ELISpot
Count cytokine-secreting cells one by one. ELISpot detects the protein released by each responding cell, so you measure the frequency of antigen-specific T cells and other secreting cells, not just the total amount of cytokine.
Complete kits with pre-coated PVDF plates, detection antibody, alkaline phosphatase conjugate, BSA and ready-to-use BCIP/NBT substrate.
Pre-titrated capture antibody and biotinylated detection antibody for coating your own plates, supplied in quantities sufficient for 10 x 96 wells.
IFN-gamma, TNF-alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, IL-17F and IL-17A/F, plus granzyme B, perforin and Fas ligand for cytotoxic responses.
An ELISA measures the total concentration of a secreted protein in a sample. An ELISpot measures how many individual cells are secreting it. Cells are cultured directly in an antibody-coated well, each secreting cell leaves a spot where its cytokine was captured, and the spots are counted.
Because the cytokine is captured the moment it leaves the cell, before it can be diluted, degraded or taken up by neighbouring cells, ELISpot can detect rare responders that an ELISA on the supernatant would miss. The two assays answer different questions and are often run side by side.
Add cells to the antibody-coated PVDF plate with or without stimulus and incubate.
Cytokine released by each responding cell is trapped by the capture antibody around that cell.
Wash the cells away, then add biotinylated detection antibody and streptavidin-enzyme conjugate.
A precipitating substrate forms a spot for each secreting cell. Count with an ELISpot reader or a stereomicroscope.
An ELISA determines the total concentration of a secreted protein. An ELISpot analyses individual secreting cells and gives the frequency of responding cells.
ELISpot is typically used to detect cytokine secretion by antigen-activated T cells, and can also be applied to other secreting cells, from peripheral blood mononuclear cells or spleen cells.
Cell density typically ranges from 1 to 2 x 10⁵ cells per well, and may need optimising. Where a high frequency of responding cells is expected, use fewer cells so that individual spots stay distinct.
In the direct method cells are stimulated in the coated wells. In the indirect method cells are stimulated first in a separate plate or flask, harvested, and then transferred to the coated wells. The indirect method suits samples with a high frequency of responding cells.
Choose a kit for a ready-to-run assay with pre-coated plates. Choose a pair if you run ELISpot regularly and prefer to coat your own PVDF plates.
Store kit reagents at 2 to 8 °C and return them to cold storage immediately after use. The expiry date is printed on the box label.