To determine the reactivity of the lymphocytes to a specific antigen, PBMCs have to be isolated of fresh blood by density gradient centrifugation, already isolated PBMCs or other single cells are stimulated directly in a microtiter plate coated with a cytokine-specific primary antibody. During the stimulation period, secreted cytokines bind specifically to the primary antibody immobilized on the membrane bottom of the plate.
After the incubation period, the plate is washed to remove the cells and unbound cytokines. A cytokine-specific alkaline phosphatase-conjugated secondary antibody is then added to detect cytokine binding. After addition of a substrate (BCIP/NBT), enzymatic conversation into an insoluble blue precipitate occurs.
Since the cytokine to be detected is bound directly at the site of secretion, the precipitate, a so-called spot, appears at the sites where activated lymphocytes were found. Each spot thus represents the cytokine secretion of a single cell. The number of spots can be interpreted as a measure of the reactivity of the immune system to a specific stimulus.
The evaluation is performed by an AID EliSpot Reader System, which enables the automated detection and quantification of the spots as well as the export of the data.
The results of an EliSpot/FluoroSpot assay can be evaluated automatically with an AID EliSpot Reader System. The AID Reader Systems acquire an image of each well of the microtiter plate. Subsequently, images are analyzed depending on assay specific Count Settings, applying individual algorithms and threshold values e.g. spot size and -intensity.
The advantages of the assay evaluation with an AID Reader Systems are:
For an EliSpot result to be valid, the controls carried must meet certain acceptance criteria.
First of all, all approaches have to be checked if the software has recognized all spots and secondly if there are artifacts like fluff or hairs in the wells.
After checking these points, the controls are checked. The blank, consisting of cell and medium only, must not contain spots (or only a few). The positive control, consisting of cells and an unspecific mitogen, should show a strong reaction to prove the reactivity of the T- or B-cells and especially to prove lymphocyte activity in immunosuppressed individuals.
In the case of antigen-specific approaches, individual threshold values are set in order to be able to judge reactivity to the specific antigen. For complex questions to clarify whether an acute or latent infection is present, an evaluation matrix is sometimes required for the evaluation.
The number of spots can be interpreted as a measure of the reactivity of the immune system to a particular stimulus, allowing an interpretation of the immune response. These results have to be coordinated with the clinical symptoms possibly with further diagnostic methods in order to establish a comprehensive, correct diagnosis. Especially in case of complex diseases, like Lyme disease or TB, where different influences/cross-reactions can occur.