title: Cytokine-specific ELISPOT assay. Single cell analysis of IL-2, IL-4 and IL-6 producing cells.
abstract: In order to assess cytokine-producing cells at the single cell level, the cytokine-specific ELISPOT assay has proven to be an important and sensitive method. The purpose of this study was to adapt this method to elucidate individual cells producing murine IL-2, IL-4 or IL-6. In order to establish these cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific cDNA transfected myeloma cell lines, e.g., X63-Ag8-653 X2, X63-Ag8-653 X4 and X63-Ag8-653 X6, respectively, were used as specific cytokine-producing cells. In the IL-2 ELISPOT assay, the coating reagent, monoclonal antibody (mAb) rat IgG2a anti-mouse IL-2 (CR #40014) was used while rabbit IgG polyclonal anti-mouse IL-2 was employed for detection of IL-2 spot forming cells (SFC). The mAbs anti-mouse IL-4, BVD4-1D11 and BVD6-24G2 were selected as capture and detection antibodies for enumeration of IL-4 SFC. For the IL-6 ELISPOT assay, anti-mouse IL-6 (MP5-20F3) mAb was used for coating and MP5-32C11 mAb was used for detection of IL-6 SFC. When IL-2 producing X63-Ag8-653 X2 cells were subjected to these three different ELISPOT assays, IL-2-specific SFC were only noted with the IL-2 ELISPOT system. In the case of IL-4 SFC, only X63-Ag8-653 X4 cells formed specific spots using the tandem of BVD4-1D11 and BVD6-24G2 mAbs. IL-6-specific spots developed in MP5-20F3 mAb pre-coated wells containing X63-Ag8-653 X6 cells, when developed with mAb anti-IL-6 (MP5-32C11). Addition of cycloheximide (50 micrograms/ml) inhibited formation of IL-2, IL-4 and IL-6 SFC by approximately 90%. When an unrelated mAb was used as detection antibody in these three different cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific SFC were not detected. Further, when concanavalin A stimulated T cells from Peyer's patch of normal mice were subjected to the respective cytokine-specific ELISPOT assay, IL-2, IL-4 and IL-6 SFC were enumerated. These results have shown that cytokine-specific IL-2, IL-4 and IL-6 ELISPOT assays have now been established and will allow analysis of the frequency of cytokine-secreting cells at the single cell level.
authors.affiliation: Department of Oral Biology, University of Alabama, Birmingham 35294.
authors.forename: K
authors.initials: K
authors.lastname: Fujihashi
authors.forename: J R
authors.initials: JR
authors.lastname: McGhee
authors.forename: K W
authors.initials: KW
authors.lastname: Beagley
authors.forename: D T
authors.initials: DT
authors.lastname: McPherson
authors.forename: S A
authors.initials: SA
authors.lastname: McPherson
authors.forename: C M
authors.initials: CM
authors.lastname: Huang
authors.forename: H
authors.initials: H
authors.lastname: Kiyono
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000096:biosynthesis*; D015847:Interleukin-4 / Q000096:biosynthesis*; D015850:Interleukin-6 / Q000096:biosynthesis*; D051379:Mice; D008809:Mice, Inbred C3H; D010581:Peyer's Patches / Q000166:cytology / Q000276:immunology; D014162:Transfection
chemical_list: D007376:Interleukin-2; D015850:Interleukin-6; D015847:Interleukin-4