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The ELI-Spot assay (enzyme-linked immunosorbent spot assay) is used for the visualization of antigen-specific immune cells that secrete specific antibodies or cytokines. Thus, the cellular immune response can be examined using an antigen-stimulated population of peripheral blood mononuclear cells (PBMC).
First, an opaque membrane is (usually made of PVDF) in a 96-well microtiter plate is coated with a mono- or polyclonal primary antibody. Thereafter, a stimulus (e.g. an antigen or peptide) and (cytokine- or antibody-secreting) immune cells are added. The stimulus leads to secretion of the cytokines or antibodies. The immobilized capture antibodies on the membrane then bind to the secreted cytokines or antibodies. Non-specific protein binding sites of the membrane are blocked by sterile FCS or BSA solutions. The direct detection is mediated by a detection antibody, which can be conjugated with e.g. alkaline phosphatase (AP) or horseradish peroxidase (HRP).
After the addition of a substrate, these enzymes catalyze a colorimetric measurable color change. Nevertheless, detection by means of fluorescence-labeled antibodies is also possible and widely used. An indirect detection is possible by using a biotin-conjugated antibody for the analyte binding. This is followed by incubation with a streptavidin-conjugated enzyme that, analogously as for the direct detection, catalyzes a color change. The individual reactive cells are visualized after a vibration-free incubation as clear points (ImmunoSpots). Shaking of the assay during incubation would lead to a lateral movement of non-adherent cells (suspension cells) and thus falsely increase the number of spots.
The analysis of ELI-Spot assay by means of a reader can be carried out both qualitatively (secreted cytokines) and quantitatively (number of labeled cells).