Over the past several years, the life science community has gained an improved understanding of the immune system–from B and T cells to molecular pathways. With new, state-of-the-art tools and technologies, immunologists now have the ability to take a holistic approach to understanding the mechanisms of immune response. These applications help us devise methods to treat immune system-related diseases and to design better vaccines to combat infectious agents and cancer.
Currently, one of the most sensitive techniques available for the detection, measurement, and functional analysis of immune cells is the enzyme-linked immunospot (ELISPOT) assay. Covance’s Translational Biomarker Solutions group uses the ELISPOT technique to evaluate test subject responses to our sponsors’ vaccines, drugs, and biological products. ELISPOT is also useful in the pre-clinical space, where Covance can assess the toxicity of investigational compounds and biologics, and evaluate the efficacy of vaccines.
The ELISPOT assay is one of the most sensitive cellular assays available for detecting and enumerating individual cells that secrete a particular protein in vitro. Based on the enzyme-linked immunosorbent assay (ELISA), the ELISPOT assay was originally developed for analyzing specific antibody-secreting cells, but has been adapted to measure the frequencies of cells that produce and secrete other effector molecules, such as cytokines.
Depending on the cytokine/factor analyzed, the ELISPOT assay is between 200 and 400 times more sensitive than a conventional ELISA assay, with the ability to detect up to 1 in 300,000 low frequency cytokine-secreting cells. Since the cytokine released in response to antigen can be mapped to a single cell, T cell responder frequencies can be calculated. The ELISPOT also provides an indication of the type of cytokine response that has been elicited and hence the type of immune response. The data generated from ELISPOT can give better quantitative and qualitative data when compared with other methods such as intracellular cytokine staining.
Applicability Across Multiple Immunology Disciplines
ELISPOT testing can be used across several disciplines of immunology, and is particularly useful in areas where the ex vivo measurement of low-frequency T cell responses is required
For instance, ELISPOT assays are useful in monitoring the frequency and profile of circulating donor-reactive T cells in organ transplantation; measuring cytotoxic T lymphocyte (CTL) activity in cancer research; measuring memory responses in infectious diseases and vaccine development; and detecting autoreactive cells (which typically occur at low frequencies) in autoimmune diseases.
ELISPOT can also be used pre-clinically to address if a test article is capable of eliciting an unwanted immune response prior to its use in humans. Such an unwanted immune response can be elicited in the form of activation of immune cells that lead to the secretion of very high levels of cytokines, a process termed as “cytokine storm.”
Maintaining ELISPOT Performance Standards
The multiple, complex steps of an ELISPOT assay require highly specialized capabilities. Following are some factors that can impact ELISPOT performance:
Cell recovery and viability: One of the most critical steps in standardizing ELISPOT is to ensure adequate cell recovery and viability after rescuing peripheral blood mononuclear cells (PBMCs) from cryopreservation. Covance’s Central Laboratory Services with its global presence can quickly prepare and cryopreserve PBMC from test subjects and ship PBMC to a single location for testing, ensuring optimal performance in functional assays such as ELISPOT.
Operator technique and proficiency: Carrying out ELISPOT assays with inexperienced staff can be time consuming and lead to variable results. Covance Translational Biomarker Solutions has two Ph.D.-level scientists and experienced associates trained specifically in ELISPOT technique, who perform these assays routinely using optimized and standardized protocols with minimal variation.
Result Variability: In order to minimize variation in results – and conversely, the ability to confidently compare subjects from different clinical trial sites – steps should be taken to ensure initial assay performance and adherence to clinical standards over time. In addition to following a standard operating procedure (SOP) for the execution of ELISPOT, our experts follow a documented training protocol, all within a Good Research Practice (GRP) environment.