title: Validation of a miniaturized assay based on IFNg secretion for assessment of specific T cell immunity.
abstract: A miniaturized method for detection of antigen induced secretion of IFNg by specific T cells cultured in 384 well plates has been recently reported. In order to confidently apply this assay to clinical investigations for monitoring of specific T cell immunity, an intralaboratory validation study has been undertaken. High reproducibility and linearity of reference curves was demonstrated. Consecutive replicate experiments handled by different operators using broad panels of recall antigens were reproducible when tested on individual biological samples. Kinetics of IFNg secretion with different antigens showed a plateau after 24h culture. Similar trends were observed with secretion of TNFa, GM-CSF and IL17, suggesting that the same kinetics can be applied if other cytokines are tested with this assay. It was demonstrated that frozen-thawed cells can be tested by cell-ELISA and that when PBMC are replaced by whole blood similar reactivity profiles were observed even though cytokine concentration was lower. T cell responses were higher in round bottom than in flat bottom wells, but these plates could not be applied to cell-ELISA as clear plates are not available for scanning. In conclusion, the assay proved flexible, since plates can be frozen at different times during the process, fresh or frozen PBMC and PBMC or whole blood could be used, and robust, since reproducibility was remarkable even when different operators performed the procedures.
authors.affiliation: Advanced Biotechnology Center, Largo Benzi 10, 16132, Genoa, Italy. lipira@email.it
authors.forename: Giusi
authors.initials: G
authors.lastname: Li Pira
authors.forename: Federico
authors.initials: F
authors.lastname: Ivaldi
authors.forename: Paolo
authors.initials: P
authors.lastname: Moretti
authors.forename: Marco
authors.initials: M
authors.lastname: Risso
authors.forename: Gino
authors.initials: G
authors.lastname: Tripodi
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Manca
mesh_terms: D000941:Antigens / Q000032:analysis* / Q000276:immunology; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology* / Q000378:metabolism; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
chemical_list: D000941:Antigens; D016207:Cytokines; D007371:Interferon-gamma