Development of a Monoclonal Antibody Against Duck IFN-γ Protein and the Application for Intracellular Cytokine Staining
Yingyi Chen,
Wei Song,
Junqiang Chen,
Chenyang Jin,
Jiewei Lin,
Ming Liao,
Manman Dai
Affiliations
Yingyi Chen
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Wei Song
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Junqiang Chen
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Chenyang Jin
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Jiewei Lin
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Ming Liao
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
Manman Dai
National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
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.
