ELISpot

The Enzyme-Linked Immunosorbent Spot (ELISpot) assay is a highly sensitive immunological assay used to quantify cytokine-secreting cells, antibody-secreting cells, or other effector molecules produced by individual cells at the single-cell level. It is often used to monitor cellular immune responses in a variety of settings, including vaccine development, infectious disease research, cancer immunotherapy, and autoimmune disease studies.

Principle:

The ELISpot assay is based on the principle of capturing secreted proteins in close proximity to the secreting cells. The process typically involves the following steps:

1. Coating: A microplate is coated with a capture antibody specific to the target protein of interest (e.g., a specific cytokine like interferon-gamma (IFN-γ) or interleukin-2 (IL-2)).

2. Cell Incubation: Cells (e.g., peripheral blood mononuclear cells (PBMCs), splenocytes) are incubated in the coated wells, often with stimulation (e.g., antigen, mitogen). During the incubation period, cells that are activated and secreting the target protein will release it into the microenvironment.

3. Capture: The secreted protein is immediately bound by the capture antibody on the plate surface, directly beneath the secreting cell.

4. Removal of Cells and Washing: The cells are washed away, leaving behind the captured protein "spots" on the plate.

5. Detection: A detection antibody, specific to the target protein but recognizing a different epitope than the capture antibody, is added. This antibody binds to the captured protein.

6. Enzyme Conjugation and Substrate Addition: The detection antibody is typically conjugated to an enzyme (e.g., alkaline phosphatase (ALP) or horseradish peroxidase (HRP)). A substrate specific to the enzyme is then added. The enzyme acts on the substrate, producing a visible precipitate at the site of protein secretion.

7. Spot Counting: The resulting spots, each representing a single cell that has secreted the target protein, are counted using an automated ELISpot reader or manually. The number of spots is often expressed as spot-forming units (SFU) per number of cells (e.g., SFU per million PBMCs).

Advantages:

• High Sensitivity: ELISpot is more sensitive than traditional ELISA because it captures the secreted protein directly at the site of secretion, minimizing dilution and degradation.

• Single-Cell Resolution: The assay allows for the quantification of individual cytokine-secreting cells, providing information about the frequency of responding cells within a population.

• Versatility: ELISpot can be used to detect a wide range of secreted proteins, including cytokines, antibodies, and other effector molecules.

Limitations:

• Semi-Quantitative: While providing valuable information about the frequency of responding cells, ELISpot is considered a semi-quantitative assay. It doesn't provide precise information about the amount of protein secreted per cell.

• Potential for Artifacts: Incomplete washing or non-specific binding of antibodies can lead to artifacts, requiring careful optimization and controls.

• Cost and Complexity: ELISpot assays can be more expensive and technically demanding compared to other immunological assays.