Radioimmunoassay Detects the Frequent Occurrence of Autoantibodies to the Mr 65,000 Isoform of Glutamic Acid Decarboxylase in Japanese Insulin-Dependent Diabetes

Abstract

Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.

Genetic Effects on Age-Dependent Onset and Islet Cell Autoantibody Markers in Type 1 Diabetes

“…IAA. In the exploratory analysis, the unadjusted frequency of IAA increased with the number of class I alleles of INS VNTR: 15% of III/III patients (95% CI 0 -30), 30% of I/III patients (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38), and 40% of I/I patients (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) were positive for IAA. Unadjusted frequencies of IAA were higher in DQ8-positive than in DQ8-negative patients: 27% of patients with no DQ8 haplotypes (95% CI 20 -34), 41% of patients with one DQ8 haplotype (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46), and 42% of patients with two DQ8 haplotypes (26 -57) were positive for IAA.…”

“…Glutamate decarboxylase autoantibodies. Antibodies to radiolabeled human Mr 65,000 glutamate decarboxylase (GADA) were quantified by fluid-phase immunoprecipitation assay using fluorographic densitometry as described (13,28) except that for 15-to 35-year-olds, GAD65 were radiolabeled by coupled in vitro transcription and translation, as described (29,30). The two assay formats correlate well, and autoantibody levels are expressed as GADA index as described elsewhere (29), using the WHO-Juvenile Diabetes Foundation (JDF) standard (31) as the positive control.…”

Normal values of first-phase insulin response to intravenous glucose in healthy Italian children and adolescents

“…The diagnosis of IDDM was made according to the guidelines of the National Diabetes Data Group [6]. In addition, a daily urinary reactive C-peptide value of less than 6.6 nmol/day [2] or anti-glutamic acid decarboxylase (GAD) antibody value of more than 8 U/ml [7] was used to define IDDM. At the time of this study, all the patients…”

“…For determination of ICA, each center sent frozen serum from all subjects to Padova University [6]. For GAD65 antibodies each center sent frozen serum to Karolinska Institute, Stockholm [7]. In each diabetes center blood samples for insulin assay were centrifuged at 4 ~ and then plasma was kept at -20 ~ until analysis.…”

Glutamic Acid Decarboxylase65 (GAD65) Antibodies and Insulin Auto-Antibodies in Japanese Patients with Nonlnsulin-Dependent Diabetes Mellitus.

“…Briefly, pEx9 (a generous gift from AE Karlsen and CE Grubin, Washington University, USA), a full-length human islet GAD65 cDNA [22] was used in an in vitro transcription and translation reaction to produce 35S-GAD65 [19]. For each analysis, all of which were conducted in triplicate, 20,000 cpm of TCA precipitable 35S-GAD65 was…”