Application of the IFN-gamma ELISPOT assay to quantify T cell responses against proteins.
The ELISPOT assay has been established for the direct ex vivo quantification of peptide-reactive T lymphocytes from peripheral blood mononuclear cells (PBMC). In this report we studied the ELISPOT assay conditions for the detection of T cell responses against protein antigens including Tetanus toxoid (TT), purified-protein-derivative (PPD) and a synthetic 30 mer peptide derived from influenza matrix protein (IMP) containing the HLA-A*0201-restricted 9 mer peptide epitope GILGFVFTL as a model for a viral protein. We found several aspects to be crucial for a sensitive detection of T cell responses against proteins including a pellet preincubation step and the monocyte concentration. Using optimized assay conditions specific CD4+ T cell responses against TT and PPD as well as CD8+ T cell responses against IMP can be quantified directly ex vivo from peripheral blood mononuclear cells.
Schmittel
A
A
Universitätsklinikum Benjamin-Franklin, Medizinische Klinik III (Hämatologie, Onkologie and Transfusionsmedizin), Freie Universität Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. schmittel@ukbf.fu-berlin.de
Keilholz
U
U
Bauer
S
S
Kuhne
U
U
Stevanovic
S
S
Thiel
E
E
Scheibenbogen
C
C
D000595:Amino Acid Sequence; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D010446:Peptide Fragments / Q000276:immunology*; D011506:Proteins / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013745:Tetanus Toxoid / Q000276:immunology*; D014373:Tuberculin / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000276:immunology*
D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D011506:Proteins; D013745:Tetanus Toxoid; D014373:Tuberculin; D014763:Viral Matrix Proteins; C098952:influenza virus membrane protein (58-66); D007371:Interferon-gamma