Development of a monoclonal antibody against duck IFN-γ protein and the application for Intracellular Cytokine Staining

Abstract

ABSTRACTInterferon-{gamma} (IFN-{gamma}), 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-{gamma} protein and to establish an ICS protocol for detecting the duck IFN-{gamma} protein. The duIFN-{gamma}-His or duIFN-{gamma}-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-{gamma} antibodies, and hybridoma cells were obtained after fusion with SP2/0 cells. A new hybridoma cell line named 24H4, which stably secreted IgG3 {kappa} subtype antibody against duck IFN-{gamma}. This monoclonal antibody (mAb) was identified by Western Blot to recognize duck IFN-{gamma} antibodies, and the indirect ELISA results showed that its ability to recognize IFN-{gamma} protein reached 0.001 g/ml. The established ICS method was used to stain PBMCs after ConA stimulation, and duck IFN-{gamma} protein was successfully detected by flow cytometry, indicating that the ICS method was successfully established. This study we use the monoclonal antibody, 24H4, provides a crucial tool for subsequent research on duck cellular immune responses.