Aβの絶対定量<sub> 1-42</sub> CSFに質量分析リファレンス測定手順を使用して
Summary
A reference measurement procedure for the absolute quantification of Aβ1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.
Abstract
Alzheimer’s disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (Aβ) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled Aβ peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF Aβ1-42 between 150-4,000 pg/mL.
Introduction
アルツハイマー病（AD）は、認知症の最も一般的な形態であり、全世界で約35万人1に影響を与えます 。広くADの病因の中心に位置するように考えられて疾患の神経病理学的特徴は、過リン酸化タウ蛋白質2の細胞内神経原線維変化および凝集したアミロイドβ（Aβ）ペプチド3からなる細胞外斑です。これに伴い、バイオマーカーによって、in vivoでのプラーク病変の評価は最近、AD 4のための研究診断基準に含まれています。 Aβ1-42のCSF測定のために、いくつかのイムノアッセイが利用可能であり、多くの臨床検査室5で使用されています。 CSF中のAβ1-42の濃度は、BRでのプラーク中のペプチドの沈着を反映し、認知正常な高齢者に比べてAD患者では約50％低くなっていますAIN 6、7。
これらのバイオマーカーは、主に免疫アッセイ（ すなわち、抗体ベースの技術）を用いて分析されているが、これらのアッセイは、マトリックス効果8によって影響され得ます。異なるテクノロジプラットフォームイムノアッセイおよびアッセイの標準化9の欠如の使用は、10は 11、12困難なグローバルなカットオフ濃度の導入を行います。分析的に検証さRMPは、理想的には、分析プラットフォーム間でより良い比較では、全体的な測定のばらつきの要因のより良好な制御が得られ、異なるアッセイプラットフォームの均一なキャリブレーションを可能にするであろう。
開発されたLC-MS / MS法を用いてAβ1-42の絶対的な定量化は、抗体ベースのTECHNに関連する問題の多くを克服しますiques。検査医学（JCTLMデータベース識別番号C11RMP9）におけるトレーサビリティのための合同委員会がRMPとしてリストされている方法は、CSFAβ1を調和させる認証標準物質（CRM）におけるAβ1-42の絶対濃度を決定するために使用されます技術および分析プラットフォーム間-42測定。説明ワークフローは、医学の他の領域内のペプチドおよびタンパク質のための候補参照方法の開発のための関連性のものでなければなりません。
Protocol
Representative Results
Discussion
記載された方法のために、代わりに代理母材を使用して、我々は、ヒトCSF中にキャリブレーションを可能にする、代用分析物アプローチ13、14、15、16を用います。サロゲートの検体のアプローチは、二つの異なる同位体標識された規格を必要とします。別の（13CAβ142）を内部標準?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was performed on behalf of the International Federation of Clinical Chemistry Working Group on CSF Proteins, which has the following composition: Kaj Blennow (Chair) and Henrik Zetterberg, Gothenburg University, Sweden; Les Shaw and Magdalena Korecka, University of Philadelphia, PA, USA; Ingrid Zegers, Institute for Reference Materials and Measurements, Geel, Belgium; Piotr Lewczuk, Universitätsklinikum Erlangen, Germany; and Rand Jenkins, PPD Bioanalytical Laboratory, Richmond, VA, USA; Randall Bateman, Washington University, MO, USA; and H Vanderstichele, Innogenetics NV, Ghent, Belgium. This work was also part of the Global Consortium for Biomarker Standardization CSF Reference Methods Working Group, which is led by Maria C. Carrillo, Ph.D., Senior Director, Medical & Scientific Relations, Alzheimer’s Association. The study was supported by The Swedish Research Council (grants #14002, #521-2011-4709, and #2013-2546); the Knut and Alice Wallenberg Foundation; Demensförbundet; and Emil and Wera Cornell’s, Aina Wallström and Mary-Ann Sjöblom’s, Gun and Bertil Stohne’s, Magnus Bergvall’s, and Gamla Tjänarinnor’s Foundations.
Materials
| Eppendorf Protein LoBind microcentrifuge tubes, 0.5 mL | Eppendorf | 022431081 | |
| Eppendorf Protein LoBind microcentrifuge tubes, 1.5 mL | Eppendorf | 022431081 | |
| Eppendorf Protein LoBind microcentrifuge tubes, 2 mL | Eppendorf | 022431102 | |
| Eppendorf Protein LoBind microcentrifuge tubes, 5 mL | Eppendorf | 0030 108.310 | |
| Eppendorf Protein LoBind Deepwell Plates 96 | Eppendorf | 951032905 | |
| Micronic 0.75 mL polypropylene tubes, V-bottom | Micronic | MPW32071BC3 | Tubes used for collecting SPE eluate, but other collection devices can be used such as 96-well plates. |
| Micronic Split TPE Capcluster for 96 ind. tubes | Micronic | MP53026 | Caps for Micronic tubes. |
| Micronic Loborack-96 white w high cover bar coded | Micronic | MPW51015BC3 | Holder for Micronic tubes. |
| Biohit Optifit tips 1.2 mL | Sartorius | 791210 | Used to reach all the way to the bottom of the LoBind deepwell plates (96-well). Other narrow tips might work as well. |
| Waters Oasis MCX 96-well µElution Plate | Waters | 186001830BA | |
| Waters Plate manifold reservoir tray | Waters | WAT058942 | |
| Waters Extraction Plate Manifold for Oasis 96-Well Plates | Waters | 186001831 | |
| Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis | Sigma-Aldrich | 30501-1L-D | |
| Acetonitrile, Far UV HPLC Gradient grade, 2.5 L | Fisher Scientific | A/0627/17X | |
| ortho-Phosphoric acid 85%, ACS,ISO,Reag. Ph Eur | Merck Millipore | 1005731000 | |
| Guanidine Hydrochloride, 500 g | Thermo Scientific | 24110 | |
| Thermo Scientific Q-exactive Hybrid Quadrupole Orbitrap Mass Spectrometer | Thermo Scientific | IQLAAEGAAPFALGMAZR | With bundled Dionex Ultimate 3000 LC & autosampler. |
| Dionex ProSwift RP-4H Monolith Column (1.0 x 250mm) | Dionex | 066640 | |
| Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated | Sigma-Aldrich | B6917 | |
| Beta-Amyloid (1-42), Ultra Pure, TFA | rPeptide | A-1002-2 | |
| 15N Beta-Amyloid (1-42), Uniformly labeled | rPeptide | A-1102-2 | |
| 13C Beta-Amyloid (1-42), Uniformly labeled | rPeptide | A-1106-2 | |
| Microplate shakers, TiMix 2 | Edmund Bühler | 6110 000 |
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