title: Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies.
abstract: Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-gamma and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent-onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that approximately 0.58% of circulating CD19(+) B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products.
authors.affiliation: Division of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Bradshaw
authors.forename: Sally C
authors.initials: SC
authors.lastname: Kent
authors.forename: Vinay
authors.initials: V
authors.lastname: Tripuraneni
authors.forename: Tihamer
authors.initials: T
authors.lastname: Orban
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: David A
authors.initials: DA
authors.lastname: Hafler
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Love
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D016207:Cytokines / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007371:Interferon-gamma / Q000032:analysis / Q000276:immunology; D015850:Interleukin-6 / Q000032:analysis / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism; D046888:Tissue Array Analysis / Q000379:methods*
chemical_list: D000906:Antibodies; D016207:Cytokines; D015850:Interleukin-6; D007371:Interferon-gamma