The Enzyme-Linked ImmunoSpot Assay (ELISpot) is a highly sensitive immunological technique designed to detect specific secreted proteins—such as cytokines or antibodies—at the single-cell level. Its key advantage lies in the ability to quantify the frequency of cells secreting a specific functional protein and to assess their activity. Compared to conventional enzyme-linked immunosorbent assays (ELISA), ELISpot offers 10–100 times greater sensitivity, making it an indispensable tool in immunological research and clinical applications.

The origins of ELISpot trace back to 1978, when Professor Don Mason at the University of Oxford developed the "spot-on-film" technique to detect antibody-secreting cells. The modern ELISpot assay was independently advanced in 1983 by J.D. Sedgwick and C. Czerkinsky, who successfully integrated enzyme-linked immunodetection principles with spot-forming techniques, substantially improving both practicality and sensitivity.

Figure 1. Timeline of the main Elispot advances. (Lima-Junior JDC, et al., 2017)

ELISpot detects cells secreting specific proteins by visualizing "spots" formed on a solid-phase support, reflecting the proportion of functionally active cells in a sample. Its applications span vaccine development, drug screening, infectious disease research, and diagnosis (e.g., tuberculosis, HIV), tumor immunology, autoimmune and allergic diseases, as well as monitoring transplant rejection.

Principle and Core Features

ELISpot is based on the sandwich immunoassay principle. The assay is performed in microplates with bottoms coated with polyvinylidene fluoride (PVDF) membranes. The workflow is as follows:

1. Specific capture antibodies are immobilized on the PVDF membrane.
2. Cells of interest (e.g., peripheral blood mononuclear cells, PBMCs) are added and stimulated. Activated cells secrete the target cytokine or antibody, which is immediately captured by the pre-coated antibody surrounding each cell.
3. After removing cells, biotinylated detection antibodies are added, binding the captured target proteins.
4. Enzyme-conjugated streptavidin is introduced, followed by substrate addition to develop insoluble colored precipitates, forming visible "spots" at the location of secreting cells. Each spot corresponds to a single functionally active cell.

Figure 2. Schematic representation of the main steps in different Elispot categories. (Lima-Junior JDC, et al., 2017)

Core features of ELISpot include:

• Ultra-high sensitivity: Capable of detecting single active cells among hundreds of thousands to millions of cells.
• Functional assessment: Reflects real-time cellular secretion rather than protein abundance alone.
• Single-cell resolution: Quantitative determination of secreting cells by spot counting.
• High throughput: 96-well plate format allows parallel screening of multiple samples or conditions.

Comparison between ELISpot and ELISA

Although both ELISpot and ELISA share enzyme-linked immunoassay foundations, they differ fundamentally in design, purpose, and applications. ELISA measures the total amount of soluble target protein in a sample, while ELISpot evaluates the frequency of cells capable of secreting the target protein, providing a semi-quantitative functional assessment.

Table 1. Key Differences between ELISpot and ELISA

Detailed ELISpot Workflow

A successful ELISpot experiment requires careful operation and quality control. The standard workflow for cytokine detection is as follows:

Figure 3. Quantification of antibody-secreting cells (ASCs) using the ELISpot assay. (Brunner C, et al., 2025)

1. Plate Preparation and Coating

• Plate type: PVDF-bottom plates (e.g., Mabtech MSIP or MAIPSWU). The porous PVDF membrane provides a high surface area for efficient antibody binding.
• Ethanol pre-treatment: Activate the hydrophobic PVDF membrane with 35–70% ethanol for 1–2 minutes, followed by thorough washing.
• Antibody coating: Add capture antibodies diluted in PBS (typically 15 μg/mL, 100 μL/well) and incubate overnight at 4°C. Keep membranes moist and handle gently.

2. Blocking

Remove coating solution, wash with PBS containing Tween-20, then incubate with 1–5% BSA or fetal calf serum (FCS) at 37°C for 1–2 hours to block unbound sites and reduce background.

3. Cell Preparation and Seeding

• Cell sources: PBMCs (most common), splenocytes, tissue-derived cells, or cell lines. Cell viability should exceed 90%.
• Cryopreservation and thawing: Standard procedures using DMSO/FCS. Wash twice post-thaw and rest at 37°C for at least 1 hour.
• Cell counting: Adjust cell numbers based on expected response frequency (e.g., 250,000 cells/well for low-frequency responses).
• Culture medium: RPMI 1640 supplemented with 10% FCS, L-glutamine, HEPES, and antibiotics. Consistency is essential.

4. Stimulation and Incubation

• Stimuli: Antigen-specific peptides, polyclonal stimulants (PHA, anti-CD3).
• Controls: Positive (polyclonal), negative (cells + medium), reagent/background controls (medium only).
• Incubation: 37°C, 5% CO₂, 16–48 hours. Avoid movement to prevent spot smearing; use aluminum foil to reduce evaporation.

5. Detection and Development

• Washing: Thorough PBS washes (5–6 times) to remove cells.
• Detection antibody incubation: Biotinylated antibodies, 37°C, 1–2 hours.
• Enzyme conjugate incubation: Streptavidin-ALP or HRP, 37°C, 1 hour.
• Substrate reaction: BCIP/NBT (ALP, blue-purple) or TMB (HRP, dark blue). Stop reaction with water at the optimized time to avoid excessive background.

6. Spot Analysis and Data Interpretation

• Drying: Plates must be fully dried before analysis (overnight or 30 min in a laminar flow hood).
• Counting: Automated ELISpot readers (e.g., Mabtech IRIS/ASTOR) with software like Apex are recommended.
• Data representation: Spots per million cells (SFC/10⁶). Positive response is determined statistically, not by simple subtraction from controls.

Applications

ELISpot is widely applied due to its unique advantages:

1. Vaccine research and evaluation: Measures antigen-specific T and B cell responses, considered a gold standard for immunogenicity assessment.
2. Infectious disease research: Used for TB (T-SPOT.TB), HIV, and COVID-19 immune monitoring, offering high specificity.
3. Tumor immunology: Evaluates tumor-specific T cell responses and monitors adoptive cell therapies (CAR-T, TIL).
4. Autoimmune and allergy research: Detects autoreactive T cells or pathogenic antibody-producing B cells to study disease mechanisms.
5. Transplant immunology: Monitors recipient responses to donor antigens, predicting rejection risk.

Advantages and Limitations

Advantages:

• Ultra-high sensitivity for low-frequency responses.
• Functional detection reflecting real cell activity.
• Single-cell resolution with direct and quantitative readout.
• High throughput for large-scale screening.
• High reliability with standardized procedures.

Limitations and Challenges:

• Highly dependent on cell viability and sample handling; thawing must be cautiously.
• Sensitive to operational variations; washing and substrate steps require precision.
• Detects only secreted proteins, not intracellular stores.
• Limited throughput for traditional ELISpot (FluoroSpot mitigates this).
• Data analysis requires careful manual oversight and statistical consideration despite automated readers.

Related Services and Products

Creative Biogene's expertise in recombinant protein expression, stable cell line generation, and viral vector packaging enables researchers to prepare high-quality reagents and cell models for ELISpot and other immune assays.

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