Serum enzyme-linked immunosorbent assay
Measuring cytokine levels in blood serum using classical enzyme-linked immunosorbent assay (ELISA) is considered a insufficiently adequate approach.
The main drawback of the method is that it does not allow differentiating between the processes of cytokine production (secretion) and their consumption by tissues. Furthermore, the blood cytokine concentration does not indicate which specific cells served as the source of its synthesis.
The ELISPOT method
A more accurate method for assessing cytokine secretion at the cellular level is ELISPOT.
- Principle of action: A cytokine (e.g., interferon-gamma, IFNγ) secreted by a specific cell is captured by specific antibodies fixed on a substrate.
- Detection: The resulting antibody-cytokine complex is detected using secondary antibodies labeled with an enzyme (peroxidase).
- Result evaluation: Using a microscope or a specialized reader, stained spots are counted. Each spot corresponds to a single cell actively secreting the studied cytokine.
This method has found wide application in scientific research, in particular, for searching and selecting T-cell epitopes during the development of new vaccines.
Briefly: answers to topical questions
What other methods besides ELISA and ELISPOT are used to assess cytokine status?
The cited sources mention the following methods:
- Intracellular cytokine staining — used to assess the ability of T-helper cells to produce specific cytokines; the study is performed by flow cytometry after stimulation with phorbol myristate acetate and ionomycin.
- Quantitative reverse transcription real-time PCR — used to assess the level of gene expression, including those encoding immunologically significant molecules.
- IGRA tests — based on the release of IFN-γ upon exposure to specific tuberculosis antigens; these include, for example, QuantiFERON-TB Gold and T-SPOT.TB.
Which specific immune system cells are studied using the ELISPOT method?
The ELISPOT method detects cytokine-secreting cells, particularly T cells; it is widely used for selecting T-cell epitopes in vaccine development. Theoretically, ELISPOT can be used to assess the antibody-producing capacity of B cells, but in vitro induction of antibody production is extremely difficult.
How does ELISPOT differ from intracellular cytokine staining in flow cytometry?
The main difference lies in the localization of the detected cytokines and the method of result detection.
| Characteristic | ELISPOT | Intracellular staining |
|---|---|---|
| Localization of cytokines | Secreted by the cell into the extracellular environment | Intracellular location |
| Detection principle | Binding to fixed antibodies on a solid phase | Staining after stimulation with phorbol myristate acetate and ionomycin |
| Result evaluation | Counting stained spots using a microscope or a device | Flow cytometry |
Class and exam questions
Why is serum ELISA considered a poor method for assessing cytokines?
ELISA only determines the total cytokine level in the blood, failing to distinguish secretion from consumption and not indicating the cellular source.
What does a single stained spot show in the ELISPOT test?
A single spot corresponds to exactly one cell that released the studied cytokine.
What else to study on this topic
- Assessment of NK cell functional activity (radiometric assays and CD107 expression)
- Assessment of the complement system state (C3 component ELISA)
- Assessment of cytotoxic T lymphocyte function
- Assessment of B lymphocyte proliferative potential (pokeweed mitogen response)
- In vivo tests in immunodiagnostics
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