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Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
Published on: November 24, 2010
Dual-color ELISPOT assay for analyzing cytokine balance
Yoshihiro Okamoto1, Mikio Nishida
1Division of Health Care Pharmacy, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya, Aichi, Japan.
Insights
A novel dual-color ELISPOT assay simultaneously distinguishes T helper 1 (Th1), T helper 2 (Th2), and T helper 0 (Th0) cells by detecting their unique cytokine profiles. This method allows for precise analysis of immune cell function in various preparations.
Area of Science:
- Immunology
- Cellular Biology
Background:
- T helper (Th) cells are crucial immune regulators.
- Th cells differentiate into functional subsets like Th1 and Th2 based on cytokine production.
- Th0 cells, precursors to Th1/Th2, secrete both Th1 and Th2 cytokines.
Purpose of the Study:
- To develop a dual-color ELISPOT assay for simultaneous analysis of three T helper cell subtypes.
- To differentiate IL-2-producing (Th1), IL-4-producing (Th2), and dual-cytokine-producing (Th0) cells in a single assay.
Main Methods:
- Utilized a dual-color enzyme-linked immunospot (ELISPOT) assay.
- Employed specific chromogenic substrates (horseradish peroxidase/amino-ethyl-carbazole/H2O2 for Th1; alkaline phosphatase/Vector blue for Th2).
- Visualized IL-2 (red), IL-4 (light blue), and dual cytokine secretion (indigo) in the same well.
Main Results:
- Successfully differentiated Th1, Th2, and Th0 cells based on distinct spot colors.
- Demonstrated simultaneous detection of IL-2 and/or IL-4 secreting cells.
- Applied the assay to murine spleen cells and human peripheral mononuclear cells.
Conclusions:
- The dual-color ELISPOT assay provides a robust method for simultaneous quantification of Th1, Th2, and Th0 cells.
- This technique enhances the ability to study T helper cell responses in immunological research.
- The assay is applicable to both murine and human immune cell samples.
Abstract:
A dual-color enzyme-linked immunospot (ELISPOT) assay enabled us to analyze three kinds of cytokine-secreting cells simultaneously. T helper (Th) cells can be subdivided into at least two distinct functional subsets based on their cytokine secretion profiles. The first type of clones (Th1) produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 or IL-5. The second type of clones (Th2) produces IL-4 and IL-5 but not IL-2 or IFN-gamma. Furthermore, the presence of the third type (Th0) cell, which is a precursor of Th1 or Th2 cells, has been demonstrated to produce both Th1- and Th2-type cytokines. The dual-color ELISPOT assay is developed to differentiate these three subtypes of Th cells in an identical well. In the system, the red spots corresponding to IL-2-secreting cells (Th1) were developed with horseradish peroxidase and amino-ethyl-carbazole/H2O2. The light blue spots corresponding to IL-4-secreting cells (Th2) were developed with alkaline phosphatase and Vector blue (chromogenic substrate for alkaline phosphatase). The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (Th0 cells) were developed with both chromogenic substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in a murine spleen cell or human peripheral mononuclear cell preparation.
