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 quantificatio n of peptide-reactive T lymphocytes from peripheral blood mononuclear cells (PBMC). In this report we studied the ELISPOT assay conditions for the det ection of T cell responses against protein antigens including Tetanus toroi d (TT), purified-protein-derivative (PPD) and a synthetic 30 mer peptide de rived from influenza matrix protein (IMP) containing the HLA-A*0201-restric ted 9 mer peptide epitope GILGFVFTL as a model for a viral protein. We foun d several aspects to be crucial for a sensitive detection of T cell respons es against proteins including a pellet preincubation step and the monocyte concentration. Using optimized assay conditions specific CD4+ T cell respon ses against TT and PPD as well as CD8+ T cell responses against IMP can be quantified directly ex vivo from peripheral blood mononuclear cells. (C) 20 01 Elsevier Science B.V. All rights reserved.