Development of a dual color enzyme-linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells.
The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies and cytokines. For simultaneous differentiation of individual cells producing interleukin-2 (IL-2) and/or interleukin-4 (IL-4) at a single cell level, a dual color ELISPOT assay has been developed. In the present system, the red spots corresponding to IL-2-secreting cells (T helper type 1, Th1 cells) were developed with a horseradish peroxidase and the amino ethyl carbazole (AEC)/H2O2. The light blue spots corresponding to IL-4-secreting cells (T helper type 2, Th2 cells) were developed with an alkaline phosphatase and the Vector blue. The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (T helper type 0, Th0 cells) were developed with both substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in crude spleen cell preparation or purified CD4 fraction. This procedure provides a useful tool for quantitatively analyzing micro-levels of dynamic immune responses.
Okamoto
Y
Y
Department of Clinical Pharmacy, Faculty of Pharmacy, Meijo University, Nagoya, Japan. yokamoto@meijo-u.ac.jp
Abe
T
T
Niwa
T
T
Mizuhashi
S
S
Nishida
M
M
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007376:Interleukin-2 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D012680:Sensitivity and Specificity; D018417:Th1 Cells / Q000276:immunology*; D018418:Th2 Cells / Q000276:immunology*
D007376:Interleukin-2; D015847:Interleukin-4