Rapid Lipid-Based Approach for Normalization of Quantum-Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples.
Rapid Lipid-Based Approach for Normalization of Quantum-Dot-Detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples.
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DOI：
10.1021/acs.nanolett.9b02232
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发表时间：
2019-11-13
期刊：
影响因子：
10.8
通讯作者：
Hu TY
中科院分区：
文献类型：
--
作者：
Rodrigues M;Richards N;Ning B;Lyon CJ;Hu TY
细胞外囊泡（EV）作为肿瘤生物标志物受到了相当大的关注，因为肿瘤衍生的EV包含有关肿瘤病理生理学的广泛信息。然而，目前的EV测定无法区分由具有稳定生物标志物表达的靶EV群体的丰度改变、稳定靶EV群体中生物标志物表达改变或由两个参数的变化引起的效应引起的EV生物标志物差异。我们现在描述了一种快速的基于纳米颗粒和染料的荧光免疫测定，可以通过将EV生物标志物水平标准化为EV丰度来区分这些可能性。在该方法中，从复杂样品（例如，血清），用亲脂性染料染色，并与特异性EV表面生物标志物的抗体缀合的量子点探针杂交。EV染料信号用于定量EV丰度和标准化EV表面生物标志物表达水平。来自恶性和非恶性胰腺细胞系的EV表现出相似的染色，并且探针与染料的比率不随EV丰度而变化，从而允许直接分析标准化EV生物标志物表达而无需单独的EV定量步骤。这种EV生物标志物标准化方法显著提高了两种胰腺癌生物标志物EV EpCAM和EV EphA2的血清水平区分胰腺癌患者与非恶性对照受试者的能力。该测定的简化的工作流程和稳健的结果适合于快速转化为临床应用，并且其模块化设计允许其通过将抗体缀合的量子点探针交换为识别不同疾病特异性EV生物标志物的那些的简单权宜之计而快速适应于定量其他EV生物标志物。
Extracellular vesicles (EVs) are of considerable interest as tumor biomarkers because tumor-derived EVs contain a broad array of information about tumor pathophysiology. However, current EV assays cannot distinguish between EV biomarker differences resulting from altered abundance of a target EV population with stable biomarker expression, altered biomarker expression in a stable target EV population, or effects arising from changes in both parameters. We now describe a rapid nanoparticle- and dyebased fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker levels to EV abundance. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic dye, and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant and nonmalignant pancreatic cell lines exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its modular design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple expedient of swapping the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker.
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影响因子：
17.1
作者：
Lee K;Fraser K;Ghaddar B;Yang K;Kim E;Balaj L;Chiocca EA;Breakefield XO;Lee H;Weissleder R
通讯作者：
Weissleder R
DOI：
10.1073/pnas.1521230113
发表时间：
2016-02-23
影响因子：
11.1
作者：
Kowal, Joanna;Arras, Guillaume;Thery, Clotilde
通讯作者：
Thery, Clotilde
影响因子：
7.4
作者：
Sarker S;Scholz-Romero K;Perez A;Illanes SE;Mitchell MD;Rice GE;Salomon C
通讯作者：
Salomon C
影响因子：
10.8
作者：
Pathak, Smita;Davidson, Marie C.;Silva, Gabriel A.
通讯作者：
Silva, Gabriel A.
影响因子：
8
作者：
Mendt, Mayela;Kamerkar, Sushrut;Kalluri, Raghu
通讯作者：
Kalluri, Raghu