Open AccessExtracellular 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
Author(s) -
Mitchell Megan I.,
BenDov Iddo Z.,
Liu Christina,
Ye Kenny,
Chow Kar,
Kramer Yael,
Gangadharan Anju,
Park Steven,
Fitzgerald Sean,
Ramnauth Andrew,
Perlin David S.,
Donato Michele,
Bhoy Emily,
Manouchehri Doulabi Ehsan,
Poulos Michael,
KamaliMoghaddam Masood,
Loudig Olivier
Publication year - 2021
Publication title -
journal of extracellular vesicles
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.94
H-Index - 68
ISSN - 2001-3078
DOI - 10.1002/jev2.12110
Subject(s) - rna , extracellular vesicles , nanoparticle tracking analysis , small rna , extracellular vesicle , nucleic acid , microvesicles , microbiology and biotechnology , titer , biology , biomarker , blot , chemistry , microrna , computational biology , virology , gene , biochemistry , virus
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.