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－　　－43Published PaperSeparation Analysis of Amyloid Fibrils Utilizing a PTFE Tube as a Separation FieldHiromichi ASAMOTO, Kyosuke NAGASHIMA, Tatsuro NAKAGAMA, Kazunori SAITOH and Hiroaki MINAMISAWAWe developed an easy-to-use analytical method for amyloid brils by high-performance liquid chromatography (HPLC) with post column fluorescent labeling. Thioflavin T (Th T), which was used widly as a labeling agent for amy-loid brils, is a traditional probe for the confirmation of amyloid bril formation. Although the traditional fluorescence spectroscopy measurements are used for monitoring the aggregation reaction, they have no applicability to separation analysis of the amyloid brils. In order to understand the fibril formation process, the development of an efficient method that enables to separate and detect the amyloid fibrils is required. In this work, Th T labeling of amyloid fibrils in 1 mM hen egg white lysozyme (HEWL) solution was performed following separation of different-sized amyloid fibrils in a PTFE tube (5000×0.50 mm i.d.) as a separation field. The fluorescence was monitored at 485 nm, with the maximum excitation wavelength at 435 nm. The novel method’s sensitivity for the detection of the amyloid brils was high and compare well with the traditional fluorescence spectroscopy measurements. In addition, the developed meth-od detected more than 5 peaks within 20 min under the optimum separation conditions. These results suggest that this analytical method is used to understand the amyloid fibril formation process.Keywords: amyloid fibril, HPLC-fluorescence detection, PTFE tubePTFEチューブを分離場とするアミロイド線維の分離分析　認知症の一つでもあるアルツハイマー病（Alzheimer＇s Disease : AD）を引き起こすタンパク質であるβ-Amyloid（Aβ）は，特定の条件下において核形成反応と伸長反応の二段階の凝集過程よりを経てアミロイド線維を形成する．この機構をより詳細に解明するにはアミロイド線維のみならず，前駆体とされるプロト線維やA・オリゴマーを含めた各凝集体のサイズ別分離を可能にする検出法が必要である．我々は分離場としてポリテトラフルオロエチレン（PTFE）チューブを組み込んだ高速液体クロマトグラフィー（HPLC）と蛍光標識化試薬を応用した新たな分離分析法を開発した．アミロイド線維は分離場から溶出された順に，蛍光検出器の直前で別流路から流れてくる標識化試薬と反応することで蛍光体となり，検出が可能となる．本法を用いることでアミロイド線維を損壊することなく，高感度に分離・検出できることがわかった．キーワード：アミロイド線維，HPLC-蛍光検出，PTFEチューブJournal （掲載誌）分析化学 (BUNSEKI KAGAKU), Vol. 66, No. 2, pp. 89～94 (2017)