Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma.
Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma.
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DOI：
10.1186/s12951-018-0372-z
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发表时间：
2018-05-02
影响因子：
10.2
通讯作者：
Valés-Gómez M
中科院分区：
文献类型：
--
作者：
López-Cobo S;Campos-Silva C;Moyano A;Oliveira-Rodríguez M;Paschen A;Yáñez-Mó M;Blanco-López MC;Valés-Gómez M
肿瘤来源的外泌体可以释放到血清中并提供有关恶性肿瘤特征的信息，然而，为了在生物样本中进行系统研究，需要更快的诊断技术，特别是对于低丰度蛋白质的检测。大多数人类癌细胞对至少一种激活免疫受体 NKG2D 的配体呈阳性，并且血浆中 NKG2D 配体的存在可能与预后相关。以 MICA 作为肿瘤源性抗原的例子，该抗原在转移性黑色素瘤中内源性表达并被招募到外泌体中，我们开发了两种基于免疫捕获的检测方法，用于检测纳米囊泡中的不同表位。尽管酶联免疫吸附测定 (ELISA) 和侧流免疫测定 (LFIA) 两种技术具有相同的理论基础，即使用捕获和检测抗体进行比色读数，但由于囊泡上存在多个表位，外泌体结合蛋白的分析会带来使用这些技术检测可溶性分子时不会出现的方法学问题。在这里，我们证明，在 ELISA 中，获得的信号与每个外泌体的表位数量成正比。在 LFIA 中，固定在金纳米颗粒中的检测抗体的量需要较低才能有效检测，否则空间位阻会导致信号较低。我们描述了外泌体中 MICA 的检测条件，并首次使用这两种技术证明了外泌体标记物（四跨膜蛋白 CD9、CD63 和 CD81）和内源表达的肿瘤源性抗原在一个囊泡中共存。该研究还表明，稀有蛋白质可以用作 LFIA 中检测抗体的靶标，其结果比非常丰富的蛋白质更好，并且可以优化血浆中蛋白质检测的条件。这些结果开启了使用高通量技术分析生物样品中肿瘤源性外泌体的存在的可能性。本文的在线版本 (10.1186/s12951-018-0372-z) 包含补充材料，可供授权用户使用。
Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques. The online version of this article (10.1186/s12951-018-0372-z) contains supplementary material, which is available to authorized users.
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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
DOI：
10.1126/science.aao0505
发表时间：
2018-03-30
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ferrari de Andrade L;Tay RE;Pan D;Luoma AM;Ito Y;Badrinath S;Tsoucas D;Franz B;May KF Jr;Harvey CJ;Kobold S;Pyrdol JW;Yoon C;Yuan GC;Hodi FS;Dranoff G;Wucherpfennig KW
通讯作者：
Wucherpfennig KW
影响因子：
11.2
作者：
Ashiru O;Boutet P;Fernández-Messina L;Agüera-González S;Skepper JN;Valés-Gómez M;Reyburn HT
通讯作者：
Reyburn HT
整理出来：外泌体负载的调节。
影响因子：
14.5
作者：
Villarroya-Beltri C;Baixauli F;Gutiérrez-Vázquez C;Sánchez-Madrid F;Mittelbrunn M
通讯作者：
Mittelbrunn M
影响因子：
4.4
作者：
Rosenberg-Hasson, Yael;Hansmann, Leo;Liedtke, Michaela;Herschmann, Iris;Maecker, Holden T.
通讯作者：
Maecker, Holden T.