Evaluation of the interferon-gamma ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood.
ELISPOT assays for detection of antigen specific HLA class I restricted T lymphocytes have recently been developed. Reliable quantitation of T cell frequencies is crucial for the monitoring of specific cancer vaccines. We have evaluated the accuracy of quantitative results obtained with the IFN-gamma ELISPOT assay and both sensitivity and specificity obtained with several pairs of antibodies was compared. The detection system was tested with IFN-gamma coupled latex beads and the quantitation of influenza specific T cells with several sets of dilution experiments. The reproducibility of the quantitative results was established. Only one pair of monoclonal antibodies had an acceptable specificity. In a serial dilution, almost 100% of IFN-gamma-coated beads could be detected. Furthermore, the number of influenza-peptide specific spots correlated closely with (a) the number of antigen specific T cells derived from an influenza-peptide specific T cell line diluted in PBMC, and (b) the number of CD8+ T cells diluted in autologous CD8-depleted PBMC. In three healthy individuals and three cohorts of healthy volunteers, the number of influenza-peptide specific spots was highly reproducible. The data presented here demonstrate that the frequency of peptide specific CD8+ T cells can be reliably determined from peripheral blood with the IFN-gamma ELISPOT assay.
Schmittel
A
A
Department of Hematology/Oncology and Rheumatology, Medizinische Klinik V, University of Heidelberg, Germany.
Keilholz
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U
Scheibenbogen
C
C
D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology*
D007371:Interferon-gamma