High throughput Surface Epitope Immunoaffinity Isolation of Extracellular Vesicles and Downstream Analysis DOI Creative Commons
Ramin Khanabdali,

Michelle Mandrekar,

Rick Grygiel

и другие.

Biology Methods and Protocols, Год журнала: 2024, Номер 9(1)

Опубликована: Янв. 1, 2024

Abstract Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods efficient high-throughput isolation analysis EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) utilizes affinity ligands, antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI represents a fit-for-purpose solution the reproducible, EVs from biofluids downstream RNA, protein, lipid biomarkers is compatible with laboratory workflows. This study evaluates new SEI method enriching subpopulations were isolated human plasma using bead-based designed on-bead EV-associated RNA protein biomarkers. Western blot confirmed presence EV markers captured nanoparticles. Mass spectrometry lysate identified over 1500 proteins, top 100 known proteins. microRNA (miRNA) sequencing followed by RT-qPCR miRNA transcripts. Using SEI, automated particle moving instruments, demonstrating equal higher yield recovery compared to manual processing. rapid, efficient, isolating enriched populations EVs; effectively reducing contamination enabling subpopulation In this study, extraction been successfully implemented. technology holds great promise advancing field research facilitating application biomarker discovery research.

Язык: Английский

High throughput Surface Epitope Immunoaffinity Isolation of Extracellular Vesicles and Downstream Analysis DOI Creative Commons
Ramin Khanabdali,

Michelle Mandrekar,

Rick Grygiel

и другие.

Biology Methods and Protocols, Год журнала: 2024, Номер 9(1)

Опубликована: Янв. 1, 2024

Abstract Extracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods efficient high-throughput isolation analysis EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) utilizes affinity ligands, antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI represents a fit-for-purpose solution the reproducible, EVs from biofluids downstream RNA, protein, lipid biomarkers is compatible with laboratory workflows. This study evaluates new SEI method enriching subpopulations were isolated human plasma using bead-based designed on-bead EV-associated RNA protein biomarkers. Western blot confirmed presence EV markers captured nanoparticles. Mass spectrometry lysate identified over 1500 proteins, top 100 known proteins. microRNA (miRNA) sequencing followed by RT-qPCR miRNA transcripts. Using SEI, automated particle moving instruments, demonstrating equal higher yield recovery compared to manual processing. rapid, efficient, isolating enriched populations EVs; effectively reducing contamination enabling subpopulation In this study, extraction been successfully implemented. technology holds great promise advancing field research facilitating application biomarker discovery research.

Язык: Английский

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