title: Improved spectrophotometric human interferon-gamma bioassay.
abstract: Interferon gamma (IFNγ) is a cytokine involved in many anti-viral and immunoregulatory processes. One of the major mechanisms through which IFNγ exerts these effects is by inducing expression of indoleamine 2,3 dioxygenase-1 (IDO1), an enzyme that catalyses the first, rate-limiting step of the kynurenine pathway. In this pathway, tryptophan can be catabolised to many products, including picolinic acid and nicotinamide adenine dinucleotide. However, in endothelial cells, the pathway ends at the production of kynurenine. This is due to little or no expression of enzymes that metabolise kynurenine. Production of kynurenine has been used as an indicator of human IDO1 activity, and hence as an hIDO1 bioassay. Due to IFNγ's ability to induce IDO1 expression, kynurenine production can also be a measure of human IFNγ (hIFNγ) bioactivity. Previously, the levels of hIFNγ have been commonly determined by anti-viral assays, high performance liquid chromatography and ELISA. Apart from their technical complexity, these assays are costly and only the anti-viral assay measures bioactive IFNγ. Here, we report the development of an improved IFNγ spectrophotometric bioassay using a human brain endothelial cell line (HBEC 5i). The method is sensitive, easy to perform and cost efficient.
authors.affiliation: School of Medical Sciences (Pathology) and Bosch Institute, Faculty of Medicine, University of Sydney, NSW 2006, Australia.
authors.forename: Felicita Fedelis
authors.initials: FF
authors.lastname: Jusof
authors.forename: Loke Tim
authors.initials: LT
authors.lastname: Khaw
authors.forename: Helen J
authors.initials: HJ
authors.lastname: Ball
authors.forename: Nicholas H
authors.initials: NH
authors.lastname: Hunt
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000378:metabolism; D007371:Interferon-gamma / Q000032:analysis*; D007737:Kynurenine / Q000032:analysis; D013053:Spectrophotometry / Q000379:methods*
chemical_list: C576710:IDO1 protein, human; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007737:Kynurenine; D007371:Interferon-gamma
keywords: Bioassay; Indoleamine 2,3-dioxygenase 1 (IDO1); Interferon gamma