Please find the link for the following protocol:
The usage of ELISPOT techniques to measure Cytokines (IFN-g, IL-4, or IL-10)
By: Dr. Mohamed Tarek Shata
Principle of the assay:
The enzyme-linked immunospot (ELISpot) assay was originally developed for the detection of individual B cells secreting antigen-specific antibodies. This method has since been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbant assay (ELISA) technique. A monoclonal antibody specific for a cytokine has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37°C CO2 incubator for a specified period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted cytokine. After washing away cells and any unbound substances, a biotinylated polyclonal antibody specific for the cytokine is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase conjugated to streptavidin is added. Unbound enzyme is subsequently removed by washing and a substrate solution (BCIP/NBT) is added. Blue-black colored precipitate forms at the sites of cytokine localization and appears as spots, with each individual spot representing an individual cytokine-secreting cell. The spots can be counted with automated ELISpot reader systems or manually, using a stereomicroscope.