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A novel ELISpot method for adherent cells.

Chantal K Turner1, Timothy M Blieden, Terry J Smith

  • 1Department of Environmental Medicine, University of Rochester, 601 Elmwood Ave, Rochester, NY 14642, USA.

Journal of Immunological Methods
|September 4, 2004
PubMed
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A new enzyme-linked immunospot (ELISpot) assay was developed for human adherent cells, enabling sensitive detection of cytokine production. This assay reveals diverse cytokine expression patterns in fibroblasts from different tissues.

Area of Science:

  • Immunology
  • Cell Biology

Background:

  • Standard enzyme-linked immunosorbent assays (ELISAs) and ELISpot assays are established for certain cell types.
  • Existing ELISpot assays are primarily used for nonadherent lymphocytes, not adherent cells like fibroblasts.
  • There was a need for a sensitive assay to quantify cytokine production by human adherent cells.

Purpose of the Study:

  • To develop and evaluate a novel enzyme-linked immunospot (ELISpot) assay for human adherent cells.
  • To assess the assay's ability to detect specific cytokines (IL-6 and IL-8) produced by primary human fibroblasts.
  • To compare the performance of the new ELISpot assay with standard ELISA methods.

Main Methods:

  • Primary human fibroblasts from four tissue types were used.
  • Cells were either unstimulated or activated with interleukin (IL)-1beta.

Related Experiment Videos

  • An ELISpot assay was developed and optimized using antibody pairs for IL-6 and IL-8.
  • Results were compared against standard ELISA measurements.

    • Main Results:

    • The developed ELISpot assay reliably detected IL-6 and IL-8 spots from as few as 10 fibroblasts.
    • Optimal detection was achieved with 50 cells per well incubated for 8 hours, with spots appearing as early as 2 hours.
    • The assay demonstrated the frequency of cytokine-producing fibroblasts varied by tissue type (e.g., lung, orbital).
    • The assay's sensitivity allows for cytokine detection in small numbers of cells, unlike standard ELISA.

    Conclusions:

    • A novel ELISpot assay is effective for quantifying cytokine production in human adherent cells.
    • Fibroblasts exhibit distinct cytokine production frequencies across different human tissues, supporting the concept of fibroblast diversity.
    • This assay provides a sensitive tool for studying cytokine-secreting adherent cells, including short-lived primary cells.