scholarly journals L1CAM is not Associated with Extracellular Vesicles in Human Cerebrospinal Fluid or Plasma

2020 ◽  
Author(s):  
Maia Norman ◽  
Dmitry Ter-Ovanesyan ◽  
Wendy Trieu ◽  
Roey Lazarovits ◽  
Emma J.K. Kowal ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Human Plasma ◽  
Extracellular Vesicles ◽  
Single Molecule ◽  
Transmembrane Protein ◽  
Gradient Centrifugation ◽  
Opportunity To Learn ◽  
Size Exclusion ◽  
Potential Candidate ◽  
Free Protein

AbstractNeuron-derived extracellular vesicles (NDEVs) present a tremendous opportunity to learn about the biochemistry of brain cells in living patients. L1CAM is a transmembrane protein expressed in neurons that is presumed to be found on NDEVs in human biofluids. Previous studies have used L1CAM immuno-isolation from human plasma to isolate NDEVs for neurodegenerative disease diagnostics. We developed a panel of ultrasensitive Single Molecule Array (Simoa) assays for known EV markers, as well as L1CAM, and applied it to study EVs in human plasma and cerebrospinal fluid (CSF). We fractionated plasma and CSF by size exclusion chromatography (SEC) and density gradient centrifugation (DGC) to separate EVs from free proteins. We observed that L1CAM did not elute in the EV fractions, but rather eluted in the free protein fractions. We found that L1CAM is present as a free protein in human plasma and CSF, possibly due to proteolytic cleavage and/or alternative splicing. We further demonstrate that the isoforms found in CSF and plasma are different. These data collectively establish that L1CAM in plasma is not EV associated and should therefore not be used for NDEV isolation. Importantly, the framework and tools described herein will allow for evaluation of other potential candidate markers for isolation of NDEVs.

scholarly journals Analysis of Cerebrospinal Fluid Extracellular Vesicles by Proximity Extension Assay: A Comparative Study of Four Isolation Kits

2020 ◽  
Vol 21 (24) ◽  
pp. 9425
Author(s):  
Sebastian Sjoqvist ◽  
Kentaro Otake ◽  
Yoshihiko Hirozane
Keyword(s):  
Cerebrospinal Fluid ◽  
Extracellular Vesicles ◽  
Proteomic Analysis ◽  
Magnetic Beads ◽  
Biomarker Discovery ◽  
De Novo ◽  
Size Exclusion ◽  
Cell Regulation ◽  
Promising Source ◽  
Proximity Extension Assay

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

scholarly journals A size-exclusion-based approach for purifying extracellular vesicles from human plasma

2021 ◽  
Vol 1 (3) ◽  
pp. 100055
Author(s):  
Patrick M. Vanderboom ◽  
Surendra Dasari ◽  
Gregory N. Ruegsegger ◽  
Mark W. Pataky ◽  
Fabrice Lucien ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Size Exclusion

scholarly journals The Separation and Characterization of Extracellular Vesicles from Medium Conditioned by Bovine Embryos

2020 ◽  
Vol 21 (8) ◽  
pp. 2942 ◽  
Author(s):  
Krishna Chaitanya Pavani ◽  
Xiaoyuan Lin ◽  
Joachim Hamacher ◽  
Wim Van Den Broeck ◽  
Liesbeth Couck ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Density Gradient ◽  
Extracellular Vesicles ◽  
Gradient Centrifugation ◽  
High Specificity ◽  
Size Exclusion ◽  
Bovine Embryo ◽  
Characterization Methods ◽  
Ribonucleoprotein Complexes ◽  
Exclusion Chromatography

Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrepTM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrepTM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02–1.04, 1.20–1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development.

scholarly journals Characterizing extracellular vesicles from cerebrospinal fluid by a novel size exclusion chromatography method

2021 ◽  
Vol 17 (S5) ◽  
Author(s):  
Yael Hirschberg ◽  
Karin Schildermans ◽  
Annemieke van Dam ◽  
Karen Sterck ◽  
Kurt Boonen ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Chromatography Method ◽  
Exclusion Chromatography

scholarly journals Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Susann Allelein ◽  
Paula Medina-Perez ◽  
Ana Leonor Heitor Lopes ◽  
Sabrina Rau ◽  
Gerd Hause ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Gradient Centrifugation ◽  
Size Exclusion ◽  
Liquid Biopsies ◽  
Antibody Microarray ◽  
Bodily Fluids ◽  
Exclusion Chromatography ◽  
Stable Source ◽  
And Function

AbstractExtracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

scholarly journals Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jik-Han Jung ◽  
Woojin Back ◽  
Junyong Yoon ◽  
Hyeonjeong Han ◽  
Ka-Won Kang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Plasma ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Soluble Proteins ◽  
Single Step ◽  
Size Exclusion ◽  
Design Parameters ◽  
Exclusion Chromatography ◽  
Porous Beads

AbstractIsolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

scholarly journals Single molecule counting coupled to rapid amplification enables detection of αSynuclein aggregates in cerebrospinal fluid of PD patients

2021 ◽  
Author(s):  
Akshay BHUMKAR ◽  
Chloe MAGNAN ◽  
Derrick LAU ◽  
Eugene Soh Wei Jun ◽  
Nicolas DZAMKO ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Single Molecule ◽  
Rapid Amplification

Extracellular Vesicles from Human Plasma Show a Distinctive Proteome and miRNome Profile in Patients with Severe Cutaneous Adverse Reactions

2021 ◽  
Author(s):  
Orlando Salinas-Jaramillo ◽  
Alejandra Monroy-Arreola ◽  
Sebastian Herrera-Noreña ◽  
Ana L. Guzmán-Ortiz ◽  
Abrahan Hernández-Hernández ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Adverse Reactions ◽  
Severe Cutaneous Adverse Reactions ◽  
Cutaneous Adverse Reactions

scholarly journals Extracellular fluid, cerebrospinal fluid and plasma biomarkers of axonal and neuronal injury following intracerebral hemorrhage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lovisa Tobieson ◽  
Henrik Zetterberg ◽  
Kaj Blennow ◽  
Niklas Marklund
Keyword(s):  
Cerebrospinal Fluid ◽  
Intracerebral Hemorrhage ◽  
Brain Tissue ◽  
Single Molecule ◽  
Tissue Injury ◽  
Extracellular Fluid ◽  
Neurofilament Light ◽  
Aβ Peptides ◽  
Post Surgery ◽  
Fluid Compartments

AbstractSpontaneous intracerebral hemorrhage (ICH) is the most devastating form of stroke. To refine treatments, improved understanding of the secondary injury processes is needed. We compared energy metabolic, amyloid and neuroaxonal injury biomarkers in extracellular fluid (ECF) from the perihemorrhagic zone (PHZ) and non-injured (NCX) brain tissue, cerebrospinal fluid (CSF) and plasma. Patients (n = 11; age 61 ± 10 years) undergoing ICH surgery received two microdialysis (MD) catheters, one in PHZ, and one in NCX. ECF was analysed at three time intervals within the first 60 h post- surgery, as were CSF and plasma samples. Amyloid-beta (Aβ) 40 and 42, microtubule associated protein tau (tau), and neurofilament-light (NF-L) were analysed using Single molecule array (Simoa) technology. Median biomarker concentrations were lowest in plasma, higher in ECF and highest in CSF. Biomarker levels varied over time, with different dynamics in the three fluid compartments. In the PHZ, ECF levels of Aβ40 were lower, and tau higher when compared to the NCX. Altered levels of Aβ peptides, NF-L and tau may reflect brain tissue injury following ICH surgery. However, the dynamics of biomarker levels in the different fluid compartments should be considered in the study of pathophysiology or biomarkers in ICH patients.

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer

2021 ◽  
Author(s):  
Luca A. Andronico ◽  
Yifei Jiang ◽  
Seung-Ryoung Jung ◽  
Bryant S. Fujimoto ◽  
Lucia Vojtech ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Single Molecule
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