Elispot

Elispot assays

1. ELISPOT ASSAYS

2. “ELISPOT represents the most sensitive technology for the detection of immune cells secreting signature proteins, such as cytokines. It therefore represents the “state-of-the-art” for the detection, measurement, and functional analysis of immune cells”

3. ELISPOT assays measure the secretory activity of individual cells. The unique sensitivity of ELISPOT assays vs. supernatant-based measurements, ( e.g ELISAs, protein/cytokine arrays, or cytokine bead arrays (CBAs) results from the fact that the analyte is captured directly around the secreting cell as it is being released, before it can be diluted in the supernatant, absorbed by receptors of adjacent cells, or degraded by proteases. ELISPOT assays are also orders of magnitudes more sensitive for detecting rare cells than flow cytometry-based techniques such as tetramer or intracytoplasmic cytokine staining (ICS). Since antigen-specific lymphocytes frequently occur in PBMC in frequencies too low for detection by other techniques, ELISPOT assays have emerged as the most sensitive and robust technique for direct ex vivo monitoring of T and B cell immunity. By measuring the frequencies of the antigen-specific cells and the types of molecules these lymphocytes secrete, ELISPOT assays not only establish the magnitude (clonal size) but also the quality (effector class, e.g. Th1, Th2, Th3, etc.) of antigen-specific immunity.

4. Principle ELISpot assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. Either a monoclonal or polyclonal antibody specific for the chosen analyte is 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 CO 2  incubator for a specified period of time. During this incubation period, the immobilized antibody, in the immediate vicinity of the secreting cells, binds secreted analyte. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for the chosen analyte 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. A blue-black colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual analyte-secreting cell. The spots can be counted with an automated ELISpot reader system or manually, using a stereomicroscope.