ELISpot and FluoroSpot
ELISpot
If one cell responds, you will find it
The enzyme-linked immunospot (ELISpot) assay is an extremely sensitive immunoassay performed in a 96-well plate format to quantify protein-secreting cells.
The assay principle is straight forward: cells are cultured and stimulated inside a plate coated with capture antibody. Cytokines, immunoglobulins, or other target proteins secreted by the cells are captured immediately upon secretion and throughout the stimulation process. After cell removal, secreted proteins are identified using a detection antibody. Visible spots form after adding a precipitating substrate. Each spot corresponds to an individual analyte-secreting cell.
With minimal manipulation of the cells, ELISpot lies closer to reality than most other cell-based immunoassays down to the single-cell level. Due to capacity to find one cell in a million, ELISpot is particularly valuable for studies of rare cell populations causing immune responses.
FluoroSpot
FluoroSpot is the multiplex version of ELISpot. Just like in the ELISpot assay, target proteins secreted by cells are captured by specific antibodies immediately after secretion and throughout the stimulation process, making it an extremely sensitive sandwich assay. The difference to ELISpot lies in the mode of detection: where ELISpot relies on an enzymatic reaction, FluoroSpot utilizes fluorescence. This opens up for multiplex analyses of several analytes simultaneously, enabling studies of cell populations with different functional profiles.
Like ELISpot, FluoroSpot is useful in e.g., vaccine research and evaluation of cancer immunotherapies, but gives a broader picture of the response by enumerating highly functional triple-secreting IFN-γ/IL-2/TNF-α cells, or killer cells secreting IFN-γ and Granzyme B simultaneously.
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