利用流式點陣儀系統偵測與Toll-Like受體相關之蛋白質

Abstract

類Toll受體 (TLRs)可以辨識侵入細胞的微生物和一些具有破壞性的訊號，進而促使免疫系統有所反應，因此被認為和許多疾病相關。所以發展可以偵測和此訊息傳遞鏈相關之蛋白質或者是蛋白質的後修飾 (例如：磷酸化、泛素化…等)具有極大臨床診斷價值。但由於生物系統中複雜的生物分子間交互作用，若僅進行單一參數的分析量測難以系統性提供整體的分子網絡動態，本研究利用以微珠為固定相之流式微陣列系統，期望於同一微量樣品中可同時進行參與TLR受體訊息傳遞途徑中相關分析物偵測，我們初步選定受不同刺激活化之類Toll受體(TLRs)所可能引發的訊息傳導路徑中之MyD88, IκB-alpha, Lipocalin-2, TNFR1和 TRAF6等五個相關之蛋白質分子進行實驗設計，最佳化之檢量線對特定目標分析物進行定量具高靈敏度，可達pg/mL數量級。最後我們也成功直接量測不需前處理之成分複雜的細胞裂解液(cell lysate)、細胞培養液(culture medium)以及病人血清樣品。而我們目前所建立之系統期許未來可以擴展於臨床診斷，用於評估作為來自不同感染或組織傷害生物標記分子之可行性。

Toll-like receptors (TLRs) are capable of sensing microorganisms and also several damage signals within cells, and thus are complicit in the pathogenesis of a multitude of diseases. Development of tools enabling focused analysis of the proteins and the corresponding posttranslational modifications (PTMs) in the associated cell signaling pathways could be of great diagnostic value. Due to the complex molecular interactions in the biological system, single parameter provides hardly systemic information for the understanding of the dynamics in the signaling networks. We report the first time, developing a microsphere-based multiplexed suspension array to simultaneously monitor five molecules (i.e. MyD88, IκB-alpha, Lipocalin-2, TNFR1 and TRAF6) which have been identified as key mediators in TLRs assocated pathways. It allows the evaluation of multiple reactions and interactions among specific proteins in a single experiment using a limited amount of sample material. And the detection limit is at pg/mL level in current setup (i.e. IκB-alpha, Lipocalin-2, TNFR1). Subsequent measurements of several “real world” biological samples such as cell lysate, culture medium or serum/plasma were also achieved. The presented strategy enables further expansion of multiplex capacity to meet the need for the analysis of complex protein-protein interaction in time-resolved pattern, and to profile the intricate regulation of TLRs associated activation caused by various stimuli during disease courses.