的な測定系については、α−ＡＮＰのホモダイマーであることから、同一のモノクローナル抗体を用いてβ−ＡＮＰを測定する測定系は報告されている（特許文献２参照）。しかし、β−ＡＮＰに特異的な抗体を用いているわけではなく、α−ＡＮＰ濃度が高くなると競合阻害を受けることになり測定系によっては感度が低くなることがあり、依然、高感度にβ−ＡＮＰのみを測定するためには、課題が残っている（特許文献２参照）。 As a measurement system specific to β-ANP, a measurement system for measuring β-ANP using the same monoclonal antibody has been reported because it is a homodimer of α-ANP (see Patent Document 2). However, antibodies specific for β-ANP are not used, and as the concentration of α-ANP increases, competitive inhibition may occur and the sensitivity may be lowered depending on the measurement system. In order to measure only ANP, a subject remains (refer patent document 2).
総ＡＮＰおよびβ−ＡＮＰが心不全の重症例で高値を示し、治療により改善すると低下することが報告されているが、これらの報告では具体的にβ−ＡＮＰ値のカットオフ値、参考基準値あるいは総ＡＮＰに対する存在比率などを用いて心不全の病態や治療効果を見ていない。加えてα型、β型、γ型の各分子型の共通領域（即ちα−ＡＮＰ）を用いる抗体と逆相高速液体クロマト（ＲＰ−ＨＰＬＣ）法を用いた測定法により確認しているために定量性に乏しく操作による変動も大きいために診断には利用されていない。 Although it has been reported that total ANP and β-ANP show high values in severe cases of heart failure and they decrease when they are improved by treatment, these reports specifically cut off the β-ANP value, reference standard value or He did not see the pathophysiology and therapeutic effect of heart failure using the ratio to total ANP, etc. In addition, because it is confirmed by the measurement method using the antibody and the reverse phase high performance liquid chromatography (RP-HPLC) method using the common region (that is, α-ANP) of α-type, β-type and γ-type molecular types It has not been used for diagnosis because it is poorly quantified and the fluctuation due to operation is large.
このように、従来は、β−ＡＮＰの分子型に非常に特異的な抗体や測定方法は見出されていなかったために、β−ＡＮＰと総ＡＮＰとの存在比から心不全の状態を判断する例はなかった。 Thus, since no antibody or measurement method that is very specific to the molecular form of β-ANP has been found conventionally, an example of determining the state of heart failure from the abundance ratio of β-ANP to total ANP There was no.
本発明の目的は、β−ＡＮＰと総ＡＮＰとの比から、心不全の進行または心不全治療の効果を判定することができる心不全の検出方法を提供することである。 An object of the present invention is to provide a method for detecting heart failure which can determine the progress of heart failure or the effect of heart failure treatment from the ratio of β-ANP to total ANP.
本発明者らは、α−ＡＮＰの逆平行ダイマーが特異的につくるジスルフィド結合を含むアミノ酸配列や構造をエピトープとして認識する抗体を獲得し、それを利用するβ−ＡＮＰの高感度測定法を完成している（特願２０１４−２１４１０３号）。そして本発明者らは、上記課題に関し鋭意検討した結果、本発明に到達した。 The present inventors have acquired an antibody that recognizes as a epitope an amino acid sequence or structure containing a disulfide bond that is specifically formed by an antiparallel dimer of α-ANP, and completed a highly sensitive assay of β-ANP using it. (Japanese Patent Application No. 2014-214103). And the present inventors arrived at the present invention as a result of earnestly examining the above-mentioned subject.
即ち本発明は以下のとおりである。
（１）試料中のβ−ＡＮＰと総ＡＮＰとの比を求めることを特徴とする、心不全の検出方法。
（２）請求項１に記載の方法において、β−ＡＮＰと総ＡＮＰとの比が、総ＡＮＰに対するβ−ＡＮＰの比（β−ＡＮＰ／総ＡＮＰ）である方法。
（３）β−ＡＮＰを特異的に測定する試薬および総ＡＮＰを測定する試薬を含むことを特徴とする、心不全の検出キット。
以下、本発明を更に詳細に説明する。
That is, the present invention is as follows.
(1) A method for detecting heart failure, which comprises determining the ratio of β-ANP to total ANP in a sample.
(2) The method according to
(3) A kit for detecting heart failure, which comprises a reagent for specifically measuring β-ANP and a reagent for measuring total ANP.
Hereinafter, the present invention will be described in more detail.
本発明において、総ＡＮＰとは、α−ＡＮＰ、β−ＡＮＰ及びγ−ＡＮＰを合計したものを表す。総ＡＮＰの測定方法としては特に限定はなく、例えば市販の免疫測定試薬等を用いて行うことができる。例えばα−ＡＮＰ、β−ＡＮＰ、γ−ＡＮＰの共通部位を認識する抗体を組み合わせた測定系を用いることができ、市販キットのように例えばα−ＡＮＰの環状部位とＣ末端部分を認識するものでも良い。 In the present invention, total ANP refers to the sum of α-ANP, β-ANP and γ-ANP. There is no limitation in particular as a measuring method of total ANP, For example, it can carry out using a commercially available immunoassay reagent etc. For example, a measurement system combined with an antibody that recognizes a common site of α-ANP, β-ANP, γ-ANP can be used, such as a commercially available kit that recognizes cyclic part and C-terminal part of α-ANP, for example. But it is good.
一方、β−ＡＮＰの測定方法も特に限定されるものではなく、例えばβ−ＡＮＰを特異的に測定する方法でもよく、また総ＡＮＰの測定値からα−ＡＮＰとγ−ＡＮＰの測定値を差し引いてもよい。β−ＡＮＰを特異的に測定する方法としては特に限定されるものではないが、β−ＡＮＰを特異的に認識することができ、α−ＡＮＰやγ−ＡＮＰを実質的に認識しない方法があげられる。好ましくはβ−ＡＮＰのジスルフィド結合を含むアミノ酸配列を認識する抗体を用いた測定系、さらに好ましくはβ−ＡＮＰのジスルフィド結合とその周辺のアミノ酸配列を認識する抗体を用いた測定系が選択される。 On the other hand, the method of measuring β-ANP is not particularly limited, and may be, for example, a method of specifically measuring β-ANP, or by subtracting the measured values of α-ANP and γ-ANP from the measured value of total ANP. May be The method for specifically measuring β-ANP is not particularly limited, but a method capable of specifically recognizing β-ANP and substantially not recognizing α-ANP or γ-ANP is given. Be Preferably, a measurement system using an antibody that recognizes an amino acid sequence containing a disulfide bond of β-ANP, more preferably, a measurement system using an antibody that recognizes a disulfide bond of β-ANP and the surrounding amino acid sequence is selected. .
β−ＡＮＰや総ＡＮＰを測定する方法として、免疫学的測定方法を用いる場合は、標識を用いることができる。標識としては、１２５Ｉ、３Ｈなどの放射性物質、西洋わさびペルオキシダーゼ、β−Ｄ−ガラクトシダーゼ、アルカリホスファターゼ（ＡＬＰ）などの酵素、フルオレッセインなどの蛍光物質、金コロイド、セレンコロイド、ルシフェリンなどの発光又は発色物質などが用いられ、標識された抗原あるいは抗体が試薬として用いられている。また、直接これらの物質を検出に用いる物質に標識せず、ビオチン−アビジン等を利用して間接的に標識してもよい。抗原に結合した標識物質を検出することは、例えば、公知の酵素免疫測定法（ＥＩＡ、ＥＬＩＳＡ）、放射免疫測定法（ＲＩＡ）、蛍光免疫測定法（ＦＩＡ）、発光免疫測定法（ＬＩＡ）又は発光酵素免疫測定法（ＣＬＥＩＡ）等により行うことができる。 When using an immunological measurement method as a method of measuring (beta) -ANP or total ANP, a label can be used. As the label, radioactive substances such as 125 I and 3 H, enzymes such as horseradish peroxidase, β-D-galactosidase, alkaline phosphatase (ALP), fluorescent substances such as fluorescein, gold colloid, selenium colloid, luciferin and the like Luminescent or chromogenic substances are used, and labeled antigens or antibodies are used as reagents. Also, these substances may not be directly labeled with the substance used for detection, but may be indirectly labeled using biotin-avidin or the like. The detection of the labeled substance bound to the antigen may be carried out, for example, by known enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA) or It can be performed by luminescence enzyme immunoassay (CLEIA) or the like.
またβ−ＡＮＰや総ＡＮＰを測定する方法として、免疫学的測定方法を用いる場合は、不溶性担体を用いることができる。不溶性担体に関しては、よく知られているガラス、ポリスチレン、ポリプロピレン、デキストランなどの物質からなるビーズ、チューブ、プレート、磁性微粒子など用いることができ、反応後にＢ／Ｆ分離可能な担体が好ましく、その材質などは問わない。また、不溶性担体と抗体（あるいはレセプター、結合蛋白質）との結合は、物理的結合あるいは化学的に中間体を介した結合等、Ｂ／Ｆ分離時に結合能が失われない方法が好ましい。 Moreover, when using an immunological measurement method as a method of measuring (beta) -ANP or total ANP, an insoluble carrier can be used. With regard to insoluble carriers, beads, tubes, plates, magnetic microparticles, etc. made of well-known glass, polystyrene, polypropylene, dextran and other substances can be used, and carriers capable of B / F separation after reaction are preferred, and their materials Etc. does not matter. Further, the binding of an insoluble carrier to an antibody (or a receptor or binding protein) is preferably a method which does not lose binding ability at the time of B / F separation, such as physical binding or binding via an intermediate chemically.
本発明における試料としては被験体由来の血液等が挙げられ、ＥＤＴＡ・アプロチニン血漿を用いるのが最も好ましい。 The sample in the present invention includes blood from a subject and the like, and it is most preferable to use EDTA.aprotinin plasma.
本発明では、β−ＡＮＰと総ＡＮＰとの比を求める。これは例えば、総ＡＮＰに対するβ−ＡＮＰの比（β−ＡＮＰ／総ＡＮＰ）であってもよく、またβ−ＡＮＰに対する総ＡＮＰの比（総ＡＮＰ／β−ＡＮＰ）であってもよい。 In the present invention, the ratio of β-ANP to total ANP is determined. This may be, for example, the ratio of β-ANP to total ANP (β-ANP / total ANP) or the ratio of total ANP to β-ANP (total ANP / β-ANP).
例えば本願実施例で用いた測定系の場合は、β−ＡＮＰ／総ＡＮＰの値が１０％以上の場合は心不全と判定される。 For example, in the case of the measurement system used in the examples of the present application, heart failure is determined when the value of β-ANP / total ANP is 10% or more.
このようにして本発明の方法により心不全を検出することができ、それにより心不全又は心不全治療の効果を判定することができる。 In this way, heart failure can be detected by the method of the present invention, whereby the effect of heart failure or heart failure treatment can be determined.
またβ−ＡＮＰを特異的に測定する試薬および総ＡＮＰを測定する試薬を含むキットで、本発明の心不全の検出を行うこともできる。β−ＡＮＰを特異的に測定する試薬および総ＡＮＰを測定する試薬としては、前述のものを使用することができる。 The detection of heart failure of the present invention can also be carried out with a kit comprising a reagent for specifically measuring β-ANP and a reagent for measuring total ANP. As the reagent for specifically measuring β-ANP and the reagent for measuring total ANP, those described above can be used.
本発明により、β−ＡＮＰと総ＡＮＰとの比から心不全の進行または心不全治療の効果を判定することが可能となった。この方法は心不全の診断に有用である。 The present invention made it possible to determine the progression of heart failure or the effect of heart failure treatment from the ratio of β-ANP to total ANP. This method is useful for the diagnosis of heart failure.
以下、実施例により本発明をさらに詳細に説明するが、本発明は本実施例により限定されるものではない。また以下の試験では、健常者以外は被験者に本研究の説明文書に基づく説明を行い、文書で同意が得られた被験者の検体を用いて行われた。 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited by the examples. In addition, in the following tests, subjects other than healthy subjects were explained based on the explanatory document of the present study, and were conducted using specimens of subjects whose consent was obtained in the document.
［対象被験者］
本実験に使用した検体としては、健常者１０例、心不全患者（急性心不全患者）６０例の血漿を用いた。急性心不全患者は、（１）入院時、（２）入院後１〜２日後、（３）入院後５〜８日後、（４）退院時の治療経過にあわせて採血した。健常者１０例は、男性５例、女性５例で、年齢は４０．３±１３．５歳（ｍｅａｎ±ＳＤ）を用いた。急性心不全患者６０例は、男性４３例、女性１７例で、年齢は７０．７±１２．５歳（ｍｅａｎ±ＳＤ）を用いた。急性心不全患者６０例の入院時および退院時におけるニューヨーク心臓協会（ＮＹＨＡ）による分類、体重、平均血圧、心臓超音波検査（左室内径短縮率）を表１に示す。血漿ＢＮＰ濃度は市販のＣＬＥＩＡキット（ルミパルスＧ１２００、富士レビオ社）を用いてＥＤＴＡ・アプロチニン血漿を測定した。血清ＮＴ−ｐｒｏＢＮＰ濃度は市販の電気化学発光免疫測定法キット（エクルーシス試薬ＮＴ−ｐｒｏＢＮＰ ＩＩ、ロシュ社）を用いて測定した。血漿レニン活性は、ラジオイムノアッセイ二抗体法を用いてＥＤＴＡ血漿を測定した。血漿アルドステロン濃度は、ラジオイムノアッセイ固相法を用いてＥＤＴＡ血漿を測定した。サイクリックＧＭＰは、サクシニル化したＥＤＴＡ血漿をラジオイムノアッセイ法を用いて測定した。血清尿素窒素濃度、血清クレアチニン濃度は、汎用生化学自動分析装置（Ｌａｂｏｓｐｅｃｔ ００８、日立製作所）を用いて測定した。
[Subject subject]
As samples used in this experiment, plasma of 10 healthy subjects and 60 cases of heart failure patients (acute heart failure patients) was used. Patients with acute heart failure were (1) hospitalized, (2) 1 to 2 days after hospitalization, (3) 5 to 8 days after hospitalization, (4) blood was drawn according to the treatment course at discharge. Ten healthy subjects were 5 males and 5 females, and the age was 40.3 ± 13.5 years (mean ± SD). For 60 acute heart failure patients, 43 males and 17 females were used, and the age was 70.7 ± 12.5 years (mean ± SD). The classification by the New York Heart Association (NYHA), body weight, mean blood pressure, and cardiac ultrasonography (left ventricular internal shortening rate) at admission and discharge of 60 patients with acute heart failure are shown in Table 1. The plasma BNP concentration measured EDTA * aprotinin plasma using the commercially available CLEIA kit (Lumipulse G1200, Fujirebio). Serum NT-proBNP concentration was measured using a commercially available electrochemiluminescence immunoassay kit (Ecclusion reagent NT-proBNP II, Roche). Plasma renin activity was measured with EDTA plasma using radioimmunoassay two antibody method. Plasma aldosterone concentrations were measured with EDTA immunoassay using radioimmunoassay solid phase method. Cyclic GMP was measured using radioimmunoassay for succinylated EDTA plasma. Serum urea nitrogen concentration and serum creatinine concentration were measured using a general-purpose biochemistry automatic analyzer (Labospect 008, Hitachi, Ltd.).
［ヒト血漿抽出物の調製］
ヒト正常血漿は市販品（ＥＤＴＡ−２Ｎａ、コージンバイオ社 １２２７１４４０）を使用した。血漿抽出物の調製には固相抽出カートリッジ（Ｓｅｐ−Ｐａｋ Ｃ１８ Ｐｌｕｓ、Ｗａｔｅｒｓ社 ＷＡＴ０２０５１５）を用いた。具体的には、固相抽出カートリッジを５ｍＬの６０％ アセトニトリル／０．１％トリフルオロ酢酸（ＴＦＡ）溶液にて洗浄し、５ｍＬの０．１％ＴＦＡ溶液にて平衡化した後、３００μＬの血漿を添加した。５ｍＬの１０％ アセトニトリル／０．１％ ＴＦＡ溶液にて２回洗浄した後、６ｍＬの４０％ アセトニトリル／０．１％ ＴＦＡ溶液にて溶出した。溶出液を濃縮した後、凍結乾燥し、反応液に溶解して測定に用いた。
[Preparation of human plasma extract]
As human normal plasma, a commercially available product (EDTA-2Na, 1221440 by Cordin Bio Inc.) was used. A solid phase extraction cartridge (Sep-Pak C18 Plus, Waters WAT020515) was used for preparation of the plasma extract. Specifically, the solid phase extraction cartridge is washed with 5 mL of a 60% acetonitrile / 0.1% trifluoroacetic acid (TFA) solution, equilibrated with 5 mL of a 0.1% TFA solution, and then 300 μL of plasma. Was added. After washing twice with 5 mL of a 10% acetonitrile / 0.1% TFA solution, elution was performed with 6 mL of a 40% acetonitrile / 0.1% TFA solution. The eluate was concentrated and then lyophilized to dissolve it in the reaction solution and used for measurement.
（１）β−ＡＮＰの測定試薬の調製
［実験用試薬等］
・固相化用緩衝液：５０ｍＭ炭酸−重炭酸緩衝液（ｐＨ９．５）
・ＰＥＧ化試薬溶液：５μＭ ｍｅｔｈｙｌ−ＰＥＧ１２−ＮＨＳ ｅｓｔｅｒ（Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社 ２２６８５）、ＰＢＳ（ｐＨ７．４）
・ブロッキング溶液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、２％ ＢｌｏｃｋＡｃｅ（ＤＳファーマバイオメディカル社 ＵＫ−Ｂ８０）、５％ ウマ血清、２０％ スクロース
・洗浄液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、０．０５％ Ｔｒｉｔｏｎ Ｘ−１００、０．０５％ ＮａＮ３
・反応液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、０．５ｍＭ ＥＤＴＡ−２Ｎａ、５％ ＢＳＡ、０．０５％ Ｔｒｉｔｏｎ Ｘ−１００、５００ＫＩＵ／ｍＬ アプロチニン（和光純薬社）、０．０５％ ＮａＮ３
・検出抗体希釈用緩衝液：２５ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１５０ｍＭ ＮａＣｌ、０．４％ ＢｌｏｃｋＡｃｅ、０．０５％ ＮａＮ３
・化学発光基質：ＣＤＰ−Ｓｔａｒ ｗｉｔｈ Ｅｍｅｒａｌｄ ＩＩ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社 Ｔ２２１６）
［ＡＬＰ標識＃３２−３の調製］
β−ＡＮＰのジスルフィド結合を含むアミノ酸配列を認識するマウスモノクローナル抗体＃３２−３（図２参照）のＡＬＰ標識にはＡｌｋａｌｉｎｅ Ｐｈｏｓｐｈａｔａｓｅ Ｌａｂｅｌｉｎｇ Ｋｉｔ−ＮＨ２（同仁化学社 Ｌ１２）を用いた。標識および標識抗体の精製は製造業者のマニュアルに従い行った。なおマウスモノクローナル抗体＃３２−３の製法は、後述の参考例に記載した。
(1) Preparation of measurement reagent for β-ANP [Reagent for experiment etc.]
· Immobilization buffer: 50 mM carbonate-bicarbonate buffer (pH 9.5)
PEGylation reagent solution: 5 μM methyl-PEG 12 -NHS ester (Thermo Scientific 22685), PBS (pH 7.4)
Blocking solution: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2% Block Ace (DS Pharma Biomedical UK-B80), 5% horse serum, 20% sucrose Washing solution: 25 mM Tris-HCl (pH 7.4) ), 150 mM NaCl, 0.05% Triton X-100, 0.05% NaN 3
Reaction solution: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM EDTA-2Na, 5% BSA, 0.05% Triton X-100, 500 KIU / mL aprotinin (Wako Pure Chemical Industries, Ltd.), 0.1. 05% NaN 3
Detection antibody dilution buffer: 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.4% BlockAce, 0.05% NaN 3
Chemiluminescent substrate: CDP-Star with Emerald II (Applied Biosystems T2216)
[Preparation of ALP Labeling # 32-3]
Alkaline Phosphatase Labeling Kit-NH 2 (Dojinka Chemical Co., Ltd. L12) was used for ALP labeling of mouse monoclonal antibody # 32-3 (see FIG. 2) that recognizes an amino acid sequence containing β-ANP disulfide bond. Purification of the labeled and labeled antibody was performed according to the manufacturer's manual. In addition, the manufacturing method of mouse monoclonal antibody # 32-3 was described in the below-mentioned reference example.
［＃１３１−７固相化プレートの作製］
本ＣＬＥＩＡ法では、ヒトα−ＡＮＰの１３〜１７残基目をエピトープとするウサギポリクローナル抗体＃１３１−７（Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ４６１；１０−１６，２０１４）（図２参照）を固相化抗体として用いた。ウサギポリクローナル抗体＃１３１−７の製法は、後述の参考例に記載した。固相化プレートの作製では、プレートへの非特異的吸着および固相化抗体のＦｃ領域への血漿由来成分の結合を低減するため、従来のＣＬＥＩＡ法と比較して、固相化抗体のＦｃ領域を標的としたポリエチレングリコール修飾（ＰＥＧ化）を追加した方法（Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ４６１；１０−１６，２０１４）を採用した。具体的には、固相化用緩衝液に溶解した＃１３１−７（３μｇ／ｍＬ、１５０μＬ）を９６穴プレート（Ｆｌｕｏｒｏ−Ｎｕｎｃ Ｍａｘｉ−Ｓｏｒｐ、Ｎｕｎｃ社 ４３７７９６）に添加し、４℃で２４時間インキュベートした。抗体溶液を除去し、ＰＥＧ化試薬溶液（１００μＬ）を添加し、室温で３０分間インキュベートした。ＰＥＧ化試薬溶液を除去し、ブロッキング溶液（２００μＬ）を添加し、室温で２時間インキュベートした。ブロッキング溶液を除去した＃１３１−７固相化プレートをデシケーターにより乾燥させ、酸素吸収剤（アズワン社 １−６６５５−０２）およびゼオライト乾燥剤（アズワン社 １−６６５５−０３）と同封して密閉し、使用時まで−２０℃にて保存した。
[Preparation of # 131-7 immobilized plate]
In the present CLEIA method, rabbit polyclonal antibody # 131-7 (Anal. Biochem., 461; 10-16, 2014) (see FIG. 2), whose epitope is 13th to 17th residues of human α-ANP, is immobilized It was used as an antibody. The preparation method of rabbit polyclonal antibody # 131-7 was described in the below-mentioned reference example. In preparation of the immobilized plate, in order to reduce nonspecific adsorption to the plate and binding of the plasma-derived component to the Fc region of the immobilized antibody, compared to the conventional CLEIA method, Fc of the immobilized antibody is used. The method (Anal. Biochem., 461; 10-16, 2014) to which region-targeted polyethylene glycol modification (PEGylation) was added was adopted. Specifically, # 131-7 (3 μg / mL, 150 μL) dissolved in a buffer for immobilization was added to a 96-well plate (Fluoro-Nunc Maxi-Sorp, Nunc 437796), and allowed to stand at 4 ° C. for 24 hours. Incubated. The antibody solution was removed, PEGylation reagent solution (100 μL) was added and incubated for 30 minutes at room temperature. The PEGylation reagent solution was removed, blocking solution (200 μL) was added and incubated for 2 hours at room temperature. The # 131-7 immobilized plate from which the blocking solution has been removed is dried by a desiccator, sealed with an oxygen absorbent (As One 1-6655-02) and a zeolite desiccant (As One 1-6655-03), and sealed. , Stored at -20 ° C until use.
［ＣＬＥＩＡ法を用いた測定手順］
マイクロプレートウォッシャー（バイオテック社 ＡＭＷ−８Ｒ）を用いて＃１３１−７固相化プレートを洗浄液（３５０μＬ）で３回洗浄した。反応液（５０μＬ）を各ウェルに添加した後、標準β−ＡＮＰ溶液（反応液に溶解した定量済の合成β−ＡＮＰ溶液、ペプチド研究所）またはヒト血漿抽出物溶液（５０μＬ）を添加し、マイクロプレートシェーカー（日伸理化社 Ｎ−７０４）を用いて振盪撹拌しながら４℃で２４時間インキュベートした。上記と同様に３回洗浄した後、検出抗体希釈用緩衝液で０．２ｎｇ／ｍＬに希釈したＡＬＰ標識＃３２−３溶液（１００μＬ）を添加し、マイクロプレートシェーカーを用いて振盪撹拌しながら室温で１時間インキュベートした。上記と同様に４回洗浄した後、化学発光基質（１００μＬ）を添加し、室温で２０分間インキュベートした。マイクロプレートルミノメーター（ＳｐｅｃｔｒａＭａｘ Ｌ、Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ社）により１秒間に生ずる発光量を測定した。各試料は異なる２ウェルで個別に測定し、その平均値より定量値を算出した。
[Measuring procedure using CLEIA method]
The # 131-7 immobilized plate was washed three times with a washing solution (350 μL) using a microplate washer (Biotech AMW-8R). After adding the reaction solution (50 μL) to each well, add a standard β-ANP solution (quantified synthetic β-ANP solution dissolved in the reaction solution, peptide laboratory) or human plasma extract solution (50 μL), The plate was incubated at 4 ° C. for 24 hours with shaking using a microplate shaker (Nisshinization N-704). After washing three times in the same manner as above, add 100 μL of ALP labeled # 32-3 solution diluted to 0.2 ng / mL with detection antibody dilution buffer, and shake at room temperature using a microplate shaker. Incubate for 1 hour. After washing four times as above, chemiluminescent substrate (100 μL) was added and incubated for 20 minutes at room temperature. The amount of luminescence generated per second was measured by a microplate luminometer (SpectraMax L, Molecular Devices). Each sample was measured separately in 2 different wells, and a quantitative value was calculated from the average value.
（２）総ＡＮＰ測定
総ＡＮＰの測定は、市販のＣＬＥＩＡキット（ＭＩ０２ シオノギ ＡＮＰ、シオノギ製薬社）を用いて測定した。
(2) Measurement of Total ANP Measurement of total ANP was performed using a commercially available CLEIA kit (MI02 Shionogi ANP, Shionogi Pharmaceutical Co., Ltd.).
（３）β−ＡＮＰ／総ＡＮＰの比の算出
健常者１０例、急性心不全患者６０例のβ−ＡＮＰと総ＡＮＰを測定した。その測定結果からβ−ＡＮＰ／総ＡＮＰの比を算出した。
(3) Calculation of ratio of β-ANP / total ANP β-ANP and total ANP were measured in 10 healthy subjects and 60 acute heart failure patients. From the measurement results, the ratio of β-ANP / total ANP was calculated.
（４）考察
健常者、急性心不全患者におけるβ−ＡＮＰ／総ＡＮＰを表１（６０例；入院時と退院時の比較）と図４（４７例；１回目〜４回目の経時的変動）に示す。
(4) Discussion The values of β-ANP / total ANP in healthy subjects and patients with acute heart failure are shown in Table 1 (60 cases; comparison at admission and discharge) and in Fig. 4 (47 cases; first to fourth time-dependent changes) Show.
急性心不全患者の総ＡＮＰとβ−ＡＮＰは治療経過とともに低下していたが、退院時（４回目）ではどちらの値も健常者よりも高い値を示していた。一方、β−ＡＮＰ／総ＡＮＰは健常者と同等の値を示していた。退院時には、心臓超音波検査における左室内径短縮率の上昇、ＮＹＨＡ分類の低下、体重の低下、平均血圧の低下で示されるように、心不全重症度が軽減したと判断されたことから、本方法で測定したβ−ＡＮＰ／総ＡＮＰが心不全の改善に相関していることが示された。また、β−ＡＮＰおよびβ−ＡＮＰ／総ＡＮＰは、既存の心不全マーカーであるＢＮＰやＮＴ−ｐｒｏＢＮＰとは異なる経時的変動を示した。腎機能の指標（血清尿素窒素、血清クレアチニン、推算糸球体濾過量）が入院時と退院時では同等であったことから（表１）、上記のＡＮＰ分子型の血漿濃度の変動は、腎機能の変化によるものではないと示された。本実施例の結果と文献等の研究に基づくと、総ＡＮＰは心不全の増悪に伴って増加してくるが、β−ＡＮＰの挙動が他のＡＮＰやＢＮＰ、ＮＴ−ｐｒｏＢＮＰとは異なる挙動を示すことから、β−ＡＮＰ／総ＡＮＰを観察することにより、心不全の進行または心不全治療の効果を判定することができる。また、ＢＮＰやＮＴ−ｐｒｏＢＮＰと併用することにより、心不全の進行の評価精度、心不全治療の効果判定の精度を上げることができると考えられる。 Total ANP and β-ANP in patients with acute heart failure decreased with the progress of treatment, but at discharge (the fourth time), both values were higher than those in healthy people. On the other hand, (beta) -ANP / total ANP showed the value equivalent to a healthy subject. At the time of discharge from the hospital, it was judged that heart failure severity was reduced as shown by an increase in left ventricular internal shortening rate in cardiac ultrasonography, a decrease in NYHA classification, a decrease in body weight, and a decrease in mean blood pressure. It has been shown that β-ANP / total ANP measured in A. has been correlated to the improvement of heart failure. Moreover, (beta) -ANP and (beta) -ANP / total ANP showed the time-dependent fluctuation | variation different from the existing heart failure markers BNP and NT-proBNP. Since the indicators of renal function (serum urea nitrogen, serum creatinine, estimated glomerular filtration rate) were the same at admission and discharge (Table 1), the above-mentioned fluctuations in plasma concentration of ANP molecular type indicate renal function It was shown that it was not due to changes in Based on the results of this example and research in the literature, total ANP increases with heart failure, but β-ANP behaves differently from other ANPs, BNPs, and NT-proBNPs. Thus, by observing β-ANP / total ANP, it is possible to determine the progression of heart failure or the effect of heart failure treatment. Moreover, it is thought that the accuracy of evaluation of the progression of heart failure and the accuracy of determination of the effect of heart failure treatment can be raised by using BNP or NT-proBNP in combination.
（５）参考例：マウスモノクローナル抗体＃３２−３及びウサギポリクローナル抗体＃１３１−７の製法
（５−１）モノクローナル抗体産生ハイブリドーマの調製とモノクローナル抗体の産生
［抗原の調製］
固相法で合成、精製した還元型ヒトα−ＡＮＰ（シグマジェノシス社製、２．１ｍｇ）を、ジスルフィド結合を形成しない状態で、マレイミド活性化キーホールリンペットヘモシアニン（３ｍｇ、Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社 ７７６０５）と結合した。これを透析し、生理的食塩水で１ｍｇ／ｍＬの溶液とした。
(5) Reference Example: Preparation of Mouse Monoclonal Antibody # 32-3 and Rabbit Polyclonal Antibody # 131-7 (5-1) Preparation of Monoclonal Antibody-Producing Hybridoma and Production of Monoclonal Antibody [Preparation of Antigen]
Maleimido activated keyhole limpet hemocyanin (3 mg, Thermo Scientific 77605) in the state of not forming a disulfide bond, reduced human α-ANP (manufactured by Sigma Genosys, 2.1 mg) synthesized and purified by the solid phase method Combined with This was dialyzed into a 1 mg / mL solution in saline.
［モノクローナル抗体産生ハイブリドーマの調製］
抗原溶液１００μＬとアジュバント１００μＬ（Ｆｒｅｕｎｄ ｃｏｍｐｌｅｔｅ ａｄｊｕｖａｎｔ，三菱化学ヤトロン社 ＲＭ６０６−１）を十分に混合して安定したエマルジョンにして、４週齢のＣ３Ｈ系マウス（４匹）の足の裏に各５０μＬずつ免疫した。これを合計５回、３日おきに実施した。最終免疫の３日後にマウス両足からリンパ節を収集し、リンパ節内の細胞を回収した。リンパ節由来細胞と増殖させたミエローマ細胞（Ｐ３Ｕ１）を２：１〜１０：１の割合で混和し、遠心して回収後、５０％ポリエチレングリコールを加えて細胞を融合させた。
[Preparation of monoclonal antibody-producing hybridoma]
100 μL of antigen solution and 100 μL of adjuvant (Freund complete adjuvant, Mitsubishi Chemical Yatron RM 606-1) are thoroughly mixed to form a stable emulsion, 50 μL each on the sole of the foot of 4 week old C3H mice (4 animals) I was immunized. This was carried out five times in total every three days. Three days after the final immunization, lymph nodes were collected from both legs of the mouse and cells in the lymph nodes were collected. Lymph node-derived cells and expanded myeloma cells (P3U1) were mixed at a ratio of 2: 1 to 10: 1, centrifuged and collected, and then 50% polyethylene glycol was added to fuse the cells.
無血清培地で洗浄後、レスキュー用ｓｕｐｐｌｅｍｅｎｔ含有の１５％ウシ胎児血清含有ＨＡＴ培地に懸濁し、９６穴プレート３枚に播種した。１〜２週間後に、ハイブリドーマのコロニー形成を確認し、各ウェルから培養上清を一部回収した。 After washing with a serum-free medium, the cells were suspended in a HAT medium containing 15% fetal bovine serum containing a supplement for rescue and seeded onto three 96-well plates. One to two weeks later, hybridoma colony formation was confirmed, and a portion of the culture supernatant was recovered from each well.
［ハイブリドーマの１次スクリーニング］
１次スクリーニングは標準的なＥＬＩＳＡ法（Ｍｅｔｈｏｄｓ ｉｎ Ｉｍｍｕｎｏｄｉａｇｎｏｓｉｓ ２ｎｄ Ｅｄｉｔｉｏｎ， Ｒｏｓｅ ａｎｄ Ｂｉｇａｚｚｉ， ｅｄｓ．， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ， １９８０； Ｃａｍｐｂｅｌｌ， ｅｔ ａｌ．， Ｍｅｔｈｏｄｓ ａｎｄ Ｉｍｍｕｎｏｌｏｇｙ， Ｗ． Ａ． Ｂｅｎｊａｍｉｎ， Ｉｎｃ．， １９６４； Ｏｅｌｌｅｒｉｃｈ， Ｍ．， Ｊ． Ｃｌｉｎ． Ｃｈｅｍ． Ｃｌｉｎ． Ｂｉｏｃｈｅｍ． ２２：８９５−９０４， １９８４； 羊土社 タンパク質実験ノート（下）岡田雅人編集 改訂第４版 ２０１１年１１月１日発行）で実施した。抗原を１μｇ／ｍＬに希釈後、９６穴プレート（ＮＵＮＣ ４６８６６７）に分注し、４℃で一晩、静置した。抗原溶液を除去後、Ｂｌｏｃｋｉｎｇ Ｂｕｆｆｅｒを１００μＬ／ｗｅｌｌで分注し、ブロッキングを行った。培養上清５０μＬを各ウェルに添加して反応させた。洗浄後、西洋わさびペルオキシダーゼで標識した抗マウスＩｇＧヤギ抗体と反応後、発色剤を添加し、４５０ｎｍの吸光度を測定した。ヘモシアニンに対する吸光度が０．２以下で、抗原に対する特異的な吸光度を０．２以上有するクローンを陽性として選択した。
[Hybridoma primary screening]
Primary screening is performed using a standard ELISA method (Methods in Immunodiagnosis 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980; Campbell, et al., Methods and Immunology, WA Benjamin, Inc., 1964 Oellerich, M., J. Clin. Chem. Clin. Biochem. 22: 895-904, 1984; Notes on protein experiments by Yodosha (bottom) edited by Masato Okada (revised 4th edition published on November 1, 2011) did. After diluting the antigen to 1 μg / mL, it was aliquoted into a 96-well plate (NUNC 468667) and allowed to stand at 4 ° C. overnight. After removing the antigen solution, blocking buffer was dispensed at 100 μL / well to perform blocking. 50 μl of culture supernatant was added to each well for reaction. After washing, after reacting with an anti-mouse IgG goat antibody labeled with horseradish peroxidase, a color former was added, and the absorbance at 450 nm was measured. Clones having an absorbance of 0.2 or less for hemocyanin and a specific absorbance of 0.2 or more for an antigen were selected as positive.
［ハイブリドーマの２次スクリーニング］
ヒトα−ＡＮＰ（ペプチド研究所製）を還元後、カルボキシアミドメチル（ＣＡＭ）化し、ＲＰ−ＨＰＬＣで精製した。精製したＣＡＭ−α−ＡＮＰをラクトペルオキシダーゼ法によりヨード１２５（１２５Ｉ）で標識し、ＲＰ−ＨＰＬＣで精製することにより、１分子の１２５Ｉで標識されたペプチドを調製した。以下の反応には、ＲＩＡ用標準バッファー（ＲＩＡバッファー、５０ ｍＭリン酸緩衝液、８０ｍＭ ＮａＣｌ、２５ｍＭ ＥＤＴＡ、０．０５％ ＮａＮ３、０．５％ Ｎ−エチルマレイミド処理済ＢＳＡ（ＳＩＧＭＡ−Ａｌｄｒｉｃｈ社 Ａ７８８８）、０．５％ Ｔｒｉｔｏｎ Ｘ−１００、ｐＨ７．４）（Ｋａｔａｆｕｃｈｉ Ｔ， ｅｔ ａｌ．， Ｊ． Ｂｉｏｌ． Ｃｈｅｍ．， ２７８：１２０４６−１２０５４， ２００３）を使用した。
[Secondary screening of hybridomas]
After reduction, human α-ANP (manufactured by Peptide Laboratories) was converted to carboxamidomethyl (CAM) and purified by RP-HPLC. The purified CAM-α-ANP was labeled with iodo125 ( 125I ) by the lactoperoxidase method, and purified by RP-HPLC to prepare one molecule of 125I- labeled peptide. For the following reactions, RIA standard buffer (RIA buffer, 50 mM phosphate buffer, 80 mM NaCl, 25 mM EDTA, 0.05% NaN 3 , 0.5% N-ethylmaleimide-treated BSA (SIGMA-Aldrich) A 7888), 0.5% Triton X-100, pH 7.4) (Katafuchi T, et al., J. Biol. Chem., 278: 12046-12054, 2003) was used.
１次スクリーニング陽性クローンの培養上清を，１／１０希釈より３倍希釈系列液を各１００μＬ作製し、これに約２０，０００ｃｐｍの１２５Ｉ標識ＣＡＭ−α−ＡＮＰ（１２５Ｉ−ＣＡＭ−α−ＡＮＰ）を含む溶液１００μＬ、ＲＩＡバッファー１００μＬを添加、撹拌し、４℃で４０時間静置した。抗体に結合した１２５Ｉ−ＣＡＭ−α−ＡＮＰの放射活性量をポリエチレングリコール分離法で分離し測定した（Ｋａｔａｆｕｃｈｉ Ｔ， ｅｔ ａｌ． 同上文献）。具体的には、リン酸緩衝生理食塩水（ＰＢＳ、５０ｍＭリン酸緩衝液、８０ｍＭ ＮａＣｌ、０．０５％ ＮａＮ３、ｐＨ７．４）を用いて作製した１００μＬの１％ ウシγ−グロブリン（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社 Ｇ５００９）溶液および５００μの２３％ポリエチレングリコール（＃６０００、ナカライテスク社 ２８２５４−８５）溶液を加え、混合し、氷上で１０分間静置した後、遠心分離（４℃、３０００ｒｐｍ、１５分間）し、上清を除去した。得られた沈殿の１分間の放射活性をγカウンター（ＡＲＣ−１０００Ｍ、Ａｌｏｋａ社）にて測定した。希釈された培養上清でも１２５Ｉ−ＣＡＭ−α−ＡＮＰに対する結合能力のあるクローン５種選択した。
The culture supernatant of the primary screening positive clone is prepared in 100 μl each in 3-fold dilutions from 1/10 dilution, to which about 20,000 cpm of 125 I-labeled CAM-α-ANP ( 125 I-CAM-α- 100 μL of a solution containing ANP) and 100 μL of RIA buffer were added, stirred, and allowed to stand at 4 ° C. for 40 hours. The amount of radioactivity of 125 I-CAM-α-ANP bound to the antibody was separated and measured by polyethylene glycol separation (Katafuchi T, et al. Supra). Specifically, 100 μL of 1% bovine γ-globulin (Sigma-Aldrich) prepared using phosphate buffered saline (PBS, 50 mM phosphate buffer, 80 mM NaCl, 0.05
［限界希釈法によるモノクローン化］
上記５種のクローンを増殖し、対数増殖期の状態でハイブリドーマを分散させ、培地で希釈後、９６穴プレートに播種した。１〜２週間後にハイブリドーマのシングルコロニーの形成が確認された段階で、各ウェルから培養上清をサンプリングし、上述の「ハイブリドーマの１次スクリーニング」に記載した方法に従い、活性を評価した。抗原に対する特異的な吸光度の強いクローンを各３種、合計１５種を選択した。
[Mono cloning by limiting dilution method]
The above five clones were grown, and hybridomas were dispersed in the logarithmic growth phase, diluted with culture medium, and then seeded in a 96-well plate. When formation of a single colony of hybridomas was confirmed after 1 to 2 weeks, the culture supernatant was sampled from each well, and the activity was evaluated according to the method described in the above-mentioned "primary screening of hybridomas". A total of 15 clones were selected, each containing 3 clones with strong specific absorbance against the antigen.
［ハイブリドーマの３次スクリーニング］
ＣＡＭ−α−ＡＮＰに加えて、α−ＡＮＰ、β−ＡＮＰ（ペプチド研究所製）をそれぞれラクトペルオキシダーゼ法により１２５Ｉで標識後、ＲＰ−ＨＰＬＣで精製し、１分子の１２５Ｉで標識されたペプチドを調製した（１２５Ｉ−α−ＡＮＰ、１２５Ｉ−β−ＡＮＰ）。
[Third-order screening of hybridomas]
In addition to CAM-α-ANP, α-ANP and β-ANP (manufactured by Peptide Laboratories) were each labeled with 125 I by the lactoperoxidase method, purified by RP-HPLC, and labeled with one molecule of 125 I The peptides were prepared ( 125 I-α-ANP, 125 I-β-ANP).
上記５種のクローンより調製した各３種のクローンについて、連続した希釈液を作製し、１２５Ｉ−ＣＡＭ−α−ＡＮＰに対する結合能力を評価し、２次スクリーニングより得られた５種のクローンより得られた各３種のクローンの中で、最も結合能力の高いクローンを選択した。 Serial dilutions were prepared for each of the three clones prepared from the above five clones, and their binding ability to 125 I-CAM-α-ANP was evaluated, and from the five clones obtained by the secondary screening Among each of the obtained 3 types of clones, a clone with the highest binding ability was selected.
これら５種について、連続した希釈液を作製し、１２５Ｉ−α−ＡＮＰ、１２５Ｉ−β−ＡＮＰを用いて２次スクリーニングに記載した方法に従い、各ペプチドに対する結合能力を評価した。 Serial dilutions were prepared for these five types, and 125 I-α-ANP and 125 I-β-ANP were used to evaluate the binding ability to each peptide according to the method described in the secondary screening.
次に、通常のＲＩＡ法に従い、１２５Ｉ−β−ＡＮＰ、１２５Ｉ−ＣＡＭ−α−ＡＮＰ、１２５Ｉ−α−ＡＮＰをトレーサーとして、それぞれについてβ−ＡＮＰ、α−ＡＮＰ、ＣＡＭ−α−ＡＮＰの標準曲線あるいは交差活性曲線を作成し、各クローンの特異性、感度を評価した。その結果、β−ＡＮＰに対して強い結合活性を有し、α−ＡＮＰ、ＣＡＭ−α−ＡＮＰとの交差性が少なく、かつＲＩＡ法において高感度にβ−ＡＮＰを測定できるクローン（＃３２−３）を選択した。 Next, in accordance with the usual RIA method, 125 I-β-ANP, 125 I-CAM-α-ANP, 125 I-α-ANP as a tracer, β-ANP, α-ANP, CAM-α-ANP, respectively. A standard curve or cross activity curve was generated, and the specificity and sensitivity of each clone were evaluated. As a result, a clone which has strong binding activity to β-ANP, less cross-reactivity with α-ANP and CAM-α-ANP, and which can measure β-ANP with high sensitivity in RIA method (# 32- 3) was selected.
［モノクローナル抗体＃３２−３の産生］
腹水採取用にはヌードマウスを使用し、アジュバントとしてプリスタンを腹腔に注射して１週間後のマウスを用いた。選定したクローン（＃３２−３）を増殖、培養し、ＰＢＳにて懸濁し、上記のマウスに１匹当たり約１×１０６細胞を腹腔に注射した。２週間後ごろになると腹水がたまるので、経過をよく観察して腹水を複数回にわたり回収した。回収する容器には、予め保存用抗凝固剤（ＡＣＤ液）を添加して凝固を抑制した。遠心により血球成分や不要物を除去し、上清を凍結保存した。
[Production of monoclonal antibody # 32-3]
Nude mice were used for ascites fluid collection, and mice one week after injection of pristane into the abdominal cavity as an adjuvant were used. The selected clone (# 32-3) was expanded, cultured, suspended in PBS, and the above mice were injected intraperitoneally with about 1 × 10 6 cells per mouse. As the ascites build up around two weeks later, the course was closely observed and ascites was collected multiple times. A storage anticoagulant (ACD solution) was previously added to the container to be collected to suppress coagulation. Blood cell components and unwanted substances were removed by centrifugation, and the supernatant was cryopreserved.
（５−２）抗体の精製
［精製モノクローナル抗体＃３２−３の調製］
腹水の上清画分からのモノクローナル抗体の精製にはＡｆｆｉ Ｇｅｌ Ｐｒｏｔｅｉｎ Ａ（ＢｉｏＲａｄ社 １５３−６１５３）を用いたプロテインＡ結合アフィニティーカラムを使用した。アフィニティーカラムへのサンプルの結合、洗浄、溶出は製造業者のマニュアルに従った。溶出液を５００μＬごとに、予め１Ｍトリス塩酸緩衝液（ｐＨ９．０）１６０μＬを加えた１．５ｍＬ容チューブに回収した。精製モノクローナル抗体を含む画分を、ＰＢＳ中で４℃、オーバーナイトにて透析した。精製した溶液中のタンパク質量をＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ（Ｐｉｅｒｃｅ社 ２３２２７）を用いて定量した。
(5-2) Purification of Antibody [Preparation of Purified Monoclonal Antibody # 32-3]
A protein A binding affinity column using Affi Gel Protein A (BioRad 153-6153) was used for purification of the monoclonal antibody from the supernatant fraction of ascites fluid. Binding of the sample to the affinity column, washing and elution were according to the manufacturer's manual. The eluate was collected every 500 μL into a 1.5 mL tube to which 160 μL of 1 M Tris-HCl buffer solution (pH 9.0) was previously added. Fractions containing purified monoclonal antibody were dialyzed overnight at 4 ° C. in PBS. The amount of protein in the purified solution was quantified using BCA Protein Assay Kit (Pierce 23227).
［精製ポリクローナル抗体＃１３１−７の調製］
ポリクローナル抗体＃１３１−７は、Ａ． Ｓａｓａｋｉ， ｅｔ ａｌ． Ｈｙｐｅｒｔｅｎｓｉｏｎ １０；３０８−３１２， １９８７に記載の方法で調製した。得られたウサギ抗血清にキャリアタンパク質として使用したサイログロブリン（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社 Ｔ１００１、１７ｍｇ／ｍＬ抗血清）およびアジュバント（Ｍ． Ｂｕｔｙｒｉｃｕｍ、ＤＩＦＣＯ社 ５２６−０２６５１、１０ｍｇ／ｍＬ抗血清）を加え、ローテーターにより撹拌しながら４℃、一晩インキュベートした後、遠心分離（４℃、１３，０００×ｇ、１５分間）し、上清を回収した。上清は直ちに上述のプロテインＡ結合アフィニティーカラムによる精製に供した。アフィニティーカラムへのサンプルの結合、洗浄、溶出は製造業者のマニュアルに従った。溶出液を５００μＬごとに、予め１Ｍトリス塩酸緩衝液（ｐＨ９．０）１６０μＬを加えた１．５ｍＬ容チューブに回収した。精製ポリクローナル抗体を含む画分を、ＰＢＳ中で４℃、一晩、透析した。精製した溶液中のタンパク質量をＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔを用いて定量した。このようにして精製ポリクローナル抗体＃１３１−７を得た。これはヒトα−ＡＮＰの１３〜１７残基目をエピトープとするウサギポリクロ―ナル抗体である（Ｎａｇａｉ Ｃ， Ｍｉｎａｍｉｎｏ Ｎ．， Ａｎａｌ． Ｂｉｏｃｈｅｍ．， ４６１：１０−１６， ２０１４）。
[Preparation of Purified Polyclonal Antibody # 131-7]
The polyclonal antibody # 131-7 is Sasaki, et al. It was prepared by the method described in
（５−３）モノクローナル抗体の特性
ヒトα−ＡＮＰおよびβ−ＡＮＰに対する精製モノクローナル抗体＃３２−３の親和定数はＲＩＡ法を用いて算出した。具体的には、ＲＩＡバッファーを用いて精製モノクローナル抗体の５倍希釈系列液（２００μＬ）を作製し、試験管内にて１２５Ｉで標識したα−ＡＮＰおよびβ−ＡＮＰ（２０，０００ｃｐｍ、１００μＬ）と混合し、４℃で４０時間インキュベートした。抗体に結合した１２５Ｉ−α−ＡＮＰまたは１２５Ｉ−β−ＡＮＰの放射活性量を上述のポリエチレングリコール分離法で分離し、測定した。１２５Ｉ−α−ＡＮＰまたは１２５Ｉ−β−ＡＮＰの結合量が５０％となる抗体濃度をＫｄ値として算出したところ、モノクローナル抗体＃３２−３はヒトα−ＡＮＰに対して１．３４×１０−８Ｍ、ヒトβ−ＡＮＰに対して１．６９×１０−１１ＭのＫｄ値を示し、この抗体がβ−ＡＮＰを選択的に認識することが実証された。
(5-3) Properties of Monoclonal Antibody The affinity constant of purified monoclonal antibody # 32-3 against human α-ANP and β-ANP was calculated using RIA method. Specifically, a 5-fold dilution series (200 μL) of purified monoclonal antibody is prepared using RIA buffer, and α-ANP and β-ANP (20,000 cpm, 100 μL) labeled with 125 I in a test tube are prepared. Mix and incubate at 4 ° C. for 40 hours. The amount of radioactivity of 125 I-α-ANP or 125 I-β-ANP bound to the antibody was separated and measured by the polyethylene glycol separation method described above. The antibody concentration at which the amount of 125 I-α-ANP or 125 I-β-ANP bound is 50% was calculated as the K d value, and monoclonal antibody # 32-3 was 1.34 × against human α-ANP. The K d value of 10 −8 M and 1.69 × 10 −11 M against human β-ANP was demonstrated, demonstrating that this antibody selectively recognizes β-ANP.
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| JPS6461500A (en) * | 1987-09-01 | 1989-03-08 | Shionogi & Co | Alpha-hanp recognizing monoclonal antibody and immunologically measuring reagent for alpha-hanp |
| JP2724315B2 (en) * | 1988-02-29 | 1998-03-09 | 塩野義製薬株式会社 | Monoclonal antibody recognizing α-ANP and immunoassay for α-ANP |
| JP2681370B2 (en) * | 1988-06-30 | 1997-11-26 | 塩野義製薬株式会社 | Immunoassay |
| JPH0667319B2 (en) * | 1989-04-19 | 1994-08-31 | 塩野義製薬株式会社 | Monoclonal antibody that recognizes the C-terminal side of ANP |
| JPH0748071B2 (en) * | 1989-05-31 | 1995-05-24 | 株式会社ミドリ十字 | hANP antibody and immunoassay for hANP |
| WO2012000119A1 (en) * | 2010-07-02 | 2012-01-05 | Prognomix Inc. | A method of determining a predisposition to atrial fibrillation in a subject |
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