[Development of the method for analysis of the cytokine balance shift associated with diseases].
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-gamma (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) 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/H(2)O(2). 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.
Okamoto
Yoshihiro
Y
Faculty of Pharmacy, Meijo University, Nagano 468-8503, Japan. yokamoto@cis.ac.jp
D000818:Animals; D001169:Arthritis, Experimental / Q000175:diagnosis*; D001171:Arthritis, Juvenile / Q000175:diagnosis*; D015415:Biomarkers / Q000032:analysis; D016207:Cytokines / Q000032:analysis* / Q000378:metabolism; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D051379:Mice; D012680:Sensitivity and Specificity; D018417:Th1 Cells / Q000378:metabolism; D018418:Th2 Cells / Q000378:metabolism
D015415:Biomarkers; D016207:Cytokines