Development of antibodies to feline IFN-gamma as tools to elucidate the cellular immune responses to FeLV.

An understanding of the nature of immune protection and the role of immune effector products such as interferon-gamma (IFN-gamma) in the control of infectious disease is fundamental to the rational design of effective vaccines and immunotherapeutic reagents. Murine monoclonal and sheep polyclonal antibodies (mAbs and pAbs) to feline IFN-gamma (fIFN-gamma) were generated firstly to facilitate further research into the role of cellular immune responses in the control of feline infectious disease, and secondly to enable evaluation of the efficacy of novel immunotherapeutic approaches. A hybridoma clone, D9, secreting IgG1 antibodies was selected for expansion and the mAbs affinity purified in vitro. Polyclonal antibodies were raised in a sheep against recombinant fIFN-gamma and affinity purified. The sensitivity of the D9 mAb and the sheep anti-fIFN-gamma pAb was determined using an indirect fIFN-gamma enzyme-linked immunosorbent assay (ELISA) and immunoblots. These antibodies were assessed for their ability to detect the production of fIFN-gamma by specific feline T cell populations ex vivo following coculture with mitogen or feline leukaemia virus (FeLV) antigens for 4 h in the presence of the protein secretion inhibitor brefeldin A (BFA). Production of fIFN-gamma was evaluated using flow cytometry to simultaneously detect PE-labelled surface molecules and fluorescein isothiocyanate (FITC)-labelled intracellular fIFN-gamma. Using this approach, our initial studies revealed an upregulation in virus-specific fIFN-gamma-secreting CD4(+)T cells in the lymph nodes of FeLV latently infected cats.