Ferret TNF-α and IFN-γ Immunoassays (2012)

Despite the prominent use of ferrets in medical research, the immune system of ferrets remains poorly characterized (Svitek & von, V, 2007).Here we describe ferret TNF-and IFN-immunoassays.Specifically, this covers the following topics:  Background: The use of ferrets in medical research  TNF-and IFN-cloning and sequencing  Expression and purification of recombinant ferret TNF-and IFN-proteins  Cytokine real-time PCR based assays  Development of IFN-and TNF-hybridoma clones  ELISA and ELISPOT assays for the ferret cytokine IFN- BackgroundThis Background describes the current use of ferrets in medical research but also includes a description of past uses.The importance of ferrets is highlighted in human disease modeling and in prophylactic and vaccine development.The various uses of ferrets in medical research demonstrate the need for immune profiling reagents and assays. Biology of the ferretThe ferret, Mustela putorius furo, is a relatively small and inexpensive animal in terms of its potential for research use, although mice remain the traditional influenza model for virus pathogenesis.www.intechopen.comTrends in Immunolabelled and Related Techniques 134Essentially, mice are low cost animals that have a broad availability of corresponding reagents for immunological investigation.Mice are easily mutated and there exists a plethora of currently available transgenic mice with immune targeted gene deletions or gene knock-ins (Belser, Szretter, Katz, & Tumpey, 2009).Two factors against the use of mice in influenza immune studies are 1) most human influenza strains must be mouse adapted prior to initiation of infection studies due to the inability of human influenza viruses to replicate in the mouse and 2) mice do not exhibit human-like clinical signs of influenza such as sneezing and temperature fluxes.In contrast to mice, ferrets do develop respiratory illnesses that are similar to human disease.Although the ferret is not considered a large laboratory animal, it is able to provide many biological samples for pathological testing during an infection study.For instance, frequent and sizable blood sampling is feasible in the ferret that is not practical in smaller rodents such as in mice and rats.Furthermore, clinical features of disease are easily observed.Such as clinical fever manifested as an elevation in body temperatures can be detected as early as 1 day following infection with many viruses.Our own as well as previously published studies have shown that high fevers can persist for many days following infection of viruses such as H1N1pdm influenza (Rowe et al., 2010b;Sweet et al., 1979;Zitzow et al., 2002).As well as fever, nasal discharge, sneezing and activity level can also be observed in ferrets infected with influenza viruses (Rowe et al., 2010b).Taken together, these clinical features along with the feasibility for blood and pathological sampling suggest the ferret to be an optimal animal for the study of human infectious diseases, including influenza viruses.For example, our group has recently used the ferret model successfully to characterize and compare immunopathology caused by several strains of currently circulating influenza A and B viruses (Huang et al., 2011).