UNIT 6.19 ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells

  1. Dennis Klinman

Published Online: 1 NOV 2008

DOI: 10.1002/0471142735.im0619s83

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Klinman, D. 2008. ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells. Current Protocols in Immunology. 83:6.19:6.19.1–6.19.9.

Author Information

  1. Cancer and Inflammation Program, National Cancer Institute, Frederick, Maryland

Publication History

  1. Published Online: 1 NOV 2008
  2. Published Print: NOV 2008


Enzyme-linked immunospot assays (ELISPOT) were initially developed to detect and quantify individual antibody-secreting B cells. As the sensitivity of this assay improved, the much smaller amounts of cytokine and chemokine produced by individual T cells became amenable to detection. ELISPOT assays utilize high-affinity antibody pairs directed against different epitopes on a single cytokine/chemokine. The critical first step involves binding the highest affinity Ab to a solid matrix. The plates are blocked to prevent nonspecific interactions, and the cells of interest incubated in the Ab-coated wells, during which time they secrete the cytokine/chemokine of interest. The secreted protein binds to the Abs immediately below the producer cell. This bound protein is recognized by a secondary enzyme-linked Ab. A colorimetric substrate is used to generate a dark precipitate or “spot” that marks the position of the protein-producing cell. The resultant spots are permanent and can be quantified visually, microscopically, or electronically. Curr. Protoc. Immunol. 83:6.19.1-6.19.9. © 2008 by John Wiley & Sons, Inc.


  • assay;
  • cytokine;
  • chemokine;
  • single cell;
  • T cell