By Dr. Kurt Sales, CSO
Introduction
Immunological assays are essential in drug development, especially for immunomodulatory therapies targeting inflammation, cancer, or autoimmune diseases. Among these assays, the Enzyme-Linked ImmunoSpot (ELISpot) assay has emerged as a critical assay for evaluating cellular immune responses. ELISpot enables the detection of cytokine-secreting cells at the single-cell level, making it a valuable tool for assessing immune function in clinical trials. This blog explores ELISpot’s role in drug development. We discuss, using a case study conducted at Agilex Biolabs and published in the Journal Immunohorizons, how discrete immune cells can be isolated using magnetic sorting beads for functional analysis in clinical trials.
The Importance of ELISpot in Drug Development
ELISpot is widely used in preclinical and clinical immunological assessments due to its high sensitivity, specificity, and ability to detect rare immune cell responses. The assay quantifies antigen-specific T-cell or B-cell responses by capturing secreted cytokines or antibodies. This capability is crucial for assessing the efficacy and safety of new therapeutics, especially in immuno-oncology, vaccine development, and autoimmune disease treatments.
Key Advantages of ELISpot
- Single-Cell Resolution: ELISpot can detect responses from individual immune cells, providing precise immune monitoring.
- High Sensitivity: The assay can identify rare antigen-specific responses, making it particularly useful in early-phase clinical trials.
- Quantitative and Qualitative Data: ELISpot provides insights into both the frequency and functionality of immune cells.
- Versatility: The assay can measure responses from T cells, B cells, and other immune cell subsets, allowing broad immunological assessments.
Application of ELISpot in Clinical Trials
ELISpot is frequently employed in different stages of clinical trials to assess immune responses to candidate drugs. Below are some key applications:
1. Vaccine Development
In vaccine trials, ELISpot is used to measure antigen-specific T-cell responses, providing early indicators of immunogenicity and potential protective immunity.
2. Immuno-Oncology
For cancer immunotherapies, such as checkpoint inhibitors and adoptive T-cell therapies, ELISpot helps in determining whether a patient’s immune system is capable of mounting an effective response against tumor antigens.
3. Autoimmune Disease Monitoring
In autoimmune diseases, ELISpot assays detect aberrant immune activation and assess the efficacy of immunosuppressive or immunomodulatory drugs.
Isolation of Immune Cells Using Magnetic Sorting Beads
To perform ELISpot effectively, high-purity immune cells are required. Magnetic-activated cell sorting (MACS) is a widely used method for isolating discrete immune cell populations from whole blood or peripheral blood mononuclear cells (PBMCs). The use of magnetic sorting beads enables rapid, high-yield, and high-purity isolation of specific immune subsets, ensuring accurate downstream functional analyses.
Magnetic Bead-Based Cell Sorting
Magnetic bead-based sorting relies on antibodies conjugated to magnetic beads that selectively bind to surface markers of target cells. A magnetic field is then applied to separate labeled cells from the rest of the population. This method allows for the enrichment of highly specific immune cell subsets, such as CD4+ and CD8+ T cells, B cells, monocytes, and dendritic cells.
Steps in Magnetic Sorting for ELISpot Analysis:
- Sample Collection: Whole blood or PBMCs are collected from study participants.
- Incubation with Magnetic Beads: Antibodies conjugated to magnetic beads bind to specific cell surface markers.
- Magnetic Separation: A magnetic field is applied, isolating target cells while non-bound cells are washed away.
- Cell Viability Assessment: Isolated cells are assessed for viability and purity before being used in ELISpot assays.
Benefits of Magnetic Sorting for ELISpot
- High Purity: Ensures minimal background noise in ELISpot assays.
- Preserved Functionality: Maintains cell viability and function, essential for accurate immune response measurement.
- Rapid and Scalable: Facilitates processing of large sample numbers in clinical trials.
Functional Analysis of Isolated Immune Cells
Once purified, immune cells can be analyzed using ELISpot to determine their functional activity in response to drug candidates. The following analyses can be performed:
1. Cytokine Secretion Assays
ELISpot detects cytokine secretion at a single-cell level, measuring responses to antigens or drug candidates. This analysis is crucial for evaluating immune activation, suppression, or dysregulation.
2. Antigen-Specific T-Cell Responses
By exposing isolated T cells to antigens or peptides, ELISpot can quantify the frequency of antigen-reactive cells, providing insights into the drug’s impact on cellular immunity.
3. B-Cell ELISpot for Antibody Production
For vaccines and monoclonal antibody development, B-cell ELISpot assesses antigen-specific antibody-secreting cells, informing on immunogenicity.
4. Drug Modulation of Immune Responses
By testing immune cells in the presence of a drug, ELISpot can determine whether the drug enhances or suppresses immune function, guiding dose selection and safety evaluations.
Integration with Other Immunological Assays
ELISpot is often combined with flow cytometry, ELISA/MSD, and multiplex cytokine assays for comprehensive immune profiling, ensuring a thorough evaluation of drug-induced immune modulation.
Case Study
Combining magnetically isolated CD45 cells with serum maintains intact drug responsiveness for ELISpot analysis in clinical trials.
At Agilex Biolabs, we frequently support a variety of immunological analyses in clinical trials. These range from multicolour flow cytometry panels to activation and proliferation assays and ELISpot analyses. Each have their own benefits and limitations. ELISpot classically utilizes peripheral blood mononuclear cell isolates from whole blood, requiring relatively high blood draw volumes and removing both granulocytes and bound drug. In our recent paper, published in Immunohorizons (Mavrangelos, C. et al. Immunohorizons 2025 Jan 27;9(3):vlae012.doi: 10.1093/immhor/vlae012), we describe a novel protocol whereby CD45 cells are magnetically isolated from human whole blood and co-incubated with serum isolated from the same subject in order to maintain responsiveness during analyses of clinical trial samples.
In our study, we spiked whole blood with infliximab, a well characterized anti-tumor necrosis factor α (TNF-α) chimeric antibody used to treat autoimmune diseases including inflammatory bowel disease, arthritis, ankylosing spondylitis, and psoriasis. Infliximab is well known to partition into serum with a reported peak serum concentration (CMAX of approximately 120µg/mL.). We aimed to assess whether serum containing infliximab at a concentration <50% of CMAX inhibits TNF-α secretion in stimulated lymphocytes isolated from whole blood. We demonstrate that magnetically isolated CD45 cells are suitable for ELISpot analysis and that drug response remains intact when these cells are combined with serum, providing proof of concept for use in longitudinal clinical trials in which blood volume may be limited and pharmacodynamic assessment requires drug response to be intact. For more detailed information on our study and how this could potentially benefit your next clinical study, the paper can be freely downloaded from PubMed or Immunohorizons (Mavrangelos, C. et al. Immunohorizons 2025 Jan 27;9(3):vlae012.doi: 10.1093/immhor/vlae012).
Conclusion
ELISpot is an indispensable tool in immunological analyses supporting clinical trials of new drugs. Its ability to detect antigen-specific immune responses at the single-cell level makes it crucial for vaccine development, immuno-oncology, and autoimmune disease research. The purification of immune cells using magnetic sorting beads further enhances assay accuracy by providing highly enriched cell populations for functional analysis. By integrating ELISpot into clinical trials, biotech and pharmaceutical companies can obtain critical immune response data, aiding in drug development, regulatory compliance, and ultimately improving patient outcomes. To learn more about how Agilex Biolabs can support your next vaccine or immune-oncology study, contact us today at BD@agilexbiolabs.com, for a confidential discussion.
