ELISpot applications

The cytokine ELISpot assay has been widely applied to investigate specific immune responses in various diseases including infections, cancer, allergies and autoimmune diseases. It has also become a standard tool in the development and monitoring of new vaccines and vaccine candidates. Often Interferon-gamma (IFN-γ) has been the analyte of choice serving as an immunocorrelate for CD8+ cytotoxic T-cell (CTL) responses (see Fig. 1).


Today, the IFN-γ ELISpot is used in many large-scale trials to monitor new HIV vaccine candidates as well as in the evaluation of vaccines and vaccine strategies for other viruses and pathogens and also tumors. The ELISpot assay is used to define vaccine efficacy by measuring the capacity to elicit potent T-cell responses. This information can then be used to evaluate suitable strategies for vaccine design as well as routes of administration. In addition to measurements of IFN-γ, ELISpot assays for other key factors of importance for cytotoxicity, such as Perforin, Granzyme B and MIP-1β have been developed to facilitate analysis of further aspects of CTL activity.


Depending on the cytokines analyzed, the ELISpot assay can be used to discriminate between different subsets of activated T cells. For example, T-helper (Th) 1 type cells are characterized by their production of IFN-γ, IL-2 and TNF-α whereas other cytokines like IL-4, IL-5 and IL-13 are typically produced by Th2 cells. Determination of Th2 type cytokines by ELISpot is of particular interest in allergy research, where Th2 responses are of major importance but most often occur at low frequencies requiring highly sensitive methods for a successful detection (see Fig. 2).


Applications of relevance include e.g. monitoring of T-cell reactivity in specific immunotherapy against atopic allergies and development of potential in vitro diagnostic tests for cell-mediated contact allergies. ELISpot detection of IL-4, the principal cytokine of Th2-type responses, is especially interesting since other methods like flow cytometry and ELISA often display insufficient sensitivity resulting in a failure to detect allergen-specific responses.


In addition to its use as a research tool, the ELISpot method has also been exploited for diagnostic purposes and recently the first approved diagnostic test based on the ELISpot platform was launched. The test which detects patients with active or latent tuberculosis infection is measuring the presence of T cells responding to defined antigens from Mycobacterium tuberculosis by IFN-γ secretion. It substitutes the commonly used skin test but adds both greater sensitivity and specificity and is unlike the skin test not confounded by prior vaccination with BCG.


Although primarily used in T-cell analysis, the ELISpot technique can basically be applied to any system where one wants to investigate protein secretion at the single cell level. The method is especially attractive in situations where the enumeration of the producing cells is critical and/or when the producing cells constitute only a small minority of the cells. An example of the latter is plasmacytoid dendritic cells which normally occur at very low frequency in the blood but are easily detected in the ELISpot based on their production of IFN-α after activation (see Fig. 3).


The ELISpot assay is suitable for performing individual tests as well as large scale trials. In contrast to e.g. tetramer analysis, an excellent method for extracting detailed information on CTL responses, screening of large sample numbers by ELISpot does not require prior identification of epitopes and their MHC class-I restriction. With a limited need for advanced laboratory equipment it has also proven to be well suited for field studies. Also, the ELISpot seems to be of similar value independent of species and in addition to assays for use with human, monkey, mouse and rat cells, reagents are available for veterinary applications enabling assessment of T-cell responses in samples of bovine, ovine, equine and porcine origin.

Peptide stimulation 200 000 cells

No stimuli 200 000 cells

Fig. 1. Human IFN-γ ELISpot analysis of peptide-reactive T cells. CD8+ T cells were isolated by positive selection with anti-CD8-coated magnetic beads (Miltenyi, Cologne, Germany). Cells were stimulated with a pool of virus-derived MHC Class I-restricted peptides (available from Mabtech as “CEF Peptide Pool”) for 16 hours. Cells cultured in medium alone (no stimuli) are shown as negative control.

Allergen stimulation 250 000 cells

No stimuli 250 000 cells

Fig. 2. Human IL-4 ELISpot analysis of allergen-reactive T cells. Peripheral blood mononuclear cells (PBMC) were stimu-lated with allergen and the number of IL-4 producing cells was determined after 36 hours. Cells cultured in medium alone (no stimuli) are shown as negative control.

CpG-ODN 250 000 cells

No stimuli 250 000 cells

Fig. 3. IFN-α response to immunostimulatory deoxynucleotides. Human PBMC were stimulated with CpG-ODN or cultured in medium alone (no stimuli).