# Development of a Monoclonal Antibody Against Duck IFN-γ Protein and the Application for Intracellular Cytokine Staining

## TL;DR

This paper describes the development of a monoclonal antibody against duck IFN-γ and its use in a new method to study immune responses in ducks.

## Contribution

The novel monoclonal antibody 24H4 and its application in intracellular cytokine staining for duck IFN-γ detection.

## Key findings

- A monoclonal antibody (24H4) against duck IFN-γ was successfully produced and characterized.
- An intracellular cytokine staining method using 24H4 was established and validated in duck PBMCs.
- The antibody detected IFN-γ with high sensitivity (0.001 μg/mL) in ELISA.

## Abstract

Interferon-γ (IFN-γ) is a crucial cytokine in the immune system and serves as an important indicator of immune response. However, there are no commercially available monoclonal antibodies against duck IFN-γ. Intracellular Cytokine Staining (ICS) is a technique used to analyze the production of cytokines within individual cells. This study developed a monoclonal antibody against duck IFN-γ protein and established an ICS method using this antibody. This study will provide a crucial tool for the research of duck cellular immunity.

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.