title: Comparison of cytokine ELISpot assay formats for the detection of islet antigen autoreactive T cells. Report of the third immunology of diabetes society T-cell workshop.
abstract: The identification of sensitive assay formats capable of distinguishing islet autoreactive T cells directly ex vivo in blood is a major goal in type 1 diabetes research. Recently, much interest has been shown in the cytokine enzyme linked immunospot assay (CK ELISpot), an assay potentially capable of fulfilling these difficult criteria. To address the utility of this assay in detecting autoreactive T cells, a 'wet' workshop was organized using the same fresh blood sample and coded antigens. Five different laboratories participated, using three distinct CK ELISpot assay formats. Samples from two subjects were pre-tested for responses to sub-optimal concentrations of tetanus toxoid, representing a low frequency recall response, and peptides from diabetes associated autoantigens GAD65, IA-2 and HSP60. All participants measured interferon-gamma production and combinations of interleukins-4, -5, -10 and -13. In the workshop 4 of 5 laboratories detected low frequency recall responses in both subjects and 3 of 5 detected at least one of the autoreactive peptide responses concordant with pre-testing. Significant assay format related differences in sensitivity and signal-to-noise ratio were observed. The results demonstrate the potential for detection of low-level autoreactive T cell responses and identify assay characteristics that will be useful for studies in type 1 diabetes.
authors: German Diabetes Research Institute at the Heinrich Heine University, Düsseldorf, Germany.
authors: Nanette C
authors: NC
authors: Schloot
authors: Guido
authors: G
authors: Meierhoff
authors: Maria
authors: M
authors: Karlsson Faresjö
authors: Patrick
authors: P
authors: Ott
authors: Amy
authors: A
authors: Putnam
authors: Paul
authors: P
authors: Lehmann
authors: Peter
authors: P
authors: Gottlieb
authors: Bart O
authors: BO
authors: Roep
authors: Mark
authors: M
authors: Peakman
authors: Timothy
authors: T
authors: Tree
mesh_terms: D000328:Adult; D001324:Autoantigens / Q000276:immunology*; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000737:chemistry / Q000494:pharmacology; D016207:Cytokines / Q000097:blood* / Q000378:metabolism; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007515:Islets of Langerhans / Q000276:immunology*; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D013744:Tetanus Toxin / Q000494:pharmacology
chemical_list: D001324:Autoantigens; D018834:Chaperonin 60; D016207:Cytokines; D013744:Tetanus Toxin