What Is a T Cell ELISpot Assay and How Does It Work?

The T cell ELISpot assay is a sensitive laboratory method used to detect and count individual cells that secrete proteins, like cytokines, upon activation. Its primary function is to quantify cellular immune responses at the single-cell level, making it useful for identifying rare cells within a larger population. The assay provides a quantitative measurement of the frequency of these active cells. This allows researchers to assess the magnitude of a T cell response to a stimulus, such as an antigen from a pathogen or a vaccine.

The Science Behind T Cell ELISpot

The T cell ELISpot assay operates on the principle of a “sandwich” immunoassay, designed to visualize proteins secreted by a single activated T cell. T cells are white blood cells that coordinate the adaptive immune response. When they recognize a specific target, or antigen, they become activated and release signaling molecules called cytokines.

The process uses a multi-well plate where the bottom of each well is coated with a “capture antibody.” This antibody is chosen to bind a specific cytokine, such as interferon-gamma (IFN-γ), a common indicator of T cell activation. A sample of T cells is added to the wells with a stimulus, causing only the T cells that recognize the antigen to activate.

These activated T cells secrete their target cytokine, which is immediately captured by the antibodies on the surface below them. This creates a localized zone of captured protein around each active cell. After an incubation period, the cells are washed away, leaving the invisible cytokine zones behind.

To visualize these zones, a “detection antibody” linked to an enzyme is added, which binds to the captured cytokine. A substrate solution is then introduced that reacts with the enzyme, producing an insoluble, colored precipitate. This reaction creates a distinct spot on the plate’s surface, marking the location of the original cytokine-secreting T cell.

Key Stages in Performing an ELISpot Assay

Conducting a T cell ELISpot assay involves a precise sequence of steps to ensure accurate results.

• Plate Preparation: The wells of a specialized microplate are coated with a capture antibody specific to the cytokine of interest. This antibody adheres to the plate’s membrane, preparing it to bind the molecules secreted by the cells.
• Cell Incubation: Live T cells are pipetted into the antibody-coated wells along with a specific stimulus, such as a peptide antigen. The plate is then placed in an incubator for 18 to 24 hours, allowing sufficient time for the T cells to respond and secrete cytokines.
• Detection Cascade: After incubation, the T cells are washed away, leaving only the secreted cytokines bound to the capture antibodies. A detection antibody is then added, which binds to the captured cytokine, followed by an enzyme conjugate that completes the molecular sandwich.
• Spot Visualization: A substrate is added to the wells, which reacts with the enzyme to produce a colored, insoluble precipitate at each location where a cytokine-secreting cell was located. Once developed, the plate is dried, and the spots are counted using an automated ELISpot reader.

Deciphering ELISpot Results

The output of an ELISpot assay is a plate with colored spots against a clear background, where each spot represents one T cell that secreted the target cytokine. The primary data point is the count of spots within each well, reported as Spot Forming Units (SFUs) per a given number of input cells. For example, a result might be 150 SFUs per million cells.

This quantitative data allows researchers to gauge the magnitude of a cellular immune response. A higher number of SFUs indicates a greater frequency of T cells responding to the specific stimulus. This metric is useful for comparing immune responses between different individuals or for tracking changes in one person’s immune response over time.

Beyond the count, the size and intensity of the spots can offer additional qualitative information. Larger, more intense spots may suggest that a T cell was secreting a higher amount of the cytokine. This provides a deeper layer of analysis, revealing not just how many cells are responding, but also how robustly.

Widespread Applications of T Cell ELISpot

The T cell ELISpot assay is applied across biomedical research and clinical diagnostics. In vaccine development, it assesses immunogenicity by measuring the frequency of T cells responding to a vaccine’s antigen. This provides direct evidence of a vaccine’s ability to elicit a cellular immune response and was used to evaluate vaccines against pathogens like SARS-CoV-2.

In infectious disease research, the assay studies T cell responses to viruses, bacteria, and parasites, and it monitors immune status in chronic infections like HIV. A diagnostic application is in testing for latent tuberculosis, where Interferon-Gamma Release Assays (IGRAs), a form of ELISpot, detect T cells reactive to tuberculosis antigens.

For cancer immunology, the assay monitors patient immune responses to treatments like checkpoint inhibitor therapy. It helps determine if the therapy is activating T cells to attack tumor cells by quantifying T cells specific for tumor-associated antigens.

The technique is also used in studying autoimmune diseases and organ transplantation. In autoimmunity, it can help identify autoreactive T cells. For transplantation, the assay assesses a recipient’s T cell reactivity against donor antigens to help monitor for the risk of organ rejection.