ELISpot and DC-ELISpot Assay to Measure Frequency of Antigen-Specific IFNγ-Secreting Cells

Methods Mol Biol. 2015:1318:79-86. doi: 10.1007/978-1-4939-2742-5_8.

Abstract

ELISpot is a highly sensitive method in immunology to enumerate cells producing a given cytokine. Cells are stimulated in a microtiter plate pre-coated with a specific anti-analyte antibody. In response to the stimulation, cells release cytokines that are bound to the anti-analyte antibody. After a washing step, which removes the cells from the wells, the location of the cytokine-releasing cell is visualized by an enzyme-labeled detection antibody and its corresponding chromogenic substrate. The end result is a set of colored spots, each of which represents an area where a cell secreting the cytokine had been located. Here we describe the standard ELISpot protocol and a variation denominated dendritic cell (DC)-ELISpot for the detection of IFNγ-secreting cells upon stimulation with oligopeptides and protein antigens, respectively.

MeSH terms

  • Alkaline Phosphatase / chemistry
  • Antibodies, Immobilized / chemistry
  • Antigens / pharmacology
  • Cell Count
  • Dendritic Cells / cytology
  • Dendritic Cells / drug effects
  • Dendritic Cells / metabolism*
  • Enzyme-Linked Immunospot Assay / methods*
  • Humans
  • Interferon-gamma / analysis*
  • Interferon-gamma / biosynthesis
  • Interferon-gamma / metabolism
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / metabolism*
  • Oligopeptides / pharmacology
  • Streptavidin / chemistry

Substances

  • Antibodies, Immobilized
  • Antigens
  • Oligopeptides
  • Interferon-gamma
  • Streptavidin
  • Alkaline Phosphatase