The body’s defense system relies on a complex network of cells that communicate constantly to detect and eliminate threats. Measuring the activity of these specialized immune cells is a significant challenge in medical science. Traditional methods often provide only an average or bulk measurement, which can obscure the responses of rare but important cell populations. The Interferon Gamma Enzyme-Linked Immunosorbent Spot (IFN-γ ELISPOT) assay is a specialized laboratory technique designed to overcome this limitation. It offers a precise and highly sensitive way to count the exact number of immune cells actively responding to a specific stimulus, such as a virus or a cancer antigen. This assay focuses on the production of a particular signaling molecule, providing a functional snapshot of cellular immunity. The high resolution of the ELISPOT method makes it an invaluable tool for researchers and clinicians.
The Core Concept of ELISPOT
The acronym ELISPOT stands for Enzyme-Linked Immunosorbent Spot, which suggests its fundamental purpose: to detect and count cells that are secreting specific molecules. This technique moves beyond simply measuring the total amount of a substance in a sample, which bulk assays like ELISA perform. Instead, ELISPOT provides a single-cell resolution, effectively identifying and localizing the individual cellular sources of an immune signal.
The IFN-γ assay specifically targets Interferon Gamma (IFN-γ), a cytokine that is a primary marker of a cell-mediated immune response. IFN-γ is a Type II interferon primarily released by activated T-cells, including cytotoxic T lymphocytes (CD8+) and T-helper 1 (Th1) cells, as well as Natural Killer (NK) cells. Its presence signals a potent immune reaction aimed at controlling intracellular pathogens, such as viruses and certain bacteria, and plays a role in anti-tumor immunity. Quantifying the frequency of IFN-γ secreting cells acts as a direct measure of the strength and specificity of the active T-cell response.
How the Assay Works
The ELISPOT process relies on a technique called a “sandwich immunoassay” performed directly on a specialized culture plate. The wells of this plate are lined with a membrane coated with a high-affinity capture antibody that is specific to the IFN-γ molecule. This immobilized antibody acts to catch any IFN-γ secreted in its immediate vicinity.
The assay begins by adding the patient’s immune cells, typically peripheral blood mononuclear cells (PBMCs), into the coated wells along with a specific antigen or peptide. If a T-cell recognizes the presented antigen, it becomes activated and starts secreting IFN-γ.
Because the cytokine is captured by the antibody on the membrane almost immediately upon release, it cannot diffuse away, which ensures that the resulting signal is localized precisely to the site of the secreting cell.
After an incubation period, the cells are washed away, leaving only the captured IFN-γ bound to the membrane. A second, biotinylated detection antibody is then introduced, which binds to the captured IFN-γ molecule, completing the “sandwich.” Next, an enzyme, such as Alkaline Phosphatase, linked to Streptavidin is added, which binds tightly to the biotin on the detection antibody. The final step involves adding a colorless substrate solution that the enzyme converts into an insoluble, colored precipitate, forming a distinct, visible spot directly on the membrane. Each colored spot represents the secretory footprint of a single, activated IFN-γ-producing T-cell.
Applications in Disease Monitoring and Vaccine Development
The sensitivity of the IFN-γ ELISPOT assay has made it a standard tool across several areas of clinical and research immunology.
Infectious Disease Diagnostics
In infectious disease diagnostics, the assay is widely used as an Interferon Gamma Release Assay (IGRA) for detecting latent tuberculosis (TB) infection. This test measures the T-cell response to specific TB-related antigens, offering an improved diagnostic method compared to older skin tests. The ELISPOT assay has also been adapted for monitoring immune responses to other pathogens, including viruses such as HIV or varicella-zoster.
Vaccine Efficacy Monitoring
The assay plays a major role in monitoring vaccine efficacy in the development of new treatments. Researchers use it in clinical trials for diseases like malaria, influenza, and COVID-19 to quantitatively measure the magnitude and durability of the T-cell response generated by a new vaccine candidate. A higher count of antigen-specific IFN-γ secreting cells indicates a stronger cellular immune memory induced by the immunization.
Cancer Immunotherapy
The ELISPOT assay is also important in cancer immunotherapy, where it helps assess a patient’s immune status and response to treatment. It can quantify the frequency of T-cells that specifically recognize and react to tumor antigens, both before and after an antitumor vaccine or other cell-based therapy. This monitoring is used to gauge whether the therapy successfully mobilized a patient’s T-cells to target the cancer.
Interpreting the Spot Counts
The data generated by the ELISPOT assay is quantified by counting the number of visible spots, with this count being referred to as Spot Forming Units (SFU). SFU measurements are normalized by dividing the spot count by the number of input cells, commonly expressed as SFU per one million cells. This normalization allows for a standardized comparison between different samples and experiments.
A higher SFU count directly correlates with a greater frequency of antigen-specific T-cells in the patient’s sample, which is interpreted as a stronger cellular immune response to the tested antigen. To determine if a response is truly significant, the SFU count in the antigen-stimulated wells must be compared to a negative control well, which contains cells but no stimulating antigen, to account for background activity. A typical threshold for a positive response may be set above a specific number, such as 50 SFU per million cells, provided it is also significantly higher than this background count.
Beyond the simple count, advanced systems can also measure the size and intensity of each spot. A larger spot may indicate that the individual cell secreted a greater quantity of IFN-γ over the incubation period, offering additional functional information beyond just the cell frequency.