8 February 2013

ELISpot assay principle

The enzyme-linked immunospot (ELISpot) assay is an extremely sensitive immunoassay performed in a 96-well plate format to quantify protein secreting cells.

The assay principle is straight forward: cells are cultured inside a plate coated with capture antibody. Cytokines, immunoglobulins, or other target proteins secreted by the cells are captured immediately after secretion and throughout the stimulation process. After cell removal, secreted proteins are identified using a detection antibody. Visible spots form after adding a precipitating substrate. Each spot corresponds to an individual analyte-secreting cell.

Identify rare cell populations

With minimal manipulation of the cells, ELISpot lies closer to reality than most other cell-based immunoassays down to the single-cell level. Due to capacity to find one cell in a million, ELISpot is particularly valuable for studies of rare cell populations causing immune responses.

Capture transient analytes

As ELISpot target analytes immediately after secretion and throughout the stimulation process, the method can detect cytokines that otherwise disappear from samples. For example, ELISpot can detect analytes that are rapidly degraded by proteases (IL-2), are quickly taken up by bystander cells (IL-4), or bind to soluble receptors (TNF-α).

Easy to scale up

ELISpot is robust and easy to perform, making it suitable for analyzing many samples in parallel or at different timepoints during a clinical trial. Standardization of the ELISpot assay has been achieved in specific settings and the method is the basis of an FDA-approved diagnostic test for tuberculosis. The assay is carried out on a 96-well plate and analyzed with an automated ELISpot reader, enabling rapid analysis of several plates in a row. 

For assessment of T cell immunity

ELISpot is commonly used to investigate antigen-specific immune responses and to discriminate between subsets of activated T cells. This is applied in studies of infectious diseases, cancer, allergies, and autoimmune diseases. In vaccine research, ELISpot is the gold standard to define vaccine efficacy by measuring the capacity to elicit T cell responses, for example by assessing IFN-γ secretion. Diagnostic assays based on ELISpot are available, including tests to detect patients with tuberculosis or SARS-CoV-2 infection by measuring IFN-γ secretion from T cells responding to defined peptides.

Also perfect for the study of antibody-secreting cells

The B cell ELISpot assay is one of few assays measuring immunoglobulins directly
upon secretion. There are two strategies: Firstly, the B cell ELISpot can be used to assess antibody-secreting cells (ASCs). Due to its sensitivity, the method enables identification of rare ASCs a specific antigen. Secondly, you can evaluate circulating antigen-specific memory B cells after polyclonal activation. The B cell ELISpot is regularly used to detect B cell responses elicited by infection or vaccination.

To get the most out of your assay, please read our Step-by-step guide to ELISpot.

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