ELISpot

The enzyme-linked immune absorbent spot (ELISpot) is a type of assay that focuses on quantitatively measuring the frequency of cytokine secretion for a single cell. The ELISpot Assay is also a form of immunostaining since it is classified as a technique that uses antibodies to detect a protein analyte, with the word analyte referring to any biological or chemical substance being identified or measured.
The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses fluorescence in order to analyze multiple analytes, meaning it can detect the secretion of more than one type of protein.
History.
Cecil Czerkinsky first described ELISpot in 1983 as a new way to quantify the production of an antigen-specific immunoglobulin by hybridoma cells.
In 1988, Czerkinsky developed an ELISA spot assay that quantified the secretion of a lymphokine by T cells.
In the same year, dual-color ELISpot was combined with computer imaging for the first time, which allowed for the enumeration and analysis of spots.
1988 also marked the first use of membrane-bottomed plates for performing these assays.
Mechanism of ELISpot.
Mechanism of FluoroSpot.
The FluoroSpot assay is very similar to the ELISpot assay. The main difference is that the FluoroSpot assay is able to analyze the presence of multiple analytes on one plate of wells, whereas the ELISpot assay can only analyze one analyte at a time. The FluoroSpot assay accomplishes this by using fluorescence rather than an enzymatic reaction for detection. The steps for a FluoroSpot assay are also similar, with a few differences.
Since the FluoroSpot assay identifies and quantifies the presence of multiple analytes, it is possible that the absorption of one analyte can affect the secretion of another analyte; this is called capture effects. The affect an analyte has on another analyte could be positive or negative (the production of the second analyte can either increase or decrease). To counteract capture effects, it is possible to use co-stimulation in order to bypass the decreased production of an analyte. This is when a second antibody that stimulates the production of the same analyte is added to the wells.
Applications of ELISpot and FluoroSpot.
The ELISpot and FluoroSpot assays can be used in many research fields: vaccine development, cancer, allergies, monocytes/macrophages/dendritic cells characterization, apolipoproteins analysis, and veterinary research. With the ELISpot, you can study antigen-specific cytokine responses, antibody specific secreting cells, tumor antigens, granzyme B and Perforin release by T cells, vaccine efficacy, epitope mapping, cytotoxic T-cell activity, detection of IL-4, IL-5, and IL-13, vaccine-induced antibody responses, antigen-specific memory B cells, and much more.
More specifically, the T-cell ELISpot assay is used to characterize T-cell subsets. This is because the assay can detect the production of cytokines IFN-y, IL-2, TNF-alpha, IL-4, IL-5, and IL-13. The first three cytokines are produced by Th1 cells, while the last three are produced by Th2 cells. Measuring T-cell responses through cytokine production also makes it possible to study vaccine efficacy.
With T-cell FluoroSpot, you can monitor tumor-infiltrating lymphocytes. You can also analyze the IFN-y cytokine and granzyme B secretion in order to assess cytotoxic T-cell responses. Both of these are used for cancer research.
With B-cell FluoroSpot, vaccine efficacy can also be observed by quantifying the secretion of IgG, IgA, and IgM before and after a vaccination. This analysis of multiple immunoglobulins is made possible because of the fluorescence method used in the FluoroSpot.