FluoroSpot Assay Principle
The FluoroSpot assay enables simultaneous measurement of different analytes secreted at the single-cell level. This method, in which mixtures of monoclonal antibodies (mAbs) with different specificities are used in a sandwich assay, is a modification of the ELISpot assay. Like the ELISpot, the FluoroSpot assay is sensitive, robust, and easy to perform; unlike the ELISpot, the FluoroSpot method employs fluorophore-labeled detection reagents. A biotinylated detection antibody is used for one analyte, and a tag-labeled detection antibody for the second (and the third). This design enables analysis of two or more analytes in the same well. The detection step is amplified by addition of specific reagents conjugated to different fluorophores. Spot analysis is performed with an automated fluorescence reader that generates separate images for the single fluorophores. Spots, derived from cells secreting more than one analyte, are identified by analysis of co-positioned spots in a digital overlay of the single-fluorescent images.
Co-stimulation with anti-CD28 can be useful in FluoroSpot for T-cells.
Anti-CD28 mAb provides a co-stimulatory signal to antigen-specific responses by binding to CD28 on T cells. Addition of an anti-CD28 mAb to the cell culture can be used to enhance antigen-specific responses. Further optimization may be necessary, depending on the cells and the stimuli which are used. Too high concentration of the anti-CD28 mAb may result in an elevation of non-specific cytokine secretion.
Cytokine absorption by coated antibodies.
Capture effects, a phenomenon where the adsorption of one cytokine may affect the secretion of other cytokines, may occur when multiple capture antibodies are coated in the same well. For example, the presence of IL-2 capture antibodies may result in reduced activation of T cells, as capturing of IL-2 reduces the overall amount of available IL-2 that may stimulate the T cells to further cytokine release. Cytokine capture may also have the opposite effect, resulting in increased production of other cytokines. For example, elevated cytokine secretion is observed when cells are deprived of IL-10.
Most often, capture effects are not a problem and, if present, they can be counteracted (see below). However, in some cytokine combinations, the capture of one cytokine may almost completely abrogate secretion of the other. One such example is IL-2 + IL-5 (see below).
Co-stimulation may counteract capture effects.
To circumvent the attenuating effect of e.g. IL-2 capture, an anti-CD28 mAb can be added to provide a co-stimulatory signal to antigen-specific responses by binding to CD28 on T cells. This measure restores e.g. IFN-γ responses, but is usually not sufficient to restore IL-5 secretion.
Possible capture effects and co-stimulation may require individual evaluation regarding the number of cells per well, the type of stimuli used and the analytes studied. As mentioned above, the anti-CD28 mAb may, in high concentrations, elevate non-specific cytokine secretion, while very low concentrations of the mAb may be insufficient to prevent the attenuation of the response. The co-stimulatory effect of the anti-CD28 mAb, as well as a possible impact on non-specific responses, can be assessed by comparing cells cultured with or without the anti-CD28 mAb.
1) IL-2 secreted by the activated T cell is captured by coated anti-IL-2 capture antibodies. 2) As a result, IL-2-stimulation of the T cell itself (autocrine stimulation) as well as nearby T cells (paracrine stimulation) is impaired, ultimately leading to (3) decreased IFN-γ secretion.
An automated reader with separate filters is recommended for analysis.
The automated reader for FluoroSpot should be equipped with filters for the fluorophores used: FITC (excitation 490 nm/ emission 510 nm), Cy3 (excitation 550 nm/emission 570 nm) and for three-color analysis also Cy5 (excitation 640 nm/ emission 660 nm). Filters should be selective for the specific wavelengths to avoid bleed-through artifacts. In order to obtain accurate measurements of cells secreting multiple analytes, it is required that the automated reader can create an overlay analysis from the filters. Fluorescent spots may fade due to excessive exposure to light and it is recommended to analyze the plates within one week of development.