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Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
Published on: November 23, 2010
ELISPOT assay to detect cytokine-secreting murine and human cells
Dennis M Klinman1, Thomas B Nutman2
1Center for Biologics Evaluation & Research, Food and Drug Administration, Bethesda, Maryland.
Insights
The enzyme-linked immunospot (ELISPOT) assay detects and quantifies single antibody-secreting B cells. Enhanced sensitivity allows detection of cells producing minimal protein amounts, visualized as permanent spots.
Area of Science:
- Immunology
- Cell Biology
Background:
- The enzyme-linked immunospot (ELISPOT) assay was developed to detect and quantify individual antibody-secreting B cells.
- Recent advancements have significantly increased the assay's sensitivity.
Purpose of the Study:
- To describe the principles and applications of the ELISPOT assay.
- To highlight the improved sensitivity of modern ELISPOT techniques.
Main Methods:
- Utilizes two high-affinity cytokine-specific antibodies targeting different epitopes.
- Employs a colorimetric reaction to generate visible spots from secreted cytokines.
- Spots are generated by individual cytokine-producing cells, serving as a cellular footprint.
Main Results:
- The modified ELISPOT assay can detect cells producing as few as 100 molecules of specific protein per second.
- The assay generates permanent spots that can be quantified visually, microscopically, or electronically.
Conclusions:
- The ELISPOT assay is a sensitive method for detecting and quantifying single antibody-secreting cells.
- The technique's high sensitivity and permanent spot generation make it valuable for immunological research.
Abstract:
The filter immunoplaque assay, otherwise called the enzyme-linked immunospot assay (ELISPOT), was initially developed to detect and quantitate individual antibody-secreting B cells. Recent modifications have improved the sensitivity of the ELISPOT assay such that cells producing as few as 100 molecules of specific protein per second can be detected. The ELISPOT assay utilizes two high-affinity cytokine-specific antibodies directed against different epitopes on the same cytokine molecule: either two monoclonal antibodies or a combination of one monoclonal antibody and one polyvalent antiserum. ELISPOT generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The spot represents a "footprint" of the original cytokine-producing cell. Spots are permanent and can be quantitated visually, microscopically, or electronically.
