title: Usage of standardized antigen-presenting cells improves ELISpot performance for complex protein antigens.
abstract: The enzyme-linked immunospot (ELISpot) assay is a widely used method for immune monitoring in cancer immunotherapy trials. In the ELISpot assay, peripheral blood mononuclear cells (PBMC) are stimulated with specific antigens, and cytokines of interest produced by individual cells are detected. In the standard procedure, T cells rely for antigen presentation on other cells like the monocyte/macrophage population present among the PBMC. Whereas oligopeptides can be added directly to the ELISpot assay without the necessity of a pre-incubation step, protein antigens must be internalized and processed by antigen-presenting cells to accomplish efficient presentation via HLA class I or II. We have studied the impact of sources for different antigen-presenting cell (i.e. PBMC-resident monocytes and monocyte-derived dendritic cells maturated with Poly I:C and PGE-2 based cocktails) on ELISpot assay performance and defined an optimized dendritic cell-based ELISpot protocol. This protocol is suitable for monitoring immune responses directed to protein antigens with higher sensitivity than the standard procedure.
authors.affiliation: Department of Hematology/Oncology, University Medical Center Freiburg, 79106 Freiburg, Germany. m.a.navarrete@lumc.nl
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Navarrete
authors.forename: Cristina
authors.initials: C
authors.lastname: Bertinetti-Lapatki
authors.forename: Ines
authors.initials: I
authors.lastname: Michelfelder
authors.forename: Hendrik
authors.initials: H
authors.lastname: Veelken
mesh_terms: D017951:Antigen Presentation; D000941:Antigens / Q000276:immunology*; D019496:Cancer Vaccines / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology*; D015232:Dinoprostone / Q000494:pharmacology; D058501:Enzyme-Linked Immunospot Assay / Q000592:standards*; D006801:Humans; D059425:Interferon-gamma Release Tests / Q000592:standards*; D016753:Interleukin-10 / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology*; D057230:Limit of Detection; D015166:Monitoring, Immunologic / Q000592:standards*; D011070:Poly I-C / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology*; D013997:Time Factors
chemical_list: D000941:Antigens; D019496:Cancer Vaccines; C508609:IL10 protein, human; D016753:Interleukin-10; D018664:Interleukin-12; D015232:Dinoprostone; D011070:Poly I-C