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Updated: May 14, 2026
Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
Published on: November 23, 2010
ELISPOT as a Functional for Biomarker Study in Cancer Immunotherapy: Applications and Future Directions
Laura R Fernández Castro1,2,3, Matias Regiart4, Francisco Gabriel Ortega-Sánchez1,2,3
1GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government PTS, Avenida de la Ilustración, 114, 18016 Granada, Spain.
Insights
The Enzyme-Linked ImmunoSpot (ELISPOT) assay is valuable for monitoring T cell responses in cancer immunotherapy. Standardization is needed for its use as a clinical biomarker.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- The Enzyme-Linked ImmunoSpot (ELISPOT) assay quantifies antigen-specific T cell responses, particularly interferon gamma (IFN-γ) secretion, at a single-cell level.
- It is widely applied in cancer research for evaluating immune responses to various immunotherapies, including vaccines, oncolytic viruses, cellular therapies, and immune checkpoint inhibitors.
Purpose of the Study:
- To systematically review and synthesize evidence on the application of ELISPOT in cancer immunotherapy research.
- To assess the utility of ELISPOT in validating antigens, monitoring T cell activity, and characterizing T cell function in preclinical and clinical settings.
Main Methods:
- A systematic qualitative synthesis of 78 studies was conducted, adhering to PRISMA guidelines.
- Studies were selected from PubMed, Scopus, and Embase, focusing on research with translational relevance in cancer immunotherapy.
Main Results:
- ELISPOT is extensively used to validate tumor antigens, monitor T cell responses during immunotherapy, and characterize T cell function.
- A limited number of studies demonstrate direct links between ELISPOT results and clinical outcomes.
- Significant variability in assay protocols and reporting hinders cross-study comparability and reproducibility.
Conclusions:
- ELISPOT is a valuable tool for immune monitoring in cancer immunotherapy research.
- Further standardization, prospective validation, and integration with other analytical methods are necessary for its clinical biomarker implementation.
Abstract:
The Enzyme-Linked ImmunoSpot (ELISPOT) assay is a highly sensitive and widely used technique for assessing antigen-specific cellular immune responses in cancer research. By enabling the quantification of cytokine secretion at the single-cell level, particularly interferon gamma (IFN-γ), ELISPOT provides a functional readout of T cell activity with applications in both preclinical and clinical settings. This systematic review presents a structured qualitative synthesis of 78 studies investigating the use of ELISPOT in cancer immunotherapy, including cancer vaccines, oncolytic viruses, cellular therapies, immune checkpoint inhibitors, and biomarker development. Studies were selected following PRISMA guidelines from PubMed, Scopus, and Embase, focusing on both clinical and preclinical research with translational relevance. The evidence indicates that ELISPOT is widely used to validate tumor-associated antigens and neoantigens, monitor antigen-specific T cell responses during cancer immunotherapy, including immune checkpoint blockade, cancer vaccines, and adoptive cell therapies, and characterize antigen-specific T cell function. However, only a limited subset of studies establishes direct associations between ELISPOT responses and clinically meaningful outcomes. In addition, substantial variability in assay protocols and reporting criteria limits cross-study comparability and reproducibility. Overall, ELISPOT remains a valuable tool for immune monitoring in cancer research. Still, its implementation as a clinically validated biomarker requires further standardization, prospective validation, and integration with complementary analytical approaches.
