Validation of Ferret Luminex Cytokine Assays with serum from ferrets infected with flu, stimulated whole blood and PBMCs

Abstract

Abstract Ferrets represent excellent models of infectious disease due to their natural susceptibility to many human respiratory viruses including influenza A virus (IAV), respiratory syncytial virus and severe acute respiratory syndrome (SARS). Ferrets can reproduce a similar course of disease and display similar symptoms of respiratory infection as humans, while mice do not exhibit key features of disease such as sneezing, nasal discharge and fever. There is pressing need to develop reagents to better understand and characterize the immune response to infection and support biomarker and countermeasure development through immunologic analyses of respiratory infections in the ferret. IL-1β, IL-2, IL-5, IL-6, IL-12 p40, IL-12p35, IL-17, IFNγ, TNFα, & IP-10 were expressed in HEK293 cells and purified. Monoclonal antibodies were generated and screened by Luminex for matched pairs to the recombinant protein and presumed positive samples. Capture antibodies were coupled to Luminex beads and detection antibodies were biotinylated. Serum from ferrets infected with flu had 10-fold higher levels of IP-10 and 3-fold higher levels of IL-6 than control serum. Stimulation of ferret whole blood with LPS showed increases in IL-2 and IL-6 at 2, 4 and 24 hrs. IL-12p40 and TNFα increased at 24hrs after stimulation with LPS. A validated cytokine panel in the study of immune related diseases in the ferret model. Supported by SBIR contract 75N93020C00022: Development of Ferret Reagents for use in the Characterization of Immune Responses to Respiratory Infections in the Ferret Model