The IL-12 p70 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, oncology, infectious diseases, autoimmune diseases and transplantation
Principle of Method
The IL-12 p70 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 a secondary biotinylated detection antibody, which is in turn recognised by streptavidin conjugated to alkaline phosphatase. PVDF-coated-well plates are then incubated with BCIP/NBT substrate. Coloured "purple" spots indicate cytokine production by individual cells.
Interleukin 12 is a cytokine that acts on T and natural killer cells, and has a broad array of biological activities. Interleukin 12 is a disulfide-linked heterodimer composed of the 35 kD cytokine receptor like subunit encoded by IL12A gene, and a 40 kD subunit encoded by IL12B.
This cytokine is expressed by activated macrophages that serve as an essential inducer of Th1 cells development. It is required for the T-cell-independent induction of interferon (IFN)-gamma, and is important for the differentiation of both Th1 and Th2 cells.
This cytokine has been found to be important for sustaining a sufficient number of memory/effector Th1 cells to mediate long-term protection to an intracellular pathogen. The responses of lymphocytes to this cytokine are mediated by the activator of transcription protein STAT4. Nitric oxide synthase 2A (NOS2A/NOS2) is found to be required for the signaling process of this cytokine in innate immunity.
Overexpression of this gene was observed in the central nervous system of patients with multiple sclerosis (MS), suggesting a role of this cytokine in the pathogenesis of the disease. The promoter polymorphism of this gene has been reported to be associated with the severity of atopic and non-atopic asthma in children.