The enzyme-linked immunospot (ELISPOT) assay took its design concept from traditional ELISA techniques and evolved over the years from a method for detecting antibodies secreted from B cells to a ...method for detecting cytokines or other soluble mediators secreted from a variety of different cell types. The ELISPOT assay allows the quantitative measurement of frequency of cytokine secreting cells at the single cell level directly ex vivo without elaborate in vitro expansion or manipulation of cell populations. The function of cells can be inferred from the pattern of cytokines secreted by cells in response to diverse antigenic stimuli and thus the ELISPOT assay has become a powerful method for monitoring immune responses in health and disease. The ELISPOT assay like the ELISA assay is relatively easy to perform and it has the promise of robustness, reliability, and reproducibility of performance for use as a diagnostic tool. The history, applications, validation process, and future challenges of the ELISPOT assay are discussed in this chapter.
The ELISpot, a heterogeneous immunoassay, is widely used for detection of low abundant analytes. It is a reliable and robust assay to monitor responses of the immune system at the single-cell level ...by capturing secreted molecules of interest with specific, membrane-bound antibodies. Those molecules are then made visible by a cascade of ELISA-related development steps. The final results are distinct spots on the membrane as an imprint of the cell secreting the captured molecules, not only allowing their quantification but also providing insight on the kinetics and strength of secretion. This chapter describes the optimized protocol steps of the ELISpot technique, important improvements and tools available for the community, and the current expansion of the technique into polyfunctional cell analysis.
Investigating the neutralizing antibody (NAb) titer against HSV-1 is essential for monitoring the immune protection against HSV-1 in susceptible populations, which would facilitate the development of ...vaccines against herpes infection and improvement of HSV-1 based oncolytic virotherapy.
In this study, we have developed a neutralization test based on the enzyme-linked immunospot assay (ELISPOT-NT) to determine the neutralizing antibody titer against HSV-1 in human serum samples. This optimized assay employed a monoclonal antibody specifically recognizing glycoprotein D to detect the HSV-1 infected cells. With this test, the neutralizing antibody titer against HSV-1 could be determined within one day by automated interpretation of the counts of cell spots. We observed good correlation in the results obtained from ELISPOT-NT and plaque reduction neutralization test (PRNT) by testing 22 human serum samples representing different titers. Moreover, 269 human serum samples collected from a wide range of age groups were tested, the average neutralizing antibody titer (log2NT50) was 8.3 ± 2.8 and the prevalence of NAbs was 83.6 % in this cohort, it also revealed that the average neutralizing antibody titer in different groups increased with the age, and no significant difference in neutralizing antibody titers was observed between males and females.
These results prove that this novel assay would serve as an accurate and simple assay for the assessment of the neutralizing antibody titers against HSV-1 in large cohorts.
The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot ...(ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-γ, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion.
Celiac disease (CD) is a common lifelong food intolerance triggered by dietary gluten affecting 1% of the general population. Gliadin-specific T-cell lines and T-cell clones obtained from intestinal ...biopsies have provided great support in the investigation of immuno-pathogenesis of CD. In the early 2000 a new in vivo, less invasive, approach was established aimed to evaluate the adaptive gliadin-specific T-cell response in peripheral blood of celiac patients on a gluten free diet. In fact, it has been demonstrated that three days of ingestion of wheat-containing food induces the mobilization of memory T lymphocytes reactive against gliadin from gut-associated lymphoid tissue into peripheral blood of CD patients. Such antigen-specific T-cells releasing interferon-γ can be transiently detected by using the enzyme-linked immunospot (ELISPOT) assays or by flow cytometry tetramer technology. This paper discusses the suitability of this in vivo tool to investigate the repertoire of gluten pathogenic peptides, to support CD diagnosis, and to assess the efficacy of novel therapeutic strategies. A systematic review of all potential applications of short oral gluten challenge is provided.
The quantification of single cell interferon-gamma (IFN-γ) release for assessing cellular immune responses using the Enzyme-linked immunospot (ELISPOT) assay is an invaluable technique in immunology. ...Peripheral blood mononuclear cells (PBMC) are stimulated in vitro with recombinant proteins, peptides and recently whole malaria organisms. Stimulation may be short term (20-36 h) or long term (cultured ELISpot, up to 7 days). ELISpot is also able to quantify other cytokines secreted by antigen-specific T-cells, such as interleukin-2, interleukin-5, and other interleukins. ELISpot is playing an important role especially in vaccine research studies.
Abstract A central, yet unresolved issue in the pathogenesis of HIV disease is the mechanism of antibody perturbation. In this study, HIV-specific memory B-cells were quantified in groups of infected ...subjects and compared with memory responses to other antigens and antibody titers. HIV-specific memory B-cell responses were vigorous in individuals with CD4+ T-cell counts > 350/μl and weak or undetectable in subjects with CD4+ T-cell numbers < 200/μl. Memory B-cell loss was permanent, because antiretroviral therapy failed to restore HIV-specific memory responses while influenza- and tetanus toxoid-specific memory B-cells remained unaffected or recovered. Antibody titers to Gag strongly correlated with memory B-cell frequencies. In contrast, Env-specific antibodies were maintained in advanced disease despite low or undetectable levels of memory B-cells. These results provide a potential mechanism by which destruction of HIV-specific CD4+ T-cells affects the humoral immune response against HIV and compromises the ability to maintain an effective antibody response.
Critical to the advancement of tumor immunotherapy is the reliable identification of responders and the quantification of the tumor-specific immune response elicited by treatments. In this regard, ...Enzyme-Linked Immunospot assay (ELISpot) is an ideal monitoring technique due to its high sensitivity, ease of execution and cost-effectiveness. Originally developed for the enumeration of B cells secreting antigen-specific antibodies, ELISpot assay has been adapted to detect and quantify cytokine-secreting immune cells present at low frequency in a variety of biological samples, including blood, in response to antigen-specific stimuli. The above-mentioned features emphasize the role of ELISpot as valuable assay for translational research and clinical applications. In the present chapter, we will focus on the use of ELISpot assay for monitoring the tumor-specific effector responses induced by different treatments in preclinical models and will provide some protocols and technical hints for its application.
The recent development of severely immunodeficient mouse strains enabled the production of new-generation humanized mice, in which major components of the human immune system are reconstituted. These ...new-generation humanized mice can be infected with human pathogenic viruses that do not infect regular mice and target cells of the hematoimmune system. Here we describe the method for preparing humanized mice, infecting them with EBV, and for their virological and immunological analyses. The results obtained from our own mouse models are briefly described.
PDF
