Abstract
Interferon-γ (IFN-γ), a member of the Type II IFN family, is a crucial cytokine in the immune system and serves as an important indicator of immune response. Intracellular cytokine staining (ICS) is a technique used to analyze the production of cytokines within individual cells, and it has a wide range of applications in the fields of immunological monitoring, vaccine trials, and the study of infectious diseases. This study aimed to prepare monoclonal antibodies against duck IFN-γ protein and to establish an ICS protocol for detecting the duck IFN-γ protein. The duIFN-γ-His or duIFN-γ-Fc gene was cloned into the pEE12.4 expression vector and expressed as a recombinant protein of size 20.2 KDa or 54.9 KDa in 293F cells. The purified recombinant proteins were inoculated into BALB/c mice to generate splenic lymphocytes capable of secreting anti-duIFN-γ antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named 24H4, which stably secreted IgG3 κ subtype antibody against duck IFN-γ, was established. This monoclonal antibody (mAb) was identified by Western blot to recognize duck IFN-γ antibodies, and the indirect ELISA results showed that its ability to recognize IFN-γ protein reached 0.001 μg/mL. The established ICS method was used to stain PBMCs after Concanavalin A (ConA) stimulation, and duck IFN-γ protein was successfully detected by flow cytometry, indicating that the ICS method was successful. In this study, we provide a crucial tool for subsequent research on duck cellular immune responses by using the monoclonal antibody 24H4.