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ELISPOT, or enzyme linked immunospot, is a technique that was developed for the detection of secreted proteins, such as cytokines and growth factors. It is most commonly used in immunology research. For example, ELISPOT can be used to understand the biological effects involved in:
ELISPOT is performed using a PVDF or nitrocellulose membrane 96-well plate pre-coated with an antibody specific to the secreted protein. Cells are added to the plate and attach to the coated membrane. Cells are then stimulated and the secreted protein binds to the antibody. Next, a detection antibody is added that binds specifically to the bound protein.
The resulting antibody complex can be detected either through enzymatic action to produce a colored substrate or with fluorescent tags. An advantage to using fluorescence is the ability to identify more than one secreted protein at a time.
The membrane can be analyzed by manually counting the spots or with an automated reader designed for this purpose. Each secreting cell appears as a spot of color or fluorescence, thus this is a quantitative method for evaluating protein secretion.
General ELISPOT procedure
In the analysis software, set the following parameters for measurement:
These parameters can be saved and used for subsequent experiments for standardized results.
We recommend reading each plate 3 times and averaging the results in order to minimize error in the measurements.
If the cells take some time to respond to stimulation, they may require pretreatment with the stimulant in a separate 96-well culture dish before transferring to the ELISPOT plate.
Experiments to detect cytokines using ELISPOT will require use of positive control. In these positive control wells, the cells should be stimulated with an agent known to induce expression of the cytokine being detected. This can then be used to compare to the negative control (no treatment or stimulation of a different secreted protein).
Ensure you are stimulating the PBMCs with the correct stimulant for detection of your target cytokine.
Typical stimulates include: