title: Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection.
abstract: The identification of parameters maximizing detection sensitivity in ELISpot assays is important to transfer this technology into the clinical setting for identifying rare Ag-specific CD8(+) T cells. We have therefore considered human IFN-gamma CD8(+) T cell responses against viral epitopes to analyze different variables which could be critical during the epitope-specific stimulation period. Two parameters were found to greatly enhance detection sensitivity (i.e., to specifically increase epitope-driven signal while keeping background noise to a minimum): use of human serum-free vs. serum-supplemented culture medium (2.4-fold median increase) and addition of low dose IL-7 (1.5-fold increase). Incorporating both of these parameters into the ELISpot procedure proved capable of greatly amplifying (35.1-fold increase) the low grade CD8(+) T cell responses directed against beta-cell epitopes of type 1 diabetes patients, as compared to a previously optimized procedure using human serum-supplemented medium and low dose IL-2. Implementation of this ELISpot procedure should expedite development of "immune staging" protocols for autoimmune as well as tumor and infectious diseases.
authors.affiliation: INSERM, U580, Necker Hospital, Paris, France.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Martinuzzi
authors.forename: Matthieu
authors.initials: M
authors.lastname: Scotto
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Enée
authors.forename: Vedran
authors.initials: V
authors.lastname: Brezar
authors.forename: Jean-Antoine
authors.initials: JA
authors.lastname: Ribeil
authors.forename: Peter
authors.initials: P
authors.lastname: van Endert
authors.forename: Roberto
authors.initials: R
authors.lastname: Mallone
mesh_terms: D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018106:CD28 Antigens / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D018929:Cell Culture Techniques / Q000379:methods; D016895:Culture Media, Serum-Free; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000032:analysis / Q000276:immunology*; D005260:Female; D015789:HLA-A2 Antigen / Q000276:immunology; D006801:Humans; D050417:Insulin-Secreting Cells / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology; D007376:Interleukin-2 / Q000276:immunology / Q000494:pharmacology; D015851:Interleukin-7 / Q000276:immunology / Q000494:pharmacology*; D008297:Male; D008875:Middle Aged; D018709:Statistics, Nonparametric
chemical_list: D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D016895:Culture Media, Serum-Free; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D007376:Interleukin-2; D015851:Interleukin-7; D007371:Interferon-gamma