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Diagnostic applications of immunology
Published in Gabriel Virella, Medical Immunology, 2019
Ajay Grover, Virginia Litwin, Gabriel Virella
The enzyme-linked immunospot (ELISpot) assay (Figure 15.11) is a highly sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single-cell level. In this assay, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of stimuli. Cytokine secreted by the cells are captured. After an appropriate incubation time, cells are removed, and the secreted molecule is detected using a detection antibody in a similar procedure to that employed by the EIA. A variety of detection systems such as enzymatic or fluorescent can be used. Since the cytokines are retained by the antibody immobilized on the test surface, the end result is visible spots on the surface where the cytokine has been captured, and if an adequate dilution is used, each spot corresponding to an individual cytokine-secreting cell. T-SPOT is a type of ELISpot assay used for tuberculosis diagnosis, which belongs to the group of interferon-γ release assays. This assay counts the number of effector T cells that produce interferon-γ in a sample of blood. This gives an overall measurement of the host immune response against mycobacteria, including Mycobacterium tuberculosis.
Immunodiagnosis of Tuberculosis Infection
Published in Peter D O Davies, Stephen B Gordon, Geraint Davies, Clinical Tuberculosis, 2014
Ajit Lalvani, Manish Pareek, Katrina Pollock
Quantitative estimates of IGRA specificity have been calculated by studying BCG-vaccinated individuals at an ultra-low risk of LTBI due to the absence of epidemiologic risk factors for tuberculosis exposure. The ELISA has been assessed in larger numbers of individuals in such studies than has the ELISpot. In recent systematic reviews and meta-analyses, the specificity of the ELISA ranged from 96%–99% (Pai, Zwerling et al. 2008; Diel, Loddenkemper et al. 2010) and 86%–93% for the ELISpot (Pai, Zwerling et al. 2008; Diel, Loddenkemper et al. 2010). On the whole, both IGRAs have consistently been shown to have a higher specificity than the TST in the immunodiagnosis of LTBI – particularly in BCG-vaccinated populations.
Molecular Analysis of Immunity
Published in Richard K. Burt, Alberto M. Marmont, Stem Cell Therapy for Autoimmune Disease, 2019
As shown in the table of T cell phenotype and function, effector T cells secrete cytokines when stimulated by cognate antigen.80,81 Thus, the frequency of antigen-specific T cells in an individual may be assessed by counting the number of cytokine-secreting T cells in an in vitro culture after antigenic stimulation. One method of achieving this is the enzyme-linked immunospot (ELISPOT) assay, a modification of the ELISA assay used for measuring antibody titers.71,72 This assay is elegant in its simplicity and high accuracy especially at low precursor antigen-specific T cell frequencies. It can be used for either CD4 or CD8 T cell responses. Cultures of T cells are stimulated with the test antigen in wells of plastic plates coated with antibodies specific for the cytokine to be assayed (usually IL-2 or IFNy). If a T cell is activated by the antigen, it will secrete cytokine which will be bound by the antibody. After an incubation period, the T cells are washed off and the remaining plate-bound cytokine may be probed and visualized with a secondary antibody. Usually, an enzymatic reaction will deposit a colored precipitate revealing the exact site of the antigen-specific T cells. Their frequency can thus be determined by counting the spots and expressing this value as a fraction of the number of T cells added to the culture. The advantage of the ELISPOT is that it uses few cells, is very sensitive, and can analyze Th1 and Th2 responses separately by using different cytokine capture antibodies. Furthermore, if the T cells are depleted of CD8+ T cells beforehand, then CD4+ and CD8+ antigen-specific T cell frequencies can be enumerated separately.
Identification of a CD8+ T-cell response to a predicted neoantigen in malignant mesothelioma
Published in OncoImmunology, 2020
Sophie Sneddon, Craig M. Rive, Shaokang Ma, Ian M. Dick, Richard J. N. Allcock, Scott D. Brown, Robert A. Holt, Mark Watson, Shay Leary, Y. C. Gary Lee, Bruce W. S. Robinson, Jenette Creaney
ELISpot assays were performed according to standard protocols.53 Briefly, wells of nitrocellulose microtiter plates were coated with 1 µg/mL anti-interferon-γ (INFγ) antibody (Mabtech, 3420-3-250) overnight at 4°C. Samples and controls were incubated in RPMI-1640 media with 10% fetal calf serum at 37°C for 20 h and tested in triplicate, where 200,000 cells were assayed per well. PBMCs and expanded T cells to be tested were pulsed with single-antigen cell lines (SALs) expressing specific HLA molecules and 10 μg/mL of mutant or wild-type peptides. SALs were a kind gift from Professors Frans Claas and Ilias Doxiadis (Academic Hospital Leiden, The Netherlands). INFγ responses were detected with 1 mg/mL biotinylated anti-INFγ antibody (Mabtech, 3420-6-250) followed by 1 µg/mL streptavidin horseradish peroxidase (Mabtech, 3310–9) and development with TMB substrate (Mabtech, 3651–10). Immune reactivity was enumerated using the AID ELISpot reader system (Autoimmun Diagnostika GmbH) running count algorithm v.3.2.x and data was generated in Spot Forming Units (SFU) per 1 × 105 cells.
MAPPs for the identification of immunogenic hotspots of biotherapeutics; an overview of the technology and its application to the biopharmaceutical arena
Published in Expert Review of Proteomics, 2018
Valerie Quarmby, Qui T Phung, Jennie R Lill
T-cell responses such as the generation of ADAs to the binding of peptides derived from a protein therapeutic to cognate T cells in the context of MHC II is a key-determining factor of immunogenicity. With the central role T cells play, it is therefore critical to employ in vitro T-cell assays to assess cell proliferation or cytokine secretion as complementary risk assessment strategies to study immunogenicity. Several different types of T-cell assays such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot-forming (ELISpot), and flow cytometry-based intracellular cytokine staining have been developed to study activation by cytokines. Each assay has its merits and limitations. Multiple cytokines can simultaneously be measured by ELISA of supernatant of cell culture to determine T-cell response upon stimulation by protein therapeutics [88,89]. However, because many different myeloid cells can secrete cytokines, the readout does not allow interrogation of which cell is responsible for the response. ELISpot is a sensitive technique that can semi-quantitatively determine the number of cells secreting a cytokine but it is limited to profiling a single cytokine per readout. Similar to ELISA, multiple cytokines can be profiled by flow cytometry-based intracellular cytokine staining but with the added advantage of correlation of cytokine secretion at a single cell level. A limiting factor to the sensitivity of the assay is the low signal-to-noise level depending on the fixation methodology employed and the sensitivity of mAbs against the cytokine to be profiled.
An overview of proteomic methods for the study of ‘cytokine storms’
Published in Expert Review of Proteomics, 2021
Paul David, Frederik J. Hansen, Adil Bhat, Georg F. Weber
Enzyme-linked ImmunoSpot is a sensitive and highly quantitative assay well known because it easily detects single-cell-producing cytokines and tracks cellular responses to distinct stimuli. In this method, the cells are incubated in a 96-well plate coated with capture antibody that detects the cytokines being released. After washing the cells, an enzyme-linked detection antibody is added and cytokine production is observed by a color reaction [77]. ELISpot was initially used for T cell function analysis; however, B cell and other immune cells are being evaluated now. It has been used for Phase I and II clinical trials for cancer therapy and hence could be a biomarker assay for vaccine development.