Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast

Powell, Michael J.; Prentice, Louise; Asawa, Takayuki; Kato, Ryoji; Sawicka, Joanna; Tanaka, Hideaki; Petersen, V. B.; Munkley, Andrea; Morgan, Sharon J.; Smith, Bernard Rees; Furmaniak, Jadwiga

doi:10.1016/s0009-8981(96)06422-4

Cited by 76 publications

(43 citation statements)

References 25 publications

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“…Purified recombinant human GAD65 expressed in yeast (Saccharomyces cerevisiae) was obtained from RSR (Cardiff, UK). The properties of this GAD65 preparation, including its reactivity with GAD autoantibodies (known to react with conformational epitopes on the GAD65 molecule), have previously been described in detail [13,14]. The N-terminal amino acids 2-45 were deleted from the preparation because N-terminally modified GAD65 was stable in solution.…”

Section: Subjects Materials and Methodsmentioning

confidence: 99%

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

et al. 2004

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Aims/hypothesis. Fulminant Type 1 diabetes is a novel subtype of Type 1 diabetes that involves the abrupt onset of insulin-deficient hyperglycaemia. This subtype appears to be non-autoimmune because of the absence of diabetes-related autoantibodies in the serum, and of insulitis in pancreatic biopsy specimens. The pathogenesis of the disease is still unknown. In this study, we investigated whether T cell autoimmune responses are involved in fulminant Type 1 diabetes. Methods. Cellular immune responses to beta cell autoantigens were studied by enzyme-linked immunospot (ELISPOT) assay in 13 fulminant Type 1 diabetic patients and 49 autoantibody-positive autoimmune Type 1 diabetic patients. Results were compared with those of 18 Type 2 diabetic patients, six secondary diabetic patients (diabetes due to chronic pancreatitis) and 35 healthy controls. Results. Nine of 13 (69.2%) GAD-reactive Th1 cells, and three of 12 (25%) insulin-B9-23-reactive Th1 cells were identified in fulminant Type 1 diabetic patients by ELISPOT, as in autoantibody-positive Type 1 diabetic patients. Four fulminant Type 1 diabetic patients possessed the highly diabetes-resistant allele DR2, three of whom had GAD-reactive Th1 cells in the periphery. Conclusions/interpretation. Peripheral immune reaction was observed in 69.2% of fulminant Type 1 diabetic patients, indicating that autoreactive T cells might contribute, at least in part, to the development of fulminant Type 1 diabetes.

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Section: Subjects Materials and Methodsmentioning

confidence: 99%

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

et al. 2004

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“…GADA were detected with commercial immunoprecipitation assays using 125 I-labeled human recombinant GAD (RSR Ltd., Cardiff, UK) (14). The lower detection limit was 0.1 U/ml.…”

Section: Laboratory Assessmentmentioning

confidence: 99%

Latent autoimmune diabetes in adults (LADA): usefulness of anti-GAD antibody titers and benefit of early insulinization

Rosário

Reis

Fagundes

et al. 2007

Arq Bras Endocrinol Metab

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Objective: To determine the clinical and laboratory parameters and the progression to insulin requirement in two groups of LADA patients separated according to GADA titers, and to evaluate the benefit of early insulinization in patients at high risk of premature beta-cell failure (high GADA titers). Methods: Among the diabetic adults seen at our service and screened for GADA at diagnosis, 54 were diagnosed with LADA and classified as having low (> 1 U/ml and < 17.2 U/ml) or high (> 17.2 U/ml) GADA titers. Fifty-four patients with type 2 diabetes (GADA-) were selected for comparison. In addition, 24 patients who had GADA titers > 20 U/ml and who were not initially insulinized were compared to 16 patients who were insulinized at diagnosis. Results: Insulin resistance was higher in the GADA-group, followed by patients with low GADA titers. BMI and the frequency of arterial hypertension, elevated triglycerides and reduced HDL cholesterol were lower in the high GADA+ group, with no difference between the GADA-or low GADA+ groups. The high GADA+ group showed a greater reduction and lower levels of C-peptide and required insulin earlier during follow-up. Patients with GADA titers > 20 U/ml and insulinized early presented no significant variation in C-peptide levels, had better glycemic control and required a lower insulin dose than patients who were insulinized later. Conclusion: We agree that patients with LADA should be differentiated on the basis of GADA titers and that patients with GADA titers > 20 U/ml benefit from early insulinization.

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“…Details of the purification, crystallization, and structural determination of human GAD65 and GAD67 have been published previously (5). We used for crystallization an NH 2 -terminally truncated form of each isoform (hereinafter referred to as GAD67 and GAD65) that lacked the first 89 and 83 residues, respectively; the NH 2 -terminal truncation facilitated purification because this region is hydrophobic and highly susceptible to proteolysis and did not affect enzymatic properties (5) nor reactivity with specific antisera (12)(13)(14). The proteins were expressed in Saccharomyces cerevisiae as fusions to a COOH-terminal hexahistidine tag and purified from the cell lysate by immobilized metal affinity chromatography and size exclusion chromatography in the presence of glutamate and PLP.…”

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confidence: 99%

COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

et al. 2008

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OBJECTIVE-To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow "molecular positioning" of epitope sites for B-and T-cell reactivity. RESEARCH DESIGN AND METHODS-Datawere assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to crossinhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. RESULTS-The COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOHterminal domain. Inclusion of epitope sites in the pyridoxal phosphate-and NH 2 -terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67.CONCLUSIONS-Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B-and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity. Diabetes 57:1293-1301, 2008 T ype 1 diabetes is characterized by autoimmune reactivity to islet cell antigens that include the 65-kDa isoform of GAD (GAD65). Of the two isoforms of GAD, GAD67 and GAD65, only GAD65 is autoantigenic, in diseases that include particularly type 1 diabetes (1,2). Importantly, the specificity of antibodies for particular epitopes on GAD65 rather than their actual levels may be a better indicator of impending or actual destruction of islet ␤-cells. Thus in genetically prone individuals, changes in the focus of autoantibody responses to epitopes of GAD65, i.e., intramolecular epitope shifts during the period of pre-diabetic insulitis, herald the onset of overt type 1 diabetes (3).B-cell/autoantibody epitopes on GAD65 engage conformational determinants that are widely distributed over the linear sequence of the three domains, NH 2 -terminal (amino acids 1-234), pyridoxal phosphate (PLP)-binding (235-442), and COOH-terminal (443-585) domains (4). Presently, unanswered questions include the precise location of epitope sites and critical binding residues for autoantibody recognition, the differing immune reactivity of the GAD65 versus the GAD67 isoform, and the failure of natural immune tolerance to GAD65 in the first place. These questions prompted the recent crystallographic s...

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Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast

Cited by 76 publications

References 25 publications

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

Latent autoimmune diabetes in adults (LADA): usefulness of anti-GAD antibody titers and benefit of early insulinization

COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity

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