Discover the power of Assay Genie ELISpot Assay Kits—offering several advantages over traditional ELISA, including exceptional sensitivity and the unique ability to detect and visualize immune responses at the single-cell level.
The enzyme-linked immunospot (ELISpot) assay is a universal method for monitoring immune responses in vitro. ELISpot is an effective immunoassay tool due to its high sensitivity. It is used for the ex vivo measurement of cytokine or antibody secreting cells at the single-cell level following activation with a suitable stimulus in vitro.
ELISpot allows for the visualization of the secretory product of individually activated or responding cells. Each cell can be detected by the assay as long as a characteristic protein is released and specific high affinity antibodies recognizing the protein are available.
The cells of interest are both cultured and activated on plates immobilised with antibodies that capture the associated target analytes released by the stimulated cells. Following target analyte capture, the presence and distribution of these molecules are detected through a sandwich ELISA. A reporter enzyme is added where the analyte is captured. Each spot that develops in the assay represents a single reactive cell.
In the ELISpot assay, cells are cultured on a 96-well plate coated with the appropriate capture antibody to the protein of interest, in the presence or absence of stimuli. Once the cells are stimulated, they release proteins (e.g. cytokines). These secreted proteins are then trapped by the immobilized antibodies coated on the surface.
Following a period of incubation, the cells are washed away, and the secreted proteins are detected using a detection antibody. The ELISpot detection process employs the same immunochemical ‘sandwich’ principle as an ELISA. In contrast to the ELISA method, ELISpot is a combination of both an immunoassay and bioassay. This is due to living cells being cultured directly within the wells of the ELISpot plate.
The detection antibody is typically a biotinylated polyclonal antibody specific for the analyte of interest. The biotinylated antibody is added to each of the wells and the wells are then washed to remove any unbound detection antibody. Next, a streptavidin-enzyme conjugate is added, or the antibody is simply conjugated to the enzyme. Unbound streptavidin-enzyme is then washed away, and a precipitating substrate is added. When a precipitating substrate is used instead of a soluble product, the result appears as visible spots. Each spot corresponds to an individual cytokine or antibody-secreting cell. The resulting spots can be quantified using an automated ELISpot reader system or manually, using a stereomicroscope (see the Figure. 1 below).
Figure 1 Schematic of ELISpot assay protocol
Frequently Asked Questions
An Enzyme-Linked Immunosorbent Assay (ELISA) determines the total concentration of the secreted signalling protein or antibody. In contrast, an ELISpot assay analyses individual cytokine or antibody secreting cells. ELISpot gives a quantitative result of the frequency of the secreting cells.
ELISpot assay is typically used to identify cytokine secretion by antigen-activated T cells and antibody secretion by B cells from peripheral blood or spleen cells.
ELISpot is the appropriate assay for determining the frequency of secreting cells, since it detects cytokines or antibody secreting cells at an individual level. Therefore, ELISpot is most suitable when used in addition to an ELISA. An ELISpot assay can be 100-400 times more sensitive than a standard ELISA.
The results of an ELISpot assay depends on high cell density. A high number of cells increases the likelihood of contact between stimulating and responding cells. The cell density should typically range between 1-2 X 105 cells per well, and this may require some optimization.
Store kit reagents between 2°C and 8°C. Immediately after use, remaining reagents should be returned to cold storage (2°C to 8°C). Expiry of the kit and reagents is stated on box front labels. The expiry of the kit components can only be guaranteed if the components are stored properly, and if in the case of repeated use of one component, the reagent is not contaminated by the first handling.
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