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基于纳米颗粒的细胞外囊泡检测方法。

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

机构信息

Department of Biochemistry, Division of Biotechnology, University of Turku, Turku, Finland.

Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland.

出版信息

Sci Rep. 2019 Jul 11;9(1):10038. doi: 10.1038/s41598-019-46395-2.

DOI:10.1038/s41598-019-46395-2
PMID:31296879
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6624270/
Abstract

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu-chelate or Eu-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.

摘要

细胞外囊泡 (EVs) 的分析通常需要从生物体液中进行繁琐且耗时的分离过程。我们开发了一种基于纳米颗粒的时间分辨荧光免疫分析 (NP-TRFIA)，该方法使用针对四跨膜蛋白家族和肿瘤相关抗原的生物素化抗体从尿液样本和细胞培养上清液中捕获 EVs，而无需进行分离。捕获的 EVs 可以通过针对四跨膜蛋白的 Eu 螯合物或基于 Eu 掺杂的纳米颗粒标记物或针对 EVs 表面糖基部分的凝集素进行检测。NP-TRFIA 证明了抗体和凝集素对 EVs 的特异性捕获和检测。与凝集素-螯合物测定相比，凝集素-纳米颗粒测定的信号与背景比值高 2-10 倍。基于纳米颗粒的测定概念允许用凝集素对尿液衍生的 EVs 进行表面糖基化分析。它还被应用于建立一种测定方法，该方法显示出在更具侵袭性的（DU145 和 PC3-EVs 上的 ITGA3 更高）与侵袭性较低的（LNCaP-EVs 上的 EpCAM 更高）PCa 细胞系衍生的 EVs 上肿瘤相关蛋白的差异表达。这种 NP-TRFIA 可作为分析和鉴定尿液和细胞培养上清液中 EVs 的简单工具。这种方法可用于鉴定患者来源的尿液 EVs 表面的疾病特异性标记物。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/99864f301bbd/41598_2019_46395_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/1ff1ad3711ee/41598_2019_46395_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/99f75768e95c/41598_2019_46395_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/9824e19183bf/41598_2019_46395_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/53827c375761/41598_2019_46395_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/b0af0037ac68/41598_2019_46395_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/99864f301bbd/41598_2019_46395_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/1ff1ad3711ee/41598_2019_46395_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/99f75768e95c/41598_2019_46395_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/9824e19183bf/41598_2019_46395_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/53827c375761/41598_2019_46395_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/b0af0037ac68/41598_2019_46395_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d9bf/6624270/99864f301bbd/41598_2019_46395_Fig6_HTML.jpg

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