L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma

Norman, Michael E.; Ter-Ovanesyan, Dmitry; Trieu, Wendy; Lazarovits, Roey; Kowal, Emma J. K.; Lee, Ju-Hyun; Chen‐Plotkin, Alice S.; Regev, Aviv; Church, George M.; Walt, David R.

doi:10.1038/s41592-021-01174-8

Cited by 168 publications

(202 citation statements)

References 17 publications

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“…The percentage of ‘on wells’ can then be converted to protein concentration by comparing to a calibration curve of recombinant protein standard. We previously developed and validated Simoa assays for the proteins CD9, CD63, and CD81, showing that they are 1–3 orders of magnitude more sensitive than the corresponding standard ELISA assays with the same pairs of antibodies ( Norman et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…One could also follow a similar approach to the one we present here with traditional ELISA or other protein detection methods, but we find that high sensitivity is often necessary for the low levels of EVs in human biofluids. We have previously shown in a direct comparison (using the same antibodies) that Simoa can detect EV markers in cases where traditional ELISA cannot, such as SEC fractions of CSF ( Norman et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

“…Simoa assays were developed and performed as previously described ( Norman et al, 2021 ). A detailed protocol is available: https://www.protocols.io/view/simoa-extracellular-vesicle-assays-bm89k9z6 .…”

Section: Methodsmentioning

confidence: 99%

“…Simoa assays can be orders of magnitude more sensitive than traditional ELISAs ( Cohen and Walt, 2019 ), which is particularly useful for EV analysis as the levels of EV proteins are often low in clinical biofluid samples ( Coumans et al, 2017b ). We have previously applied Simoa to the investigation of L1CAM, a protein thought to be a marker of neuron-derived EV, showing it is not associated with EVs in plasma and CSF ( Norman et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

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Framework for rapid comparison of extracellular vesicle isolation methods

Ter-Ovanesyan

Norman

Lazarovits

et al. 2021

eLife

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Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Framework for rapid comparison of extracellular vesicle isolation methods

Ter-Ovanesyan

Norman

Lazarovits

et al. 2021

eLife

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“…Immunoaffinity capture of TEX from plasma has been introduced as an approach to the pulldown of TEX based on the use of Abs specific for the antigens selectively expressed or markedly overexpressed by cancer cells and carried by TEX [20]. Immunocapture-based exosome isolation from body fluids has been extensively used in diseases other than cancer, including neurological diseases, where Ab-based capture is broadly used for the isolation of neuron-derived L1CAM bearing EVs (NDEVs) [21]. In cancer, TEX separated from non-TEX are expected to serve as a liquid tumor biopsy that faithfully recapitulates molecular and genetic features of parental cancer cells.…”

Section: Rationale For Sev Fractionation Into Tex and Non-texmentioning

confidence: 99%

Diversity of Extracellular Vesicles (EV) in Plasma of Cancer Patients

Whiteside¹,

Ferrone²

2022

Physiology

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Extracellular vesicles (EVs) are produced by all cells and are found in all body fluids. They function as intercellular messengers that carry and deliver signals regulating cellular interactions in health and disease. EVs are emerging as potential biomarkers of diseases and responses to therapies, and much attention is being devoted to understanding their role in physiological as well as pathological events. EVs are heterogenous in their origin, size, molecular characteristics, genetic content and functions. Isolation of EV subsets from plasma and characterization of their distinct properties have been a limiting factor in ongoing efforts to understand their biological importance. Here, we discuss the immunoaffinity-based strategies that are available for isolating distinct subsets of EVs from plasma and provide a road-map to their successful immunocapture and molecular profiling, with special attention to tumor-derived EVs or TEX.

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Exosome Processing and Characterization Approaches for Research and Technology Development

et al. 2022

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Exosomes are extracellular vesicles that share components of their parent cells and are attractive in biotechnology and biomedical research as potential disease biomarkers as well as therapeutic agents. Crucial to realizing this potential is the ability to manufacture high‐quality exosomes; however, unlike biologics such as proteins, exosomes lack standardized Good Manufacturing Practices for their processing and characterization. Furthermore, there is a lack of well‐characterized reference exosome materials to aid in selection of methods for exosome isolation, purification, and analysis. This review informs exosome research and technology development by comparing exosome processing and characterization methods and recommending exosome workflows. This review also provides a detailed introduction to exosomes, including their physical and chemical properties, roles in normal biological processes and in disease progression, and summarizes some of the on‐going clinical trials.

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L1CAM is not associated with extracellular vesicles in human cerebrospinal fluid or plasma

Cited by 168 publications

References 17 publications

Framework for rapid comparison of extracellular vesicle isolation methods

Framework for rapid comparison of extracellular vesicle isolation methods

Diversity of Extracellular Vesicles (EV) in Plasma of Cancer Patients

Exosome Processing and Characterization Approaches for Research and Technology Development

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