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    首頁> 外文期刊>Biochimica et Biophysica Acta. General Subjects >A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients
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    A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients

    機譯:高通量檢測膀胱癌患者尿液中Tyr磷酸化蛋白的方法

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    摘要

    Background Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine. Methods The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated. Results Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform. Conclusions A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker. General significance The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.
    機譯:背景膀胱癌具有脫落性腫瘤細胞及其尿液成分的特殊性,代表了檢測診斷標志物的寶貴機會。我們以前使用半定量方法證明了膀胱癌組織中Tyr磷酸化蛋白(TPPs)的水平大大增加,并且在患者的尿液樣本中也可以檢測到可溶性TPPs。盡管初步評估顯示出非常有前途的特異性和靈敏度,但該方法的準確性不足和通量極低使新標記物的診斷評估中斷了。為了克服這個問題,我們開發了一種具有高靈敏度和準確性的定量方法來測量尿液中的TPP。方法將固定化金屬親和色譜法(IMAC)微型化至96孔格式。比較評估了基于發光,可見光和紅外熒光抗體的檢測方法。結果由于它們的豐度低,我們證明磷蛋白富集步驟和非常靈敏的檢測方法都需要檢測尿液樣品中的TPP。為了追求膀胱癌篩查計劃所需的高通量,可重復性和成本控制,我們在自動化平臺中將分析前的IMAC程序與高靈敏度檢測階段(紅外熒光或化學發光)相結合。結論高通量方法可用于尿樣中高靈敏度TPP水平的測定,目前已用于大型臨床試驗,以建立TPPs作為膀胱癌標志物的診斷和預測能力。一般意義新方法代表了第一種定量和高通量的尿液中TPPs測定方法。

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