title: Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles.
abstract: Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.
authors.affiliation: Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.
authors.forename: Anush
authors.initials: A
authors.lastname: Arakelyan
authors.affiliation: Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.
authors.forename: Wendy
authors.initials: W
authors.lastname: Fitzgerald
authors.affiliation: Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health.
authors.forename: Sonia
authors.initials: S
authors.lastname: Zicari
authors.affiliation: Laboratory of Atherothrombosis, Cardiology Department, Moscow State University of Medicine and Dentistry.
authors.forename: Murad
authors.initials: M
authors.lastname: Vagida
authors.affiliation: Sidra Medical and Research Center; jgrivel@sidra.org.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Grivel
authors.affiliation: Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health; margolil@helix.nih.gov.
authors.forename: Leonid
authors.initials: L
authors.lastname: Margolis
mesh_terms: D054058:Acute Coronary Syndrome / Q000097:blood; D000906:Antibodies / Q000737:chemistry; D000956:Antigens, Viral / Q000032:analysis*; D015415:Biomarkers; D016022:Case-Control Studies; D003716:Dengue Virus / Q000276:immunology / Q000472:pathogenicity; D000067128:Extracellular Vesicles / Q000737:chemistry / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005455:Fluorescent Antibody Technique / Q000379:methods; D015497:HIV-1 / Q000276:immunology / Q000472:pathogenicity; D054884:Host-Pathogen Interactions; D006801:Humans; D058185:Magnetite Nanoparticles / Q000737:chemistry; D014771:Virion / Q000737:chemistry / Q000276:immunology*
chemical_list: D000906:Antibodies; D000956:Antigens, Viral; D015415:Biomarkers; D058185:Magnetite Nanoparticles