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
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: schlosse@uni-greifswald.de
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.
