Cytokine-specific ELISPOT assay. Single cell analysis of IL-2, IL-4 and IL-6 producing cells
K Fujihashi1, J R McGhee, K W Beagley
1Department of Oral Biology, University of Alabama, Birmingham 35294.
Insights
This study established sensitive cytokine-specific ELISPOT assays for enumerating single cells producing IL-2, IL-4, and IL-6. These validated assays enable precise analysis of cytokine-secreting cell frequencies in biological samples.
Area of Science:
- Immunology
- Cell Biology
Background:
- The Enzyme-Linked ImmunoSpot (ELISPOT) assay is a sensitive method for detecting cytokine-producing cells at the single-cell level.
- Adapting ELISPOT for specific cytokines like IL-2, IL-4, and IL-6 is crucial for detailed immunological studies.
Purpose of the Study:
- To develop and validate cytokine-specific ELISPOT assays for murine Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-6 (IL-6).
- To enable the enumeration of individual cells secreting these key cytokines.
Main Methods:
- Utilized cytokine-specific cDNA transfected myeloma cell lines (X63-Ag8-653 X2, X4, X6) as positive controls.
- Employed specific monoclonal antibodies (mAbs) for capture and detection in IL-2, IL-4, and IL-6 ELISPOT assays.
- Validated assay specificity using unrelated mAbs and assessed cytokine production inhibition with cycloheximide.
Main Results:
- Successfully established distinct ELISPOT assays for IL-2, IL-4, and IL-6, demonstrating high specificity.
- Confirmed that cycloheximide significantly inhibited cytokine spot formation (approx. 90%).
- Enumerated IL-2, IL-4, and IL-6 spot-forming cells (SFC) in concanavalin A-stimulated T cells from mouse Peyer's patches.
Conclusions:
- Cytokine-specific ELISPOT assays for IL-2, IL-4, and IL-6 have been successfully established.
- These validated assays allow for the accurate analysis of cytokine-secreting cell frequencies at the single-cell level.
- The developed methods provide a valuable tool for immunological research involving cytokine production.
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.
