ELISpot Assay: Applications and Challenges

Christine Bain, Ph.D. April 17, 2025

Initially developed to assess the frequency of circulating antigen-specific Antibody-Secreting Cells (ASC), ELISpot has become a vital tool for quantifying antigen-reactive T-cells by measuring secreted immune mediators such as cytokines or key molecules involved in cell-mediated cytotoxicity. Compared to other assays for monitoring cell-mediated immunity (CMI), such as ELISA (cytokine release) or flow cytometry (intracellular cytokine staining /ICS or tetramer/multimer staining), ELISpot assay offers greater sensitivity and detailed single-cell analysis without the need for complex cell sorting. These advantages have made it a widely used tool in both clinical and research settings.

What is an ELISpot assay, and How Does It Work?

An ELISpot assay can be thought of as a sandwich ELISA performed on cells, most commonly, cryopreserved peripheral blood mononuclear cells (PBMCs). Antigen-reactive PBMCs secrete the analyte of interest, which is captured by a specific antibody coated on the membrane of a 96-well plate. After the cells are washed away, a detection antibody is added to reveal the secreted protein. The resulting immune complexes, corresponding to spot-forming cells, are then counted using an automated reader.

The key reagents for an ELISpot assay include capture & detection antibodies specific to the target analyte (e.g., cytokine or antibody) as well as stimulating agents (e.g., antigen-derived peptides, recombinant proteins, or whole pathogens) used to activate the cells. However, even with optimal key reagents, reliable data cannot be obtained if the responding cells are not healthy and “happy”.

Common Challenges in ELISpot Assay and How to Overcome Them

ELISpot technology is highly versatile, enabling a wide range of applications. Depending on the assay format, ELISpot can differentiate between various types of circulating T cells: effector T cells can be detected by ex vivo ELISpot, while ELISpot after in vitro expansion will identify circulating central memory T cells. Despite its many advantages, ELISpot does present some challenges, which we have learned to address through extensive experience in the field.

Ensuring Robustness in ELISpot Assays Through Appropriate Controls

Like any cell-based assay, ELISpot exhibits higher variability than ligand binding assays (LBAs) used to measure soluble analytes. Therefore, the inclusion of appropriate control samples is essential, not only for the proper development and validation of the method, but also for detecting potential method drift or technical issues during clinical studies. Samples from the general population, screened for positive responses to commonly encountered infectious agents or mandatory vaccines, can serve as controls.

Optimizing ELISpot Assay Protocol for Consistency

To ensure optimal results, it is important to use high-quality cells and plate them at an appropriate density to allow effective contact with both the antigen and the capture antibody. For ex vivo ELISpot, the typical incubation time ranges from 18 to 24 hours, though it can be extended depending on the expected response levels. Stimulating agents should be titrated to achieve optimal immune activation with minimal background noise.

Applications of the ELISpot Assay in Research and Clinical Settings

The Interferon-gamma (IFN-g) ELISpot is the most widespread format for monitoring either wanted or unwanted immunogenicity. It is a powerful tool for understanding natural immunity to infection, measuring T-cell responses upon vaccination, or dissecting immunological processes underlying immune-mediated non-communicable diseases.

Vaccine Development

ELISpot helps predict the clinical benefit of vaccine-based therapeutic interventions, known as “wanted immunogenicity,” which refers to a vaccine’s ability to induce antigen-specific T cell responses. This applies to preventive vaccines for acute infectious diseases like flu, HIV, Tuberculosis (TB) or Malaria, as well as therapeutic vaccines aimed at breaking tolerance and boosting immune responses in chronic infections (HBV, HCV, HPV…) or non-communicable diseases such as cancer, diabetes, and Alzheimer’s disease.

Cell and Gene Therapies

ELISpot is also valuable for detecting “unwanted immunogenicity” to ensure efficacy of cell and gene therapies (CGT) or safety for vaccines directed to antigens, expressed in vital organs such as the brain. It can identify immune responses against components like the adeno-associated virus (AAV) capsid, the encoded transgene product [1], or the chimeric antigen receptor (CAR) in CAR-based cell therapies [2], which could impact treatment outcomes. Additionally, unwanted Th-1 T-cell immunogenicity is closely monitored for vaccine targeting neuronal proteins, as it has been linked to harmful side effects in patients with Alzheimer’s disease after the administration of one of the early Amyloid-beta vaccines [3].

KCAS Bio’s Proven Track Record in ELISpot Assays for Immunogenicity Evaluation

Since the founding of our company, we have gained significant experience with a range of pathologies and innovative therapeutic products through non-clinical studies in non-human primates and clinical studies. Our journey began with therapeutic vaccines, including viral vector- and DNA-based vaccines targeting viral antigens for chronic infectious diseases like HCV, HBV, and HPV. In these diseases, characterized by impaired natural adaptive immunity and low frequencies of antigen-specific T cells, obtaining samples that react to disease-related antigens has been one of the biggest challenges in method development. To overcome this, we’ve relied on a network of clinicians, biobanks, and close interactions with our clients to source antigen-reactive samples.

Since then, we have expanded our expertise to support the development of tumor-associated antigen (TAA)- or personalized neoantigen-based vaccines for both hematological and solid tumors, as well as prophylactic T-cell vaccines for acute viral infections like flu and SARS-CoV-2. More recently, we have focused on active immunization strategies aimed at inducing antibodies against neurotoxic proteins in Alzheimer’s disease, monitoring for potentially harmful T cell responses.

Dealing with poor-quality samples in the early stages of our work was a significant challenge, driving us to find ways to secure high-quality starting material for our ELISpot analyses. Over the years, we have sharpened our capabilities, establishing, for some clinical studies, a network of labs near clinical centers, trained and qualified to process and cryopreserve PBMC samples within 8 hours of blood draw. We even managed, during the COVID-19 lockdown, to successfully train and qualify laboratory technicians remotely, using virtual glasses, for preparing PBMC for a phase 1 clinical study in Alzheimer’s disease.

Let’s discuss how our successful track record in ELISpot assays can help you explore and document the immunogenicity of your therapeutic candidate or product.

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