ELISPOT Protocol

The Enzyme Linked Immunospot technique was developed by Cecil Czerkinskdy in 1983. ELISPOT is used for the detection of secreted proteins, such as cytokines and growth factors. It is therefore used primarily in immunology research in the following areas: • Transplantation – prediction of infectious risk • Vaccine development (IFN gamma) • Th1/Th2 analysis • Autoimmune disease • Cancer – tumor antigens • Allergy • Viral infection monitoring and treatment Cells are grown in 96 well plates with antibody-coated PVDF or nitrocellulose membranes to the wells. First, the membrane is coated with primary antibody. Next, the cells are grown on this membrane and the secreted protein of interest will bind to the antibody. When this is detected using secondary antibody, the protein will be seen in the locality of that secreting cells as a spot of colour (one spot = 1 cell). The membranes are then scanned in and analyzed using one of various software programs that are available for this purpose. These are able to quantify the number and percentage of cells secreting the protein. Therefore, immune responses to various stimuli can be quantified and compared.