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ELISpot Assay Principle

Schematic illustration of the ELISpot assay principle.
Schematic illustration of the principle of the ELISpot assay.

The enzyme-linked immunospot (ELISpot) assay is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. In this assay, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli. Proteins, such as cytokines, that are secreted by the cells will be captured by the specific antibodies on the surface. After an appropriate incubation time, cells are removed and the secreted molecule is detected using a detection antibody in a similar procedure to that employed by the ELISA. The detection antibody is either biotinylated and followed by a streptavidin-enzyme conjugate or the antibody is directly conjugated to an enzyme. By using a substrate with a precipitating rather than a soluble product, the end result is visible spots on the surface. Each spot corresponds to an individual cytokine-secreting cell.


The ELISpot assay captures the presence of cytokines immediately after secretion, in contrast to measurements that are skewed by receptor binding or protease degradation. The assay is considered as one of the most sensitive cellular assays available. The limit of detection typically achieved can be 1 in 100,000 cells. The high sensitivity of the assay makes it particularly useful for studies of the small population of cells found in specific immune responses.

Mabtech has worked extensively for more than 20 years to optimize the ELISpot protocol. New proteins are continuously added to the range of analytes that can be analyzed by ELISpot.

Easy to perform

The ELISpot assay is carried out in a 96-well plate, and an automated ELISpot reader is used for analysis. The assay is therefore easy to perform and allows rapid analysis of a large number of samples. It is also robust and suitable for large-scale trials and for field studies. Plates pre-coated with the capture antibodies and one-step detection reagents offer additional advantages. Standardization of the ELISpot assay in specific settings is well described and the method is the basis of an FDA-approved diagnostic test, the T-spot test for tuberculosis.

The ELISpot technique is not limited to measurement of cytokines; it is also suitable for almost any secreted protein where single-cell analysis is of interest. The method was originally developed to quantify immunoglobulin-secreting cells.

ELISpot brochure