The Elispot assay is designed to enumerate cytokine producing cells in a single cell suspension. This method has the advantage of requiring a minimum of in-vitro manipulations allowing cytokine production analysis as close as possible to in-vivo conditions in a highly specific way. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation, and the follow-up of such frequency during a treatment and/or a pathological state. Elispot assay constitutes an ideal tool in the TH1 / TH2 response, vaccine development, viral infection monitoring and treatment, cancerology, infectious diseases, autoimmune diseases and transplantation.
This Elispot assay is based on sandwich immuno-enzyme technology. Cell secreted cytokines or soluble molecules are captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured cytokines are revealed by tracer antibodies and appropriate conjugates.
After cell stimulation, locally produced cytokines are captured by a specific monoclonal antibody. After cell lysis, trapped cytokine molecules are revealed by a secondary biotinylated detection antibody, which is in turn recognised by streptavidin conjugated to alkaline phosphatase. PVDF-bottomed-well plates are then incubated with BCIP/NBT substrate. Colored "purple" spots indicate cytokine production by individual cells.
Interferon gamma is a dimerized soluble cytokine that is the only member of the type II class of interferons.
In contrast to interferon alpha and interferon beta which can be expressed by all cells, Interferon gamma is secreted by T lymphocytes and NK cells only. Also known as immune interferon, Interferon 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.
Interferon gamma has antiviral, immunoregulatory, and anti-tumour properties. It alters transcription in up to 30 genes producing a variety of physiological and cellular responses. Activation by Interferon gamma is achieved by its interaction with a heterodimeric receptor consisting of IFNGR1 & IFNGR2 (interferon gamma receptors). Interferon gamma binding to the receptor activates the JAK-STAT pathway. In addition, Interferon gamma activates APCs and promotes Th1 differentiation by upregulating the transcription factor T-bet.
Interferon gamma is the hallmark cytokine of Th1 cells (Th2 cells produce IL-4). NK cells and CD8+ cytotoxic T cells also produce Interferon gamma. Interferon gamma suppresses osteoclast formation by rapidly degrading the RANK adaptor protein TRAF6 in the RANK-RANKL signaling pathway, which otherwise stimulates the production of NF?B.