Enzyme-Linked Immunospot: An Alternative Method for the Detection of Interferon Gamma in Johne's Disease

Abstract

Abstract. To date, the sensitivity of the interferon gamma (IFN-c) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN-c detection in the early stages of infection, an alternate assay needs to be developed. The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive technique for the detection of cytokines and has the potential to improve the diagnosis of JD. Of the variables examined, choice of capture antibody and the method by which the peripheral blood mononuclear cells were isolated significantly affected the ability to enumerate IFN-c-secreting cells. The ELISPOT assay was as sensitive as or better than the IFN-c ELISA at detecting ovine JD and could also detect disease at early time points postinoculation. The IFN-c ELISPOT could distinguish infected from unexposed animals; however, neither the IFN-c ELISA nor the ELISPOT assay could distinguish between sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis and those exposed to the bacterium but diagnosed as uninfected at necropsy.

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle

“…A combination of lipid transport molecules (van den Elzen et al, 2005) and a selected recombinant antigen pool (Mikkelsen et al, 2011a) could enhance the IFN-γ response at the same time conserving high specificity. Using an enzyme-linked immunospot (ELISPOT) assay, a highly sensitive technique to detect cytokine release, could be an alternative way to improve response detection (Begg et al, 2009). For the animals used in the present study, the IFN-γ responses using PPD-A and L5P did not enable to define a specific status which corresponded to that obtained from the humoral response and faecal culture.…”

Lymphoproliferative and Gamma Interferon Responses to Stress-Regulated Mycobacterium avium subsp. paratuberculosis Recombinant Proteins

“…The IFN-␥ enzyme-linked immunosorbent assay (ELISA) was performed as previously described (25,26). A 96-well MaxiSorp microplate (Nunc, Denmark) was coated with mouse monoclonal anti-bovine IFN-␥ antibody (IFN 6.19) (50 l/well), generously provided by Gregers Jungersen (National Veterinary Institute, Denmark), at a final concentration of 1.5 g/ml in phosphate-buffered saline (PBS).…”

Can early host responses to mycobacterial infection predict eventual disease outcomes?

“…when the animals were sacrificed. The IFNγ assay was carried out using whole blood cultured with MAP-specific antigen (316v) for 48 hrs as previously described (Begg et al, 2009). Reagents for the in-house IFNγ ELISA were: capture antibody, IFN 6.19 (a generous gift from Dr Gregers Jungersen); detection antibody, CC302biotin (Serotec); conjugate, Streptavidin horseradish peroxidase (Vector Labs); substrate, 3,3´,5,5´-tetramethylbenzidine (TMB) (Pierce).…”