ICS vs. ELISPOT

Blood cells need to be killed for detection via intracellular cytoplasmic staining, (ICS) whereas they survive ELISPOT assays unaffected. These cells can thus be reused for subsequent testing in other platforms (including ICS itself) or the can be frozen for retesting. If necessary, ELISPOT assays even permit serial testing of the very same cells in different assay formats. This optional maximization of sample resources can be critical when conducting clinical trials in which the number of peripheral blood mononuclear cells available is a limiting factor.

In ICS, cells are killed at some supposedly ideal time point and the intracellular cytokine content is measured. In ELISPOT assays however, the cytokine is continuously captured around the secreting cell during incubation time. This generates the characteristic ELISPOT spots long after the secretion process has halted. This basic feature makes ELISPOT assays largely independent of the secretion kinetics of the individual cells. This is valuable because the kinetics of T cell cytokine secretion is unsynchronized even when a single T cell clone is tested. This principle of nonsychronization is even more relevant when using a potential polyclonal population of effector cells reacting to the diverse APC population.

In addition, ELISPOT assays lend themselves more thorougly to high-throughput testing. The initial phases of both ICS and ELISPOT assays are identical whenit comes to setting up the activation cultures. In addition, the subsequent processing of the cells, including the washing steps, require about the same amount of time. However, the actual data acquisition and analysis phases are much shorter for ELISPOT assays. Typically, ELISPOT footprints generated by 300,000 PBMCs per well can be analyzes from a single image with an acquisition and analysis time of about three seconds per 300,000 cells.