The EliSpot (Enzyme Linked Immuno Spot) assay is designed to detect cell-mediated immune reactions to stimuli such as antigens to characterize the immune status and is based on the detection of cytokines or antibodies secreted during an antigen-dependent T- or B-cell reaction.
The B-cell EliSpot assay is designed to detect B-cell-mediated immune reactions to stimuli such as antigens/ bacterial lysate to characterize the immune status and is based on the detection of antibodies during an antigen-dependent B-cell reaction.
In contrast to the T-cell EliSpot, antigens are usually immobilized on the PVDF membrane for stimulation, to which specific antibodies secreted by B cells bind. This binding is determined by enzyme- or fluorophore-coupled detection antibodies.
The FluoroSpot, like the EliSpot, is used to detect cell-mediated immune reactions to stimuli such as antigens to assess the immune status and is based on the same basic principles. The FluoroSpot offers the advantage that multiple analytes can be analyzed in one run. This is made possible by the use of fluorescent dye conjugated antibodies. This allows the simultaneous detection of several cytokines in one approach by selecting non-overlapping spectral ranges of fluorescent dyes.
Finally, the evaluation is performed using an AID EliSpot Reader System, which enables the detection and quantification of fluorescent spots including the differentiation of cell sub-populations like single-, double- or triple-secreting cells.
The enumeration of cytokine or antibody secreting cells is possible through the immobilization of capturing antibodies on PVDF membrane, the stimulation of isolated cells in particular PBMCs with an antigen. Through the recognition of the antigen by a B- or T-cell, specific cytokines / antibodies are released and they bind on the immobilized antibodies. With a secondary-enzyme- or fluorophore-conjugated antibody a successful binding is determined. Each resulting spot stands for one cytokine- or antibody-secreting cell.