Description
Introduction: Interferon-gamma (IFN-gamma) is a dimerized soluble cytokine that is the only member of the type II class of interferons. This interferon was originally called macrophage-activating factor. The IFN-gamma monomer consists of a core of six alpha-helices and an extended unfolded sequence in the C-terminal region. This is shown in the structural models below. The alpha-helices in the core of the structure are numbered 1 to 6. Full length IFN-gamma is 143 amino acids in length, the models are 126 amino acids in length. Affinity for the glycosaminoglycan heparan sulfate resides solely within the deleted sequence of 17 amino acids. In contrast to interferon-alpha and interferon-beta which can be expressed by all cells, IFN-gamma is secreted by Th1 cells, Tc cells, dendritic cells and NK cells. Also known as immune interferon, IFN-gamma is the only Type II interferon. It is serologically distinct from Type I interferons and it is acid-labile, while the type I variants are acid-stable. IFN-gamma has antiviral, immunoregulatory, and anti-tumour properties. It alters transcription in up to 30 genes producing a variety of physiological and cellular responses. Amongst the effects are: Increase antigen presentation of macrophages. Activate and increase lysosome activity in macrophages. Suppress Th2 cell activity. Cause normal cells to increase expression of class I MHC molecules. Promotes adhesion and binding required for leukocyte migration Promotes NK cell activity. Activation by IFN-gamma is achieved by its interaction with a heterodimeric receptor consisting of IFNGR1 & IFNGR2 (interferon gamma receptors). IFN-gamma binding to the receptor activates the JAK-STAT pathway. In addition, IFN-gamma activates APCs and promotes Th1 differentiation by upregulating the transcription factor T-bet. IFN-gamma is the hallmark cytokine of Th1 cells (whereas Th2 cells produce IL-4 and Th17 cells produce IL-17). NK cells and CD8+ cytotoxic T cells also produce IFN-gamma. IFN-gamma suppresses osteoclast formation by rapidly degrading the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway, which otherwise stimulates the production of NFkappaB.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to IFN-gamma. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated monoclonal antibody preparation specific for IFN-gamma and incubated. Then substrate solution A and B are added to each well. Only those wells that contain IFN-gamma, HRP-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of IFN-gamma in the samples is then determined by comparing the O.D. of the samples to the standard curve