The principles of ELISpot
ELISpot is used to quantify protein-secreting cells.
In ELISpot, a cell suspension is added to the wells of a plate coated with protein-specific capture antibody. Protein secreted by the cells is immediately captured by these antibodies during the entire stimulation period. The cells are removed and then biotinylated protein-specific detection antibody is added followed by a streptavidin-enzyme conjugate. Visible spots are formed after adding a precipitating substrate.
The plate is then read using an ELISpot reader where each spot corresponds to a responding analyte-secreting cell.
What are the benefits of ELISpot?
Identify rare cells
ELISpot is a cell-based immunoassay used to quantify analyte-secreting cells at the single-cell level. Due to its capacity to identify one cell in a million, ELISpot is considered one of the most sensitive cellular assays available. This makes the method particularly valuable for studies of rare cell populations. With minimal manipulation of the cells, ELISpot offers a closer representation of reality compared to many other cell-based immunoassays.
Easy to scale up
ELISpot is robust and easy to perform, making it suitable for analyzing many samples in parallel or at different time points during a clinical trial. The reproducibility of the method has been repeatedly shown in numerous proficiency tests. Standardization of the ELISpot assay has been achieved in specific settings and the method is the basis of an FDA-approved diagnostic test for tuberculosis.
The assay is conducted in a 96-well plate and is analyzed with an automated ELISpot reader, allowing for rapid analysis of multiple plates consecutively.
Capture transient analytes
As ELISpot captures target proteins immediately after secretion, the method can detect cytokines that otherwise are too diluted or disappear
from samples. For example, ELISpot can detect analytes that are rapidly degraded by proteases (IL-2), are quickly taken up by bystander cells (IL-4), or bind to soluble receptors (TNF-α).
ELISpot applications and usage
T cell profiling
ELISpot is commonly used to investigate antigen-specific immune responses and to discriminate between subsets of activated T cells. This is applied in studies of infectious diseases, cancer, allergies, and autoimmune diseases. In vaccine research, ELISpot is the gold standard to define vaccine efficacy by measuring the capacity to elicit T cell responses, for example by assessing IFN-γ secretion. Diagnostic assays based on ELISpot are available, including tests to detect patients with tuberculosis or SARS-CoV-2 infection by measuring IFN-γ secretion from T cells responding to defined peptides.
Human IFN-γ ELISpot
IFN-γ secretion by peripheral blood mononuclear cells (PBMCs) incubated overnight without stimuli or with anti-CD3, phytohaemagglutinin (PHA) , or purified protein derivative (PPD).
B cell profiling
The B cell ELISpot assay is one of few assays measuring immunoglobulins directly upon secretion. There are two strategies: First, B cell ELISpot can be used to assess antibody-secreting cells (ASCs). Due to its sensitivity, the method enables the identification of rare ASCs to a specific antigen. Secondly, you can evaluate circulating antigen-specific memory B cells after polyclonal activation. B cell ELISpot is thus regularly used to detect B cell responses elicited by infection or vaccination.
Human IgG and IgA ELISpot
PBMCs were collected before and after vaccination against influenza and secretion of influenza A (H1N1)-specific IgG and IgA was analyzed by ELISpot.
Vaccine immune profiling
ELISpot assays are a robust method for assessing vaccine-induced immunity by quantifying antigen-specific T and B cell responses through detecting cytokines like IFN-γ and IL-2 or immunoglobulin secretion. The assay allows researchers to gauge a vaccine's immunogenicity and potential protective effects, providing critical insights into their performance and function. Our white paper takes a deeper look into ELISpot's role in vaccine development, offering a detailed perspective on its application in shaping vaccine-mediated immunity.
Mabtech's ELISpot kits, utilized in studies of a Nobel laureate caliber, enable precise immune monitoring, capturing the activity of T cells through key markers like IFN-γ or Granzyme B. This technology has been instrumental in advancing cancer immunotherapy research, offering insights into how the immune system targets and destroys cancer cells. Explore our white paper to see how ELISpot has facilitated this groundbreaking work, spotlighting the assay's critical role in the development of new cancer treatments.
Our kit formats
We offer ELISpot kits in different formats for different research needs: from flexible kits that include the core reagents to complete pre-coated kits made for studying the immune response to specific pathogens. Find all our ELISpot kits here.
The future of ELISpot analysis
It is possible to analyze ELISpot plates with the naked eye but to save (a considerable amount of) time and minimize errors, an automated reader is highly recommended.
Mabtech ASTOR 2 is tailor-made for ELISpot analysis. It allows for a plug-and-play workflow: No calibration is needed thanks to the fixed camera and an automatic XY table. In addition, the spot-counting algorithm RAWspot is based on signal processing and therefore able to identify spots reliably up to 3,000 spots per well.
Our ELISpot kits appear in numerous publications ranging from vaccine development to cancer research and diagnostics. Below is a list of some of our favorites. Read them all in our Publication database.
Gebre et al., Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine, Nature 2022
Marsh et al., ChAdOx1 nCoV-19 (AZD1222) vaccine candidate significantly reduces SARS-CoV-2 shedding in ferrets, NPJ Vaccines 2021
Te Kamp et al., Comparable Long-Term Rabies Immunity in Foxes after IntraMuscular and Oral Application Using a Third-Generation Oral Rabies Virus Vaccine, Vaccines (Basel) 2021
Gasser et al., The mechanistic and functional profile of the therapeutic anti-IgE antibody ligelizumab differs from omalizumab, Nat Commun. 2020
Sicard et al., Donor-specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients, Am J Transplant. 2020