Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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CD8-Positive T-Lymphocytes / cytology
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CD8-Positive T-Lymphocytes / immunology
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Cell Count
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Cell Separation / methods
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Epitopes, T-Lymphocyte / immunology
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Histocompatibility Antigens Class I / immunology
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Humans
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Immunoenzyme Techniques / methods
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Interferon-gamma / immunology*
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Leukocytes, Mononuclear / cytology
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Leukocytes, Mononuclear / immunology
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Molecular Sequence Data
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Monocytes / cytology
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Peptide Fragments / immunology*
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Proteins / immunology
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T-Lymphocyte Subsets / cytology
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T-Lymphocyte Subsets / immunology
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T-Lymphocytes / cytology
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T-Lymphocytes / immunology*
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Tetanus Toxoid / immunology*
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Tuberculin / immunology*
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Viral Matrix Proteins / immunology*
Substances
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Epitopes, T-Lymphocyte
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Histocompatibility Antigens Class I
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Peptide Fragments
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Proteins
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Tetanus Toxoid
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Tuberculin
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Viral Matrix Proteins
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influenza virus membrane protein (58-66)
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Interferon-gamma