1. Validation of a double-color ELISpot assay of IFN-γ and IL-4 production in human peripheral blood mononuclear cells.
- Author
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Dapporto, Francesca, De Tommaso, Domiziana, Marrocco, Camilla, Piu, Pietro, Semplici, Claudia, Fantoni, Giulia, Ferrigno, Ilaria, Piccini, Giulia, Monti, Martina, Vanni, Francesca, Razzano, Ilaria, Manini, Ilaria, Montomoli, Emanuele, and Manenti, Alessandro
- Subjects
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MONONUCLEAR leukocytes , *T helper cells , *DETECTION limit - Abstract
The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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