or measuring, a biotinylated detection antibody is added (this is specific for one type of analyte that you wish to quantify) and then tag-labeled detection antibodies are added for the second and third types of analytes being studied. Fluorophore-labeled Conjugates: Instead of adding a streptavidin-enzyme conjugate, the detection of multiple analytes is amplified in the FluoroSpot with the use of fluorophore-labeled anti-tag antibody and streptavidin-fluorophore conjugate. A fluorescence enhancer solution is also added during this step in order to enhance the signals later used when analyzing the fluorescence colors in the wells. This fluorescence is what makes it possible for the FluoroSpot to analyze and compare multiple analytes, unlike the ELISpot. Analysis: Since the FluoroSpot relies on the use of fluorescence and not an enzymatic reaction, there is no need for a step that adds a substrate to react with enzymes (as needed for the ELISpot). The last step for the FluoroSpot assay is to analyze the fluorophores under an automated fluorescence reader that has separate filters for the different fluorophores being analyzed. These filters should be selected for the specific wavelengths of the fluorophores if you want accurate measurements. 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,