JP6813503B2 - バイオマーカーとしての遊離ヒストンタンパク質 - Google Patents
バイオマーカーとしての遊離ヒストンタンパク質 Download PDFInfo
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- JP6813503B2 JP6813503B2 JP2017559913A JP2017559913A JP6813503B2 JP 6813503 B2 JP6813503 B2 JP 6813503B2 JP 2017559913 A JP2017559913 A JP 2017559913A JP 2017559913 A JP2017559913 A JP 2017559913A JP 6813503 B2 JP6813503 B2 JP 6813503B2
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6875—Nucleoproteins
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/50—Determining the risk of developing a disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
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Description
(i)サンプルに対して化学物質または生化学物質の添加などの前処理を実施する;
(ii)サンプルに対して還元および/またはアルキル化反応を実施する;
(iii)MS分析の前かつトリプシン消化の後にクロマトグラフィ法でサンプルを分離する;
(iv)イムノアフィニティ装置(例えばThermo Scientific MSIA(登録商標)技術)を用いてサンプルを豊富にするか、除去カラムを用いて望まない成分を減少させる;
(v)少なくとも1つの内部参照基準を添加する。その参照基準は、検出すべきペプチドまたはタンパク質を同位体で標識したバージョンである。
ヒストンH4アミノ酸配列に関係するペプチドが、Thermo Fisher Scientific社(ウルム、ドイツ国)、またはNew England Peptide社(ガードナー、マサチューセッツ州、アメリカ合衆国)、またはJPT Peptide Technologies社によって化学的に合成されて精製された(90%超)。免疫化ペプチドは、スルホ-MBS(m-マレイミドベンゾイル-N-ヒドロキシスクシンイミドエステル)を用いてウシ血清アルブミン(BSA、Sigma Aldrich社)に共役させるか、追加のN末端システインを介してスカシガイのヘモシアニン(KLH、Thermo Fisher Scientific社)に共役させた。抗体のアフィニティ精製に用いるペプチドは、免疫化に用いるペプチドと比べて1つまたは2つの追加のC末端アミノ酸を含むとともに、SulfoLinkカップリング樹脂に固定化するための追加の1個のN末端システインを含んでいた。
H4-LI-9(ヒストンH4のアミノ酸22〜30、配列番号10)、H4-EE-13(ヒストンH4のアミノ酸52〜63、配列番号15)、H4-RR-13(ヒストンH4のアミノ酸67〜78、配列番号17)、H4-RG-9(ヒストンH4のアミノ酸92〜99、配列番号21)に対するヒツジのポリクローナル抗体を標準的な手続き(EP 1488209 A1、EP 1738178 A1を参照のこと)に従って生成させた。簡単に述べると、MBSを用い、追加の1個のN末端システインを介してペプチドを担体タンパク質KLHにカップルさせた。以下のスキームに従い、共役体を用いてヒツジを免疫化した:最初に100μgの共役体(質量は、共役体のペプチド部分を意味する)を用いてヒツジを免疫化し、その後は4週間の間隔で50μgを用いて追加免疫化した。最初に免疫化してから4ヶ月後、300 mlの抗血清をヒツジから取得した。
H4-GR-17(ヒストンH4のアミノ酸2〜17、配列番号8)、H4-LI-9(ヒストンH4のアミノ酸22〜30、配列番号10)、H4-EE-13(ヒストンH4のアミノ酸52〜63、配列番号15)、H4-RR-13(ヒストンH4のアミノ酸67〜78、配列番号17)、H4-TK-13(ヒストンH4のアミノ酸80〜91、配列番号19)、H4-RG-12(ヒストンH4のアミノ酸92〜102、配列番号22)、完全長ヒストンH3(Sigma Aldrich社 SRP0177、配列番号36)に対するモノクローナル抗体を標準的な手続き(Lane, R. D. J Immunol Methods、1985年、第81巻(2):223〜228ページ)によって生成させた。この研究では、リンパ球-骨髄腫細胞混合物をフソゲン(融合因子)に曝露する時間が全ハイブリドーマコロニーの収率とモノクローナル抗体を分泌したハイブリドーマコロニーの収率に及ぼす影響を評価した。Sp2/0骨髄腫細胞とFOX-NY骨髄腫細胞を、時間をさまざまに変えて、ヒツジ赤血球細胞で免疫化したマウス脾臓リンパ球と融合させた。最適な融合時間は、フソゲン(5.0 mlのKodak 1450 PEG、0.5 mlのジメチルスルホキシド、4.5 mlのリン酸緩衝化食塩水、pH 7.0)を37℃で45秒間かけて細胞混合物に添加することであった。融合プロセスは、混合物を50 mlのRPMI-1640に徐々に希釈することによって停止させた。10分後、細胞を遠心分離し、フィーダーマクロファージとともに選択培地に再懸濁させて培養した。一般的なより長時間の融合技術と比較すると、この手続きにより、融合パートナーとしてSp2/0細胞を用いたときには生成するハイブリッドの数が約5倍になり、FOX-NY細胞を用いたときには生成するハイブリッドの数が約30倍になる。どちらの場合にも、実質的にすべてのウエルがモノクローナル抗体を分泌するハイブリッドコロニーを含んでいる。この高効率の融合技術は、弱い免疫原に対するモノクローナル抗体を生成させるのに、またはより強い免疫原を用いて免疫化に必要な時間を短くするのに非常に有用である可能性がある。
図4に示した標準曲線を作成するのに用いた組み換え完全長ヒストンH4を、Roche社から市販されているELISAで試験した。Roche細胞死検出ELISA(#11544675001)は、細胞ライセートの細胞質分画に含まれるモノヌクレオソームとオリゴヌクレオソームを特異的に求める設計であるという事実にもかかわらず、ヒストンH2A、H2B、H3、H4はヌクレオソームの八量体コアを形成しているという理由で、そのELISAの性能を血清サンプルとEDTA血漿サンプルで試験した。このアッセイは、哺乳動物のヒストンH2A、H2B、H3、H4に対する抗体とDNAに対する検出抗体で被覆した微量滴定プレートを含んでいる。キットは標準材料を含んでいないため、天然のヌクレオソーム(BPS Bioscience社 #52015)を用いてマトリックスの濃度を評価した。図1に示したヒストンH4の濃度を調べたのとまったく同じサンプルを用いると、プロカルシトニンのレベルが高い患者では、ヌクレオソームのレベルは、上昇するか、検出不能であった(図6)。したがってヌクレオソームのレベルと遊離ヒストンH4のレベルの間には相関がないことを結論できる(図7)。それに加え、ゼロ血清に添加した市販の分析物、すなわち、組み換えヒトヒストンH2A(Sigma Aldrich社 #SRP0406、配列番号23)、組み換えヒトヒストンH3(Sigma Aldrich社 #SRP0177、配列番号36)、ヒストンH4(Sigma Aldrich社 #SRP0178、配列番号1)、天然のヒトヌクレオソーム(BPS Bioscience社 #52015)を試験した。表3に、BRAHMSヒストンH4 LIAまたはRoche細胞死検出ELISAによってどの分析物を検出できたかを掲載してある。ヒストンH4 LIAはヒトヒストンH4に特異的であるため、ヒストンH2AとH3は検出せず、重要なことだが、天然のヌクレオソームも検出しなかった。これは、ヒストンH4がヌクレオソームの八量体コアの一部になっているとき、本発明に記載したイムノアッセイで用いる抗体によって認識されるヒストンH4のエピトープには近づけないことを示唆している。しかしヒストンH4が溶液中で遊離していると、ヒストンH4 LIAは、血清とEDTA血漿を含むマトリックス中のヒストンH4のレベルを検出することができる。逆に、Roche細胞死検出ELISAは、ヌクレオソームを検出するが、どのヒストンも検出しない。
SRMは、非常に複雑な背景(例えば血液由来の血清または血漿)で検出可能な所定のフラグメンテーション特性を持つ以前に選択された標的タンパク特異的ペプチドを標的とした検出と定量のためのMSに基づく技術である。
個々のペプチドフラグメントに関する三連四重極質量分析器でのSRM/MRMアッセイ
SRMは、非常に複雑な背景(例えば血液由来の血清または血漿)で検出可能な所定のフラグメンテーション特性を持つ以前に選択された標的タンパク特異的ペプチドを標的とした検出と定量のためのMSに基づく技術である。
A.サンプルの調製
血漿サンプル(12μl)を氷の上で解凍し、50μlの8 M尿素/2.5% n-プロパノール/200 mMトリス-HCl/10 mM DTT pH 8.5と混合した。サンプルを37℃で1時間インキュベートした。次にサンプルを、5μlの500 mMヨード酢酸を含む1 M炭酸水素アンモニウム用いてアルキル化し、暗所で室温にて1時間インキュベートした。次に、残ったアルキル化剤を3.3μlの500 mM DTTと反応させた。次にサンプルを260μlの50 mMトリス-HCl、5 mM CaCl2緩衝液 pH 8で希釈した。次に150μlのトリプシン(Pierce社、25 mM酢酸の中に20μg)を各サンプルに添加し、トリプシン:タンパク質の比を1:50にした。サンプルを37℃で18時間放置して消化させた。発見操作のために300 ngのサンプルを50 mm×1 mm 1.9μmのHypersil Goldカラム(Thermo Fisher Scientific社)に装填した後、質量分析を実施した。
SRM/MRM質量分析からの唯一のSRM/MRMシグネチャピーク面積の関数として検出されるヒストンタンパク質のペプチドフラグメントの量が特定のサンプルに含まれるタンパク質の相対量と絶対量を示すようにするため、SRM/MRM分析を実施した。
一定量の軽いペプチドを内在性分析物の上部に添加して内部標準を作成し、可変量の重いペプチドを添加して一連の濃度標準を作成した。曲線の作成に用いる変化する重いペプチドの信号には未知量の内在性分析物からの寄与がないため、測定が定量的に意味を持つようになる点である定量限界(LOQ)値を求めることが可能である。このLOQに対する分析物の応答は、同定可能であり、離散的であり、20%の精度かつ80〜120%の正確さで再現可能である。
SRM/MRM質量分析からの唯一のSRM/MRMシグネチャピーク面積の関数として検出されるヒストンタンパク質のペプチドフラグメントの量が特定のサンプル中のタンパク質の相対量と絶対量を示すようにするため、SRM/MRM分析を実施した。
以下に、本発明のいくつかの項目を列挙する。
被験体の生体サンプルに含まれる遊離ヒストンタンパク質またはそのペプチドフラグメントを検出することを含んでいて、
前記遊離ヒストンタンパク質またはそのペプチドフラグメントの存在が、前記疾患または病状の存在を示している方法。
(i)前記遊離ヒストンタンパク質の第1のエピトープに特異的な第1の抗体、またはその抗原結合フラグメントまたは誘導体、ならびに
(ii)前記遊離ヒストンタンパク質の第2のエピトープに特異的な第2の抗体、またはその抗原結合フラグメントまたは誘導体
と接触させる工程と、
b)前記遊離ヒストンタンパク質に対する前記2つの抗体、またはその抗原結合フラグメントまたは誘導体の結合を検出する工程を含むイムノアッセイ法である、項1〜11のいずれか1項に記載の方法。
b)MS分析の前かつトリプシン消化の後に、そのサンプルをクロマトグラフィ法で分離する工程を含む、項22または23に記載の方法。
Claims (11)
- 遊離ヒストンタンパク質又はそのペプチドフラグメントを分析する方法であって、
質量分析及び/又は免疫アッセイを用いて、被験体の生体サンプルに含まれる遊離ヒストンタンパク質またはそのペプチドフラグメントを検出する
ことを含み、ここで前記遊離ヒストンタンパク質またはそのペプチドフラグメントの存在が、感染性と非感染性の病因に関係する炎症性反応に関わる疾患または病状の存在を示し、ここで前記遊離ヒストンタンパク質又はそのペプチドフラグメントがヒストンH4であり、そしてここで配列番号1の遊離ヒストンH4のアミノ酸残基22〜102にわたる配列におけるエピトープ又はペプチド断片が検出され、ここで前記遊離ヒストンタンパク質が、ヌクレオソーム又は好中球細胞外トラップ(NET)に組み込まれていない、前記方法。 - a)サンプルを、以下の:
(i)前記遊離ヒストンタンパク質の第1のエピトープに特異的な第1の抗体、またはその抗原結合フラグメントまたは誘導体、ならびに
(ii)前記遊離ヒストンタンパク質の第2のエピトープに特異的な第2の抗体、またはその抗原結合フラグメントまたは誘導体
と接触させる工程と、
b)前記遊離ヒストンタンパク質に対する前記2つの抗体、またはその抗原結合フラグメントまたは誘導体の結合を検出する工程
を含むイムノアッセイ法である、請求項1に記載の方法。 - 前記第1および/または第2のエピトープが、配列番号1のアミノ酸残基22〜102の配列中に存在するヒストンH4のエピトープである、請求項2に記載の方法。
- 前記第1のエピトープが、配列番号1のアミノ酸残基22〜30、または配列番号1の残基67〜78、または配列番号1の残基92〜102の配列中に存在し、および/または前記第2のエピトープが、配列番号1のアミノ酸残基46〜102の配列中に存在する、請求項3に記載の方法。
- 前記遊離ヒストンタンパク質またはそのペプチドフラグメントを質量分析(MS)によって検出する、請求項1に記載の方法。
- 前記MS分析法が、反応モニタリング(SRM)、多重反応モニタリング(MRM)、並列反応モニタリング(PRM)のいずれかである、請求項5に記載の方法。
- a)前記サンプルをトリプシンで消化させる工程と;
b)MS分析の前かつトリプシン消化の後にそのサンプルをクロマトグラフィ法で分離する工程を含む、請求項5または6に記載の方法。 - 前記遊離ヒストンタンパク質がヒストンH4であり、配列番号2、3、4、5、6、7からなるグループから選択されるヒストンH4の少なくとも1つのペプチドフラグメントを検出して定量する、請求項5〜7のいずれか1項に記載の方法。
- 前記疾患または病状の選択を、感染性と非感染性の病因に関係する炎症性反応、又は介入措置を含む疾患または病状から行なう、請求項1〜8のいずれか1項に記載の方法。
- 前記感染性と非感染性の病因に関係する炎症性反応が、非感染性SIRS、細菌性、ウイルス性、真菌性の敗血症、重篤な敗血症、敗血症ショック、腹膜炎、出血および/または組織損傷を含む外傷関連障害、火傷損傷、急性膵臓炎、虚血再灌流障害、心筋梗塞、心原性ショック、脳卒中、急性中毒、血栓塞栓症から選ばれる、請求項9に記載の方法。
- 前記介入措置が、心肺バイパスや腫瘍治療から選ばれる、請求項9に記載の方法。
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