Over the past several years, the scientific community has made tremendous progress in advancing our understanding of the immune system, from the basic functions of its various components to molecular pathways that operate within those components. With new, state-of-the-art tools and technologies, immunologists now have the ability to better understand the mechanisms of immune response to various antigens, thereby aiding them in the development of novel approaches to treat immune-system-related diseases and better design 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 uses the ELISPOT technique in applications such as evaluation of vaccine efficacy and immunogenicity of biological products.
Benefits of ELISPOT assays
The ELISPOT assay is one of the most sensitive in vitro cellular assays for detecting and enumerating individual immune cells that secrete soluble mediators in response to a stimulus. The ELISPOT assay was originally developed for analyzing specific antibody-secreting cells based on the enzyme-linked immunosorbent assay (ELISA) but it has been adapted to measure the frequencies of cells that produce and secrete other effector molecules such as cytokines.
Depending on the soluble factor analyzed, the ELISPOT assay is between 200 and 400 times more sensitive than a conventional ELISA assay. It has 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 determined using this technique. ELISPOT assays also provide an indication of the type of cytokine response that has been elicited and hence the type of immune response. ELISPOT assays can yield 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 disease.
Biological therapeutic agents such as antibodies that are widely used to treat various diseases including cancer and autoimmunity are capable of inducing unwanted immune responses. Such an unwanted immune response can be manifested in the form of activation of immune cells that can lead to the secretion of very high levels of inflammatory cytokines, a phenomenon termed as ‘cytokine storm’. A ‘cytokine storm’ can have serious consequences including death. ELISPOT assays can also be used to assess if a biologic is capable of eliciting a ‘cytokine storm’ prior to its use in animals and humans.
Maintaining ELISPOT Performance Standards
The multiple, complex steps of an ELISPOT assay require highly specialized capabilities. The following are some factors that can impact ELISPOT assay performance.
Cell recovery and viability: One of the most critical steps in standardizing an ELISPOT assay is to ensure adequate cell recovery and viability after rescuing peripheral blood mononuclear cells (PBMCs) from cryopreservation. With its global presence, Covance’s Central Laboratory Services can quickly prepare and cryopreserve high quality PBMCs from test subjects and ship PBMCs to a single location for testing, ensuring optimal performance in functional assays such as ELISPOT.
Operator Technique and Proficiency: Carrying out ELISPOT assays can be laborious and can yield variable results. Covance has experienced scientists and associates trained specifically in ELISPOT technique, who perform these assays routinely using optimized and standardized protocols with minimal assay variability.
Result variability: In order to minimize variability in results and improve 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 assays, Covance experts follow a documented training protocol, under a set of guiding ‘quality principles’. Our assay validation parameters ensure optimal assay performance and high quality data generation.
ELISPOT expertise, in concert with global reach, is an important capability for your CRO partner of choice. For more information, please visit Covance.com.