Diagnostic applications of immunology
Gabriel Virella in Medical Immunology, 2019
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
Peter D O Davies, Stephen B Gordon, Geraint Davies in Clinical Tuberculosis, 2014
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
Richard K. Burt, Alberto M. Marmont in 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.
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.
Intravenous delivery of the toll-like receptor 7 agonist SC1 confers tumor control by inducing a CD8+ T cell response
Published in OncoImmunology, 2019
Fulvia Vascotto, Jutta Petschenka, Kerstin C. Walzer, Mathias Vormehr, Magdalena Brkic, Stefan Strobl, Roman Rösemann, Mustafa Diken, Sebastian Kreiter, Özlem Türeci, Ugur Sahin
ELISpot assay was carried out as previously described.31 In brief, responder cells were cultured overnight at 37°C in Multiscreen 96-well plates (Millipore) coated with anti-IFNγ antibody (10 µg/mL, clone AN18, Mabtech) and cytokine secretion was detected with an anti–IFNγ antibody (1 µg/mL, clone R4-6A2, Mabtech) after T-cell stimulation with 2 µg/mL gp70 AH1 or VSV peptide. For analysis of antigen specificity of tumor-infiltrating lymphocytes tumors were digested as described above. Dead cells were removed via density gradient centrifugation (Ficoll-Paque PREMIUM 1,084, GE Healthcare) followed by removal of CT26 tumor cells via plastic adhesion (2–3 h at 37°C) and of dead cells via magnetic beads (Miltenyi). Counted leukocytes were finally re-stimulated with syngeneic peptide-pulsed BMDC. For analysis of T-cell responses in the blood, PBMC were isolated via density gradient centrifugation, counted and re-stimulated by addition of peptide. All samples were tested in duplicates or triplicates and spots counted using Cellular Technology Ltd. Immunospot Reader.
ASTX660, an antagonist of cIAP1/2 and XIAP, increases antigen processing machinery and can enhance radiation-induced immunogenic cell death in preclinical models of head and neck cancer
Published in OncoImmunology, 2020
Wenda Ye, Sreenivasulu Gunti, Clint T. Allen, Youji Hong, Paul E. Clavijo, Carter Van Waes, Nicole C. Schmitt
Additional ex vivo experiments combining tumor cells and TIL in an environment devoid of dendritic cells demonstrated that ASTX660 enhances cytotoxic TIL-dependent killing across multiple cell lines, suggesting additional mechanistic pathways for enhancement of anti-tumor immunity that are independent from ICD. Consistent with our results, a prior study showed that knockdown of neuronal apoptosis inhibitory protein (NAIP), another member of the IAP family, sensitized prostate cancer cells to antigen-specific T-cell killing.44 As a possible mechanism for this enhanced T-cell killing, we hypothesized that ASTX660 might enhance antigen presentation on tumor cells, facilitating T lymphocyte recognition and subsequent cytotoxic killing. Further experiments showed that ASTX660 + TNFα reliably increased the expression of HLA-A,B,C across multiple human HNSCC cell lines in vitro, and ASTX660 + XRT increased murine MHC class I expression on tumor cells in vivo. In addition, ELISpot analysis demonstrated a prominent antigen-specific functional immune response to the combination treatment. ASTX660 + XRT, but neither agent alone, significantly enhanced clonal proliferation of antigen-specific TIL generating IFN-γ, clarifying possible immune-stimulating mechanisms of ASTX660 and XRT.
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