Determination of glutamic acid decarboxylase (GAD65) in pancreatic islets and its in vitro and in vivo degradation kinetics in serum using a highly sensitive enzyme immunoassay

Article type: Research Article

Authors: Schlosser, Michael^; | Walschus, Uwe^; | Klöting, Ingrid | Walther, Reinhard

Affiliations: Department of Medical Biochemistry and Molecular Biology, Ernst Moritz Arndt University of Greifswald, Karlsburg, Germany | Institute of Pathophysiology, Ernst Moritz Arndt University of Greifswald, Karlsburg, Germany | Department of Laboratory Animal Science, Ernst Moritz Arndt University of Greifswald, Karlsburg, Germany

Note: [] Corresponding author: Dr. M. Schlosser, Department of Medical Biochemistry and Molecular Biology, Ernst Moritz Arndt University of Greifswald, Greifswalder Str. 11c, D-17495 Karlsburg, Germany. Tel.: +49 3834 8619178; Fax: +49 3834 8680118; E-mail: [email protected]

Abstract: 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. The in vitro half-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, the in vivo half-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.

Keywords: GAD65 determination, ELISA, pancreatic islets, half-life, streptozotocin

Journal: Disease Markers, vol. 24, no. 3, pp. 191-198, 2008