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Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
Published on: November 24, 2010
Enzyme-linked immunospot assays provide a sensitive tool for detection of cytokine secretion by monocytes
M Kouwenhoven1, V Ozenci, N Teleshova
1Neuroimmunology Unit, Division of Neurology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden. mathilde.kouwenhoven@neurotec.ki.se
Insights
This study optimized Enzyme-Linked Immunospot (ELISPOT) assays to accurately count monocytes secreting key immune signaling proteins like Interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-alpha). The findings establish ELISPOT as a valuable tool for cytokine research and immune response analysis.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Monocytes and macrophages are crucial for immune response regulation through cytokine secretion.
- Key cytokines like IL-6, TNF-alpha, IL-10, and IL-12 modulate Th1/Th2 immune balance.
- Accurate enumeration of cytokine-secreting cells is vital for understanding immune dynamics.
Purpose of the Study:
- To optimize Enzyme-Linked Immunospot (ELISPOT) assay conditions for detecting and quantifying monocytes that secrete specific cytokines.
- To establish optimal incubation times and cell numbers for enumerating IL-6, TNF-alpha, IL-10, and IL-12 secreting monocytes.
- To determine baseline levels of these cytokine-secreting monocytes in healthy individuals.
Main Methods:
- Preparation of monocytes from healthy human blood.
- Adaptation and optimization of Enzyme-Linked Immunospot (ELISPOT) assays.
- Enumeration of monocytes secreting Interleukin 6 (IL-6), Tumor Necrosis Factor alpha (TNF-alpha), Interleukin 10 (IL-10), and Interleukin 12 (IL-12).
Main Results:
- Optimal monocyte incubation time was determined to be 24 hours.
- Specific optimal cell numbers per well were identified for each cytokine assay (e.g., 2,000 for IL-6, 1,000 for TNF-alpha).
- Prevalence of cytokine-secreting monocytes in healthy subjects was quantified (e.g., IL-6: 10% ± 5%, TNF-alpha: 12% ± 12%).
Conclusions:
- ELISPOT assays are a sensitive and specific method for enumerating cytokine-secreting monocytes.
- Optimized protocols enable reliable quantification of monocytes producing IL-6, TNF-alpha, IL-10, and IL-12.
- This methodology can likely be extended to enumerate monocytes secreting other relevant proteins and cytokines.
Abstract:
Blood monocytes as well as tissue-differentiated macrophages play a pivotal role in controlling immune reactions. Monocytes regulate the extent, nature, and duration of immune responses by secretion of cytokines. Interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), IL-10, and IL-12 are of particular interest, since IL-12 shifts the immune response towards a Th1 type, facilitating the production of, e.g., TNF-alpha and IL-6, while IL-10 counteracts Th1 responses and promotes the production of Th2-related cytokines such as IL-4. A tight regulation of these four cytokines keeps the balance and decides whether Th1 or Th2 will predominate in immune reactions. Enzyme-linked immunospot (ELISPOT) assays are among the most-sensitive and -specific methods available for cytokine research. They permit ex vivo identification of individual cells actively secreting cytokines. In the present study we prepared monocytes from healthy subjects' blood and adapted ELISPOT assays to define optimal conditions to detect and enumerate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12. The optimal time for monocyte incubation was 24 h, and optimal monocyte numbers (in cells per well) were 2,000 for IL-6, 1,000 for TNF-alpha, 50,000 for IL-10, and 100,000 for enumeration of IL-12 secreting monocytes. Among healthy subjects, 10% +/- 5% of the monocytes secreted IL-6, 12% +/- 12% secreted TNF-alpha, 0.1% +/- 0.1% secreted IL-10, and 0.2% +/- 0.3% secreted IL-12 (values are means +/- standard deviations). In conclusion, ELISPOT assays constitute a valuable tool to enumerate monocytes secreting IL-6, TNF-alpha, IL-10, and IL-12 and probably to enumerate monocytes secreting other cytokines and proteins.
