Multiplexed protein analysis using encoded antibody-conjugated microbeads.
Multiplexed protein analysis using encoded antibody-conjugated microbeads.
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使用编码的抗体缀合微珠进行多重蛋白质分析。
DOI：
10.1098/rsif.2010.0594
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Huang,Xiaohua
中科院分区：
文献类型：
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作者：
Theilacker,Nora;Roller,EricE;Barbee,KristopherD;Franzreb,Matthias;Huang,Xiaohua
我们描述了一种使用荧光编码微珠进行蛋白质多重分析的方法。我们的方法的灵敏度与酶联免疫吸附测定法的灵敏度相当，而只需要5微升的样品体积。链霉亲和素包被的1 µm微珠由不同强度水平的荧光团组合编码。作为概念的证明，我们证明了27个微珠群体可以很容易地编码的亲和共轭使用三种不同的生物素化荧光染料的每一个的三个强度水平。四个编码的微珠群体进一步与生物素化的捕获抗体缀合，然后组合并固定在微流体流动池中用于多重蛋白质分析。使用四个独特编码的微珠群体，我们表明，癌症生物标志物和三种细胞因子蛋白可以在皮克每毫升范围内通过荧光显微镜在一个单一的测定进行定量分析。我们的方法将允许使用集成的微流体装置和自动化样品处理来制造用于多重蛋白质分析的高密度、基于珠的抗体阵列。
We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 µl sample volumes are needed. Streptavidin-coated, 1 µm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.