Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.
Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release (EV-CATCHER): A customizable purification assay designed for small-RNA biomarker identification and evaluation of circulating small-EVs.
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DOI：
10.1002/jev2.12110
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发表时间：
2021-06
影响因子：
16
通讯作者：
Loudig O
中科院分区：
文献类型：
--
作者：
Mitchell MI;Ben-Dov IZ;Liu C;Ye K;Chow K;Kramer Y;Gangadharan A;Park S;Fitzgerald S;Ramnauth A;Perlin DS;Donato M;Bhoy E;Manouchehri Doulabi E;Poulos M;Kamali-Moghaddam M;Loudig O
封装在小细胞外囊泡 (EV) 内的循环核酸提供了源自患病组织的生物标志物的远程细胞快照，但选择性分离至关重要。目前基于实验室的纯化技术依赖于小型电动汽车的物理特性，而不是其遗传的细胞指纹。我们建立了一种称为 EV-CATCHER 的高度选择性纯化方法，最初设计用于通过下一代测序对低丰度小 RNA 货物进行高通量分析。我们通过从掺入人血浆的小鼠小 EV 中特异性分离小 RNA 并对其进行测序，证明了其选择性。使用蛋白质印迹、纳米粒子追踪和透射电子显微镜来验证和量化完整小型 EV 的捕获和释放。作为灵敏检测循环 miRNA 的原理验证，我们使用最近住院的一小群 Covid-19 患者的样本，将用 EV-CATCHER 纯化的小 EV 血清子集的小 RNA 测序数据与全血清的数据进行了比较。我们仅在小型 EV 中发现并验证了 hsa-miR-146a 和 hsa-miR-126-3p 随疾病严重程度而显着下调。另外，我们使用具有高抗尖峰 IgG 滴度的康复 Covid-19 患者的恢复期血清，证实了通过 EV-CATCHER 纯化的一小部分小 EV 血清在体外针对 SARS-CoV-2 的中和特性，正如最初在超速离心小 EV 中观察到的那样。总而言之，我们的数据强调了 EV-CATCHER 的敏感性和多功能性。 “一种可定制的、低背景、高亲和力的测定，用于在小 RNA 测序之前对循环小细胞外囊泡进行特异性免疫捕获和释放，以识别 miRNA 生物标志物或进行体外评估：EV-CATCHER 测定（通过 AnTibody of CHoice 进行细胞外囊泡捕获和酶促释放）”。
Circulating nucleic acids, encapsulated within small extracellular vesicles (EVs), provide a remote cellular snapshot of biomarkers derived from diseased tissues, however selective isolation is critical. Current laboratory‐based purification techniques rely on the physical properties of small‐EVs rather than their inherited cellular fingerprints. We established a highly‐selective purification assay, termed EV‐CATCHER, initially designed for high‐throughput analysis of low‐abundance small‐RNA cargos by next‐generation sequencing. We demonstrated its selectivity by specifically isolating and sequencing small‐RNAs from mouse small‐EVs spiked into human plasma. Western blotting, nanoparticle tracking, and transmission electron microscopy were used to validate and quantify the capture and release of intact small‐EVs. As proof‐of‐principle for sensitive detection of circulating miRNAs, we compared small‐RNA sequencing data from a subset of small‐EVs serum‐purified with EV‐CATCHER to data from whole serum, using samples from a small cohort of recently hospitalized Covid‐19 patients. We identified and validated, only in small‐EVs, hsa‐miR‐146a and hsa‐miR‐126‐3p to be significantly downregulated with disease severity. Separately, using convalescent sera from recovered Covid‐19 patients with high anti‐spike IgG titers, we confirmed the neutralizing properties, against SARS‐CoV‐2 in vitro, of a subset of small‐EVs serum‐purified by EV‐CATCHER, as initially observed with ultracentrifuged small‐EVs. Altogether our data highlight the sensitivity and versatility of EV‐CATCHER. ‘A customizable, low background, high‐affinity assay for specific immuno‐capture and release of circulating small extracellular vesicles prior to small‐RNA sequencing for identification of miRNA biomarkers or in‐vitro evaluation: The EV‐CATCHER assay (Extracellular Vesicle Capture by AnTibody of CHoice and Enzymatic Release)’.
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