scholarly journals Determination of Glutamic Acid Decarboxylase (GAD65) in Pancreatic Islets and ItsIn VitroandIn VivoDegradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

2008 ◽  
Vol 24 (3) ◽  
pp. 191-198 ◽  
Author(s):  
Michael Schlosser ◽  
Uwe Walschus ◽  
Ingrid Klöting ◽  
Reinhard Walther
Keyword(s):  
Glutamic Acid ◽  
Pancreatic Islets ◽  
Glutamic Acid Decarboxylase ◽  
Half Life ◽  
Autoimmune Diabetes ◽  
Degradation Kinetics ◽  
Acid Decarboxylase ◽  
Gad65 Autoantibodies

Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. Thein vitrohalf-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, thein vivohalf-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65.

scholarly journals Pathogenic Roles of Glutamic Acid Decarboxylase 65 Autoantibodies in Cerebellar Ataxias

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Hiroshi Mitoma ◽  
Mario Manto ◽  
Christiane S. Hampe
Keyword(s):  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
In Vitro Studies ◽  
Acid Decarboxylase ◽  
Pathogenic Role ◽  
Cerebellar Ataxias ◽  
Glutamic Acid Decarboxylase 65

Reports suggesting a pathogenic role of autoantibodies directed against glutamic acid decarboxylase 65 (GAD65Abs) in cerebellar ataxias (CAs) are reviewed, and debatable issues such as internalization of antibodies by neurons and roles of epitopes are discussed. GAD65 is one of two enzymes that catalyze the conversion of glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). A pathogenic role of GAD65Ab in CAs is suggested by in vivo and in vitro studies. (1) Intracerebellar administration of cerebrospinal fluid (CSF) immunoglobulins (IgGs) obtained from GAD65Ab-positive CA patients impairs cerebellar modulation of motor control in rats. (2) CSF IgGs act on terminals of GABAergic neurons and decrease the release of GABA in cerebellar slices from rats and mice. (3) Absorption of GAD65Ab by recombinant GAD65 diminishes the above effects, and monoclonal human GAD65Ab (b78) mimic the effects of CSF IgGs in vivo and in vitro. Studies using GAD65-KO mice confirm that the target molecule is GAD65. (4) Notably, the effects of GAD65Ab depend on the epitope specificity of the monoclonal GAD65Ab. Taken together, these results indicate that epitope-specific GAD65Ab-induced impairment of GABA release is involved in the pathogenesis of GAD65Ab-positive CA and support the early detection of GAD65Ab-associated CA to initiate immunotherapy before irreversible neuronal death in the cerebellum.

Diagnostic sensitivity of immunodominant epitopes of glutamic acid decarboxylase (GAD65) autoantibodies in childhood IDDM

Diabetologia ◽  
1996 ◽  
Vol 39 (9) ◽  
pp. 1091-1098 ◽  
Author(s):  
A. Falorni ◽  
M. Ackefors ◽  
C. Carlberg ◽  
T. Daniels ◽  
B. Persson ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
Diagnostic Sensitivity ◽  
Acid Decarboxylase ◽  
Gad65 Autoantibodies ◽  
Childhood Iddm ◽  
Immunodominant Epitopes

scholarly journals Evaluation of Two Nonisotopic Immunoassays for Determination of Glutamic Acid Decarboxylase and Tyrosine Phosphatase Autoantibodies in Serum

2004 ◽  
Vol 50 (8) ◽  
pp. 1378-1382 ◽  
Author(s):  
Xavier Palomer ◽  
Dídac Mauricio ◽  
José Rodríguez-Espinosa ◽  
Edgar Zapico ◽  
Carme Mayoral ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
Clinical Laboratory ◽  
Autoimmune Diabetes ◽  
Tyrosine Phosphatase ◽  
Acid Decarboxylase ◽  
Time Resolved ◽  
Radiobinding Assay

Abstract Background: Autoantibodies for the 65-kDa form of glutamic acid decarboxylase (GAD65) and protein tyrosine phosphatase-like protein (IA-2) are measured for risk prediction and diagnosis of autoimmune diabetes mellitus. There is a lack of adequate nonisotopic alternatives to the most widely used method for both autoantibodies, which is a radiobinding assay (RBA). Methods: We compared two commercially available immunoassays, an ELISA and a time-resolved immunofluorometric assay (TR-IFMA), with RBA. Results: We found excellent agreement between the RBA and ELISA for measurement of GAD65 autoantibodies (GADAs); they showed comparable analytical precision in the cutoff range and achieved similar diagnostic specificity. The ELISA identified more GADA-positive individuals among patients with new-onset type 1 diabetes than did the RBA [89% (95% confidence interval, 78–95%), vs 71% (58–82%); P <0.03]. For IA-2 autoantibodies (IA-2As), only the TR-IFMA achieved analytical performance and diagnostic accuracy comparable to that of the RBA. These results with the GADA ELISA and IA-2A TR-IFMA were consistent with those obtained blindly in the Diabetes Antibody Standardization Program 2003. The performance of the GADA TR-IFMA and IA-2A ELISA was unsatisfactory, and these tests were not subjected to clinical evaluation. Conclusions: The GADA ELISA and IA-2A TR-IFMA behave comparably with RBA and are thus suitable for use in the clinical laboratory.

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