A rapid alternative method to evaluate T-cell hybridoma activation using an improved cytokine (IL-2) secretion assay

T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation because they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in culture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensitivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay. The assay used flow cytometry detection and employed the hen egg lysozyme (HEL)-specific 3A9 hybridoma as a model. The original murine IL-2 secretion assay protocol from Miltenyi Biotec® was tested and modified; the conjugated capture antibody (anti-CD45-anti-IL-2) was added together with the stimulus at the beginning of the antigen presentation assay instead of after antigenic stimulation. With this modification, the percentage of detectable CD4⁺IL-2⁺ cells following HEL stimulation rose from 4.5% with the original protocol (0.8% without stimulus) to 94.1% (0.8% without stimulus) with the newly proposed method under the conditions evaluated in this study. This modification allowed us to evaluate the activation of hybridomas directly and more rapidly (~18h) than the reference method that assayed CTLL-2 cell proliferation using the MTT reduction assay (~48h). In conclusion, the proposed method offered a rapid alternative for screening T-cell hybridomas and evaluating their antigen-specific activation.