Collier Mary E W, Allcock Natalie, Sylvius Nicolas, Cassidy Jordan, Giorgini Flaviano
Department of Genetics and Genome Biology, University Road, University of Leicester, Leicester, UK, LE1 7RH.
Electron Microscopy Facility, Hodgkin Building, Lancaster Road, University of Leicester, UK, LE1 7HB.
bioRxiv. 2025 May 14:2025.05.13.653678. doi: 10.1101/2025.05.13.653678.
The isolation of neuron-derived extracellular vesicles (nEVs) from biofluids offers the potential to discover novel biomarkers to aid in diagnosis and treatment of psychiatric and neurodegenerative diseases. A few studies have used anti-NCAM antibody-bead-based immunocapture to enrich nEVs from plasma, some with little method validation. We therefore examined in detail this method for nEV enrichment.
EVs were isolated from SH-SY5Y cell-conditioned media by precipitation, or from plasma using size exclusion chromatography. EVs were characterised using nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and immunoblot analysis. SH-SY5Y-EVs were incubated with anti-NCAM immunocapture beads and examined by flow cytometry, immunoblot analysis and scanning electron microscopy (SEM). Immunocaptured plasma-derived EVs were examined using a sensitive NCAM ELISA, SEM and qPCR for miRNAs.
Characterisation of SH-SY5Y-derived and plasma-derived EVs revealed the expected size distributions of EVs using NTA, the presence of EV markers using immunoblot analysis, and a cup-shaped morphology using TEM. Anti-NCAM beads, but not anti-L1CAM or IgG beads, captured NCAM-positive SH-SY5Y-EVs as shown by flow cytometry and immunoblot analysis. Both SH-SY5Y and plasma-derived EVs were visualised on the surface of anti-NCAM immunocapture beads using SEM. A sensitive NCAM ELISA detected NCAM antigen in plasma-derived EVs immunocaptured on anti-NCAM beads. qPCR analysis of plasma-derived EVs detected many miRNAs in total plasma-EVs with high expression of hsa-miR-16-5p, hsa-miR-451a and hsa-miR-126-3p. However, only between two and seven miRNAs were detected in EVs captured on anti-NCAM-beads from three blood donors. Finally, tissue distribution analysis of miRNAs from plasma-derived EVs on anti-NCAM beads revealed that these miRNAs are enriched in tissues or organs such as blood vessels, lung, bone, thyroid and heart, but were not enriched for brain-derived miRNAs. Discussion: This study indicates that anti-NCAM beads can efficiently enrich NCAM-positive EVs from cell culture conditioned media. However, nEV levels in small volumes of plasma are possibly too low to enable efficient anti-NCAM immunocapture for subsequent miRNA analysis. Other neuron-specific markers with high expression levels on nEVs are therefore required for processing patient samples where plasma volumes are likely to be low, and to allow efficient isolation of nEVs in clinical studies for subsequent cargo analysis.
从生物流体中分离神经元衍生的细胞外囊泡（nEVs）为发现有助于精神疾病和神经退行性疾病诊断与治疗的新型生物标志物提供了可能。一些研究已使用基于抗NCAM抗体磁珠的免疫捕获法从血浆中富集nEVs，其中一些研究的方法验证较少。因此，我们详细研究了这种nEVs富集方法。
通过沉淀从SH-SY5Y细胞条件培养基中分离细胞外囊泡（EVs），或使用尺寸排阻色谱法从血浆中分离。使用纳米颗粒跟踪分析（NTA）、透射电子显微镜（TEM）和免疫印迹分析对EVs进行表征。将SH-SY5Y-EVs与抗NCAM免疫捕获磁珠孵育，并通过流式细胞术、免疫印迹分析和扫描电子显微镜（SEM）进行检测。使用灵敏的NCAM酶联免疫吸附测定（ELISA）、SEM和miRNA的定量聚合酶链反应（qPCR）检测免疫捕获的血浆来源的EVs。
对SH-SY5Y来源和血浆来源的EVs进行表征发现，使用NTA可观察到EVs预期的大小分布，免疫印迹分析显示存在EV标志物，TEM显示为杯状形态。流式细胞术和免疫印迹分析表明，抗NCAM磁珠而非抗L1CAM或IgG磁珠捕获了NCAM阳性的SH-SY5Y-EVs。使用SEM可在抗NCAM免疫捕获磁珠表面观察到SH-SY5Y和血浆来源的EVs。灵敏的NCAM ELISA检测到抗NCAM磁珠上免疫捕获的血浆来源EVs中的NCAM抗原。对血浆来源的EVs进行qPCR分析，在总血浆-EVs中检测到许多miRNA，其中hsa-miR-16-5p、hsa-miR-451a和hsa-miR-126-3p高表达。然而，在来自三名献血者的抗NCAM磁珠捕获的EVs中仅检测到2至7种miRNA。最后，对血浆来源的抗NCAM磁珠上的EVs的miRNA进行组织分布分析，发现这些miRNA在血管、肺、骨、甲状腺和心脏等组织或器官中富集，但未富集脑源性miRNA。
本研究表明，抗NCAM磁珠可有效从细胞培养条件培养基中富集NCAM阳性的EVs。然而，少量血浆中的nEVs水平可能过低，无法进行有效的抗NCAM免疫捕获以用于后续的miRNA分析。因此，对于血浆量可能较低的患者样本处理，以及在临床研究中高效分离nEVs以进行后续的货物分析，需要其他在nEVs上高表达的神经元特异性标志物。
