title: A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples.
abstract: Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors--allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function.
authors.affiliation: Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Boston, MA, United States. meackerman@partners.org
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Ackerman
authors.forename: Brian
authors.initials: B
authors.lastname: Moldt
authors.forename: Richard T
authors.initials: RT
authors.lastname: Wyatt
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Dugast
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McAndrew
authors.forename: Stephen
authors.initials: S
authors.lastname: Tsoukas
authors.forename: Stephanie
authors.initials: S
authors.lastname: Jost
authors.forename: Christoph T
authors.initials: CT
authors.lastname: Berger
authors.forename: Gaia
authors.initials: G
authors.lastname: Sciaranghella
authors.forename: Qingquan
authors.initials: Q
authors.lastname: Liu
authors.forename: Darrell J
authors.initials: DJ
authors.lastname: Irvine
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.forename: Galit
authors.initials: G
authors.lastname: Alter
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D019138:Antibodies, Blocking / Q000276:immunology; D000918:Antibody Specificity; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D005434:Flow Cytometry / Q000379:methods*; D006031:Glycosylation; D015658:HIV Infections / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D066298:In Vitro Techniques; D007251:Influenza, Human / Q000276:immunology; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D009000:Monocytes / Q000276:immunology; D010587:Phagocytosis; D017452:Receptors, IgG / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D014612:Vaccines / Q000276:immunology
chemical_list: D000906:Antibodies; D019138:Antibodies, Blocking; D016207:Cytokines; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins; D014612:Vaccines