ELISpot Assay: Versatile Applications and Common Challenges to Overcome

Christine Bain, Ph.D. November 29, 2023

ELISpot (Enzyme-linked immunosorbent spot) is an immunoassay-based technique used to quantify cells that are capable of secreting proteins, including cytokines, cytotoxic molecules, and antibodies, when stimulated with the appropriate antigen. Initially designed to monitor the frequency of circulating antigen-specific memory B-cells, it is now commonly used to determine the frequency of antigen-reactive T-cells by measuring the secretion of specific immune mediators at a single-cell level.

What is the principle of the ELISpot?

Roughly, an ELISpot assay is a sandwich ELISA on cells. Upon stimulation with a specific antigen, the analyte of interest is immediately captured by a specific antibody coated on a membrane of the 96-well plate. After washing off the cells, the secreted protein is revealed with a detection antibody, and resulting immune complexes, corresponding to spot-forming cells, are enumerated by an automated reader.

The applications of ELISpot assay are diverse

ELISpot allows measuring a wide range of cellular immune functions, including cytolytic activity and the secretion of one or multiple cytokines like IFN-γ and IL4 or other bio-active molecules like granzyme B, a specific indicator of Cytotoxic T lymphocytes (CTL) and Natural Killers (NK) cytotoxic ability. It is a very powerful tool used in the context of infectious and autoimmune diseases, allergies, organ transplantation, cancer research, and vaccine development.

It serves to predict the clinical benefit of vaccine-based therapeutic interventions, or what is called “wanted immunogenicity” which is the ability of a vaccine to trigger antigen-specific immune responses. This is the case for preventive vaccines for acute infectious diseases such as flu, COVID-19, RSV but also therapeutic vaccines, aiming at breaking tolerance and enhancing immune responses to established diseases such as chronic infections or non-communicable diseases such as cancer, diabetes or Alzheimer’s disease…

ELISpot can also help spot “unwanted immunogenicity” for safety purposes, for instance, in cell and gene therapies (CGT), to detect immune responses to the adeno-associated virus (AAV) capsid, the encoded functional transgene product [1], or the chimeric antigen receptor in CAR-based therapies [2], that may affect treatment outcome. Unwanted immunogenicity by Th-1 T-cells is also carefully monitored as it has caused deleterious side effects in patients with Alzheimer’s disease after administration of one of the first Amyloid-beta vaccine [3].

A technology with many advantages but some major challenges to tackle

As described above, ELISpot technology is incredibly versatile, allowing for a wide range of applications. In addition, depending on the assay format, ELISpot can differentiate different types of circulating T cells: effector T cells will be detected by direct ex-vivo ELISpot while in vitro expansion prior to ELISpot will be needed to identify circulating memory T cells. Furthermore, the ELISpot technique is highly sensitive, enabling a detailed observation of the consequences of immune therapy. Despite its numerous advantages, ELISpot presents some challenges that we have learned to tackle through our extensive experience in the field.

Like any cell-based assay, ELISpot shows higher variability than an immunoassay for measuring a soluble analyte. Having appropriate control samples is of utmost importance for proper development and validation of the method but also to ensure no method drift or technical issues arise during the clinical study. When our goal is to document a specific response to a vaccine in the context of commonly encountered viruses, like flu, samples with and without pre-existing positive responses are easily accessible from healthy blood donors who have been exposed to seasonal viruses.

This becomes more challenging when the antigens being tested do not typically induce positive responses in healthy individuals. This is the case in immuno-oncology, where the antigens are either tumor-associated antigens (TAAs) to which the general population is tolerant or neoantigens which are patient-specific. This is also the case when attempting to document the absence of a response in the case of unwanted immunogenicity, to neurotoxic proteins in AD, or any gene replacement therapies. In these settings, positive control samples cannot be easily found in the general population. One alternative is to develop and validate the method using a surrogate antigen, such as the human cytomegalovirus (HCMV) pp-65 structural protein, possibly completed by in-study validation, provided that positive responses are induced. One crucial parameter is to define a positivity threshold on samples, collected from randomly selected healthy donors, stimulated with the antigen of interest.

Since the inception of the company, we have gained experience with multiple pathologies and disruptive therapeutic products in non-clinical non-human primate and clinical studies.

We have earned our first stripes with therapeutic vaccines, based on viral vectors or DNA, encoding viral antigens, developed for chronic infectious diseases (HCV, HBV, HPV). In these pathologies, characterized by impaired adaptive immunity, thus low frequencies of antigen-specific T cells, getting samples reacting to disease-related antigens is one of the biggest challenges for method development. For that, we have been relying on a network of clinicians and biobanks, as well as tight interactions with our clients, to source antigen-reactive samples.

Since then, we have been supporting the development of TAA- or personalized neoantigen-based vaccines in hematological and solid tumors, prophylactic T-cell vaccines for acute viral infections such as flu or SARS-CoV-2 viruses, and lately active immunization to induce antibodies specific for neurotoxic proteins in AD.

Dealing with samples of poor quality has been a recurring challenge for our company in the early days, it has been a driver to work on ways to secure the starting material of our ELISpot analyses. Over the years, we have gained a lot of experience in this area, and for some clients, we have successfully established a network of labs, close to the clinical centers and trained for processing samples according to a standardized protocol, ensuring that samples are processed quickly and with the utmost quality.

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

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