Description
Key facts
Sample types:Suspension cells,
Reacts with:Human,
Assay type:Sandwich (qualitative),
Assay Platform:Reagents
Product details:
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, oncology, 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.
The dual colour ELISPOT allows you to monitor the production of two cytokines simultaneously in the same well.
Principle of the Method
After cell stimulation, locally produced cytokines are captured by IFN gamma and Granzyme B specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti-IFN gamma FITC conjugated antibody and a biotinylated anti-Granzyme B antibody. Those are in turn recognised by anti-FITC HRP and streptavidin-AP conjugates. PVDF-bottomed-well plates are then incubated first with AEC substrate buffer, washed and subsequently incubated with BCIP/NBT. Coloured red/brownish spots indicate IFN gamma production while Granzyme B is revealed by blue/purple spots.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-+4°C, Appropriate long-term storage conditions-+4°C, Storage information-+4°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Interferon Gamma (IFN-γ) and Granzyme B are important components of the immune response. IFN-γ also known as Type II interferon is a dimerized soluble cytokine with a mass of around 20 kDa. It is primarily expressed by activated T cells and natural killer (NK) cells. Granzyme B is a serine protease with a mass of approximately 32 kDa found in the granules of cytotoxic T lymphocytes (CTLs) and NK cells. Both these proteins are critical for host defense especially in viral infections and cancer.
Biological function summary
IFN-γ activates macrophages enhances antigen presentation and orchestrates the Th1 immune response by inducing the expression of MHC class II molecules. It is not part of any protein complex. Granzyme B on the other hand induces apoptosis in target cells by cleaving caspases and various cellular substrates. It operates as part of the cytotoxic granule complex alongside perforin which forms pores in target cell membranes to facilitate Granzyme B entry.
Pathways
IFN-γ plays an important role in the JAK-STAT signaling pathway which is fundamental for its action in immune modulation. Through JAK-STAT it works closely with STAT1 a specific transcription factor. Granzyme B is involved in the induction of apoptosis through the mitochondrial pathway where its activity intersects with Bid a pro-apoptotic Bcl-2 family member. These proteins contribute significantly to the regulation and execution of immune responses.
IFN-γ and Granzyme B are implicated in autoimmune diseases and cancer. Elevated levels of IFN-γ link to autoimmune pathologies like rheumatoid arthritis often correlated with proteins such as TNF-α. Granzyme B has associations with cancer prognosis; its expression levels can affect tumor cell death and immune surveillance. In certain cancers upregulation of inhibitors like PI9 can prevent Granzyme B function leading to tumor evasion from the immune system.
