Autoantibodies to GAD, IA-2 and insulin in ICA-positive first-degree relatives of children with type 1 diabetes: a comparison between parents and siblings.

BACKGROUND: Islet cell antibodies (ICA) represent a heterogenous group of autoantibodies to diabetes-associated antigens, including glutamic acid decarboxylase (GAD) and the IA-2 protein. The objectives of the present study were to compare the prevalence of autoantibodies to known biochemically characterized autoantigens between ICA-positive non-diabetic parents and siblings of children with type 1 diabetes and to evaluate how such antibodies explain ICA reactivity. METHODS: The presence and levels of GAD antibodies (GADA), IA-2 antibodies (IA-2A) and insulin autoantibodies (IAA) were analyzed in the sera of 184 ICA-positive first-degree relatives (79 parents and 105 siblings). RESULTS: The prevalences of GADA (61.9% in siblings vs 32.9% in parents), IA-2A (55.2% vs 15.2%) and IAA (41.0% vs 0%) were increased among ICA-positive siblings relative to ICA-positive parents (p<0.001). The siblings had higher ICA titers (p<0.001) than the parents but tended to have lower GADA levels (p=0.12). IA-2A levels did not differ between the two groups. IA-2A levels explained a higher proportion of the ICA reactivity in the siblings than in the parents (44% vs 12%, p=0.004), and GADA levels had the same tendency (27% vs 10%, p=0.11). In a multiple regression analysis, GADA and IA-2A were found to explain together 16% of the ICA reactivity in parents and 49% in siblings (p=0.003 for the difference). CONCLUSIONS: These results indicate that the increased frequency of additional diabetes-associated autoantibodies in ICA-positive siblings when compared to their ICA-positive parents may reflect the increased risk of progression to clinical type 1 diabetes previously reported in young ICA-positive relatives. We conclude that ICA immunofluorescence is not only due to GADA and IA-2A, but there are other additional antigens contributing to the ICA reactivity. Antibodies to such antigens appear to be more common among adults than in children.

Time-resolved fluorometric assay for detection of autoantibodies to glutamic acid decarboxylase (GAD65).

BACKGROUND: Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process. METHODS: Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay. RESULTS: The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4140 WHO units/mL, and no hook effect was observed up to 41,400 WHO units/mL. The intraassay CV was 2.1-6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4-7.0%, 9.8-13%, and 12-14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to perform the measurements was shorter. At a cutoff with 99% specificity, the new assay and the radiobinding assay were positive in 71 and 67 patients, respectively. CONCLUSIONS: The new assay provides a rapid and sensitive nonradioactive method applicable for large-scale screening for beta-cell autoimmunity. It has a broad linear analytical range, is easy to perform and automate, and has sensitivity and specificity comparable to those for the conventional radioisotope assay.

Prevalence and fate of type 1 diabetes-associated autoantibodies in cord blood samples from newborn infants of non-diabetic mothers.

BACKGROUND: Cord blood samples were collected from 1002 consecutive births at Turku University Hospital to study the prevalence and fate of type 1 diabetes-associated autoantibodies in newborn infants of unaffected mothers. METHODS: The samples were analysed for cytoplasmic islet cell antibodies (ICA), autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA), autoantibodies to the protein tyrosine phosphatase related IA-2 antigen (IA-2A), insulin autoantibodies (IAA) and HLA DQB1 genotypes. RESULTS: ICA were detected in 27 cord blood samples (2.7%), with a median of 6 (range 4-34) JDF units. GADA were found to be positive (> or =6.6 RU) in six samples (0.6%), with a median of 66 (range 19-125) RU. IA-2A (> or =0.43 RU) were observed in three samples (0.3%), with a median of 1.3 (range 0.8-57) RU, while only one cord blood sample (0.1%) tested positive for IAA (> or =1.56 nU/ml) with a value of 5.4 RU. Maternal or gestational age, sex and birth weight of the infant were not related to antibody prevalence or titres. Altogether there were 29 infants with antibody positivity in their cord blood (2.9%). Five of these (0.5%) tested positive for two antibodies (ICA and GADA), and one was positive for all four antibodies measured. All nine infants from whom follow-up samples were available became antibody negative by the age of 15 months, and in all but one case inverse seroconversion occurred by the age of 9 months. CONCLUSIONS: Around 3% of infants of non-diabetic mothers in Finland have diabetes-associated autoantibodies at birth, and these antibodies disappear at the latest by the age of 15 months.