Validation of a double-color ELISpot assay of IFN- γ and IL-4 production in human peripheral blood mononuclear cells

J Immunol Methods. 2023 Nov 29:113588. doi: 10.1016/j.jim.2023.113588. Online ahead of print.ABSTRACTThe 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.PMID:38040193 | DOI:10.1016/j.jim.2023.113588

Source: Journal of Immunological Methods - December 1, 2023 Category: Allergy & Immunology Authors: Francesca Dapporto Domiziana De Tommaso Camilla Marrocco Pietro Piu Claudia Semplici Giulia Fantoni Ilaria Ferrigno Giulia Piccini Martina Monti Francesca Vanni Ilaria Razzano Ilaria Manini Emanuele Montomoli Alessandro Manenti Source Type: research