title: T-cell antigen discovery (T-CAD) assay: a novel technique for identifying T cell epitopes.
abstract: The identification of T cell epitopes is a critical step in evaluating and monitoring T cell mediated immune responses. Here, we describe a novel technique for simultaneously identifying class I and class II MHC restricted epitopes using a one-step protein purification system. This method uses Ni/chelate coated magnetic beads and magnetic separation to isolate poly-histidine tagged recombinant antigen from bacterial lysates. These beads, once coated with antigen, are also used to deliver antigen to APC where it is processed and presented to T cells. A colorimetric assay and ovalbumin specific, lacZ inducible, T cell hybridomas were used to validate the system. Further, using PSA specific hybrids, generated from T cells isolated from PSA secreting tumors, both class I and class II MHC restricted epitopes of PSA were identified. Additional characterization has shown that these peptides contribute significantly to the overall PSA specific response in vivo, and may represent the dominant epitopes of PSA.
authors.affiliation: Department of Microbiology and Immunology and the Cancer Center, University of Rochester Medical Center, Box 704, Rochester, NY 14642, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Turner
authors.forename: C S
authors.initials: CS
authors.lastname: Abdul-Alim
authors.forename: R A
authors.initials: RA
authors.lastname: Willis
authors.forename: T L
authors.initials: TL
authors.lastname: Fisher
authors.forename: E M
authors.initials: EM
authors.lastname: Lord
authors.forename: J G
authors.initials: JG
authors.lastname: Frelinger
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D000938:Antigen-Presenting Cells / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006825:Hybridomas; D008213:Lymphocyte Activation; D051379:Mice; D008863:Microspheres; D010455:Peptides / Q000235:genetics; D017430:Prostate-Specific Antigen / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D017384:Sequence Deletion; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
chemical_list: D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D017430:Prostate-Specific Antigen