ELISPOT Assay (Dot ELISA)

Theoretical Background / Context of ELISpot immunoassay

• ELISPOT, or enzyme-linked immunospot, is a technique that was developed for the detection and quantification of cells that secrete proteins, such as cytokines and growth factors. 
• ELISPOT assay protocol is most commonly used in immunology research. For example Transplantation, vaccine development (IFNγ), viral infection monitoring, and treatment, Th1/Th2, T cell regulation, and monocyte and dendritic cell analyses. 
• EliSPOT technique can also be used to study autoimmune diseases, allergies, and cancer.

ELISPOT Assay Principle of Work

• ELISPOT analysis is performed using a PVDF (or nitrocellulose) membrane 96-well plate, which is pre-coated with an antibody specific to the secreted protein. 
• Samples (including cells) are added to the plate. Cells are then stimulated to secrete their proteins. 
• The secreted protein binds to the coating antibody. A detection antibody is then added. It is specific to the bound protein. 
• Detection of the antibody complex can be done either enzymatically by the production of a colored substrate, or,  fluorescently. 
• Fluorescent tags can identify more than one secreted protein at a time.
• Each secreting cell appears as a spot of color or fluorescence, this allows quantitative evaluation of cells secreting proteins. 
• The membrane can be analyzed either by manually counting the spots or by an automated reader.