Cloning, expression and immunoassay detection of ferret IFN- γ

Abstract

SummaryFerrets (Mustela putorius furo) develop symptoms upon influenza infection that resemble those of humans, including sneezing, body temperature variation and weight loss. Highly pathogenic strains of influenza A, such as H5N1, have the capacity to cause severe illness or death in ferrets. The use of ferrets as a model of influenza infection is currently limited by a lack of species-specific immunological reagents. Interferon gamma (IFN-γ) plays a key role in the development of innate and adaptive immunity and the regulation of Th1-type immune responses. Here we describe the cloning of the full-length cDNA for ferret IFN-γ. Multiple sequence alignment of the predicted amino acid sequence with those of other species indicates that the predicted ferret protein shares the highest identity with Eurasian badger IFN-γ. We raised two hybridoma clones expressing monoclonal antibodies against recombinant ferret IFN-γ capable of detecting IFN-γ protein derived from mitogen-stimulated ferret PBMCs by immunoblotting, ELISA and ELISPOT assay. Finally, an ELISA utilizing the ferret-specific antibodies detected elevated levels of IFN-γ in serum samples from H3N2 influenza A-infected ferrets.