病態・疾患に連動するサイトカインの新規解析法による迅速な病態把握の試み

抄録

  A dual-color enzyme-linked immunospot (ELISPOT) assay enabled us to analyze three types 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-2 (IL-2) and interferon-γ (IFN-γ), 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-γ. Furthermore, the presence of the third type (Th0) of 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 was 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 aminoethylcarbazole/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 types 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.