Immune monitoring technology primer: the enzyme-linked immunospot (Elispot) and Fluorospot assay

Description of the technology Elispot is a workhorse for functional analysis of single immune cells. Originally established for the enumeration of specific antibody-secreting B cells in 1983 [1], the technology has been expanded to the analysis of T cells, NK cells and monocytes. The assay is typically carried out in 96 well plates with a nitrocellulose or PVDF membrane, which is coated with an antibody that is directed against the analyte of interest. Cells and stimulants are added to the plate, and secreted analyte is captured in the immediate surroundings of the cell. After cell removal, bound analyte is made visible via the addition of a biotinylated secondary antibody, avidinenzyme complex and substrate that precipitates onto the membrane and forms spots which can be automatically enumerated. Each spot represents one cell that secreted the analyte. The most common analytes investigated today are cytokines (IFNɣ, IL-2, IL-5, IL-10, IL-17, Granzyme B, TNF, GM-CSF and many more) and immunoglobulins. Others can also be evaluated, such as chemokines (e.g., CXCL8) or apolipoproteins. The hallmark of the assay is its sensitivity allowing the detection of antigen-specific immune cells in very low frequencies. In contrast to other assays, Elispot measures cells that actually secrete analytes without impairment by receptor binding or protease activity. The assay is easy to perform and transferable, and can be adapted to high throughput. While it was historically limited to the assessment of one parameter per assay, it has now been expanded to the simultaneous assessment of 2 or even 3 parameters in one well by the introduction of fluorescent dyes in the detection cascade (Fluorospot), allowing the identification of up to 7 subsets of immune cells in