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.
Abcam 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.
IL6 is a pro-inflammatory cytokine secreted by T cells and macrophages to stimulate immune response to trauma, especially burns or other tissue damage leading to inflammation.
IL6 signals through a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL6R alpha chain (CD126), and the signal-transducing component gp130 (also called CD130). CD130 is the common signal transducer for several cytokines including leukemia inhibitory factor, ciliary neurotropic factor, oncostatin M, IL11 and cardiotrophin-1, and is almost ubiquitously expressed in most tissues. In contrast, the expression of CD126 is restricted to certain tissues. As IL6 interacts with its receptor, it triggers the gp130 and IL6R proteins to form a complex, thus activating the receptor. These complexes bring together the intracellular regions of gp130 to initiate a signal transduction cascade through certain transcription factors, Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs).
In addition to the membrane-bound receptor, a soluble form of IL6R (sIL6R) has been purified from human serum and urine. Many neuronal cells are unresponsive to stimulation by IL6 alone, but differentiation and survival of neuronal cells can be mediated through the action of sIL6R. The sIL6R/IL6 complex can stimulate neurites outgrowth promote survival of neurons, hence may be important in nerve regeneration through remyelination.