scholarly journals Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

1995 ◽  
Vol 43 (1) ◽  
pp. 53-59 ◽  
Author(s):  
L Li ◽  
J Jiang ◽  
W A Hagopian ◽  
A E Karlsen ◽  
M Skelly ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Beta Cells ◽  
Glutamic Acid Decarboxylase ◽  
Autoimmune Diabetes ◽  
Polyclonal Antibodies ◽  
Acid Decarboxylase ◽  
Alpha Cells ◽  
Specific Antibodies ◽  
Pancreatic Islet Beta Cells ◽  
Irrelevant Peptide

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

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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