Family history of type 1 diabetes and autoantibodies to the islet antigens insulin (IAA), glutamate decarboxylase (GADA), and the protein tyrosine phosphatase-like protein IA-2 (IA-2A) are strong predictors of type 1 diabetes, but the rate of progression to diabetes in multiple islet autoantibody-positive relatives varies widely. We asked whether detailed characterization of islet autoantibodies that included determination of titer, epitope specificity, and IgG subclass would improve diabetes prediction in a large cohort of autoantibodypositive relatives. The study shows a strong association between risk and high titer, broad antibody responses to IA-2 and insulin. The highest risks were associated with high-titer IA-2A and IAA, IgG2, IgG3, and/or IgG4 subclass of IA-2A and IAA, and antibodies to the IA-2-related molecule IA-2␤. Using models based on these antibody characteristics, autoantibody-positive relatives can be classified into groups with risks of diabetes ranging from 7 to 89% within 5 years. Diabetes 53: 384 -392, 2004 A utoantibodies to islet cell antigens such as insulin (IAA), the 65-kDa isoform of glutamate decarboxylase (GADA), and the protein tyrosine phosphatase (PTP)-like antigen IA-2 (IA-2A) are markers of the autoimmune process that precedes type 1 diabetes (1-9). At-risk relatives can be identified on the basis of positivity for these autoantibodies. Diabetes risk is highest in relatives with more than one islet autoantibody (4 -11) or with high-titer islet cell antibodies (10,12), suggesting that the intensity of the humoral response may reflect the stage of ␤-cell destruction. It has however been shown that a proportion of relatives with multiple islet autoantibodies do not develop diabetes for many years (6,13), indicating that additional tests are necessary for accurate prediction of diabetes. IgG subclass and the epitope specificity of autoantibodies may reflect qualitative and quantitative differences in the autoimmune response (14 -16), and their measurement could, therefore, improve our ability to predict diabetes. We have examined islet autoantibody titer, epitope specificity and IgG subclass in prospectively followed islet autoantibodypositive first-degree relatives of patients with type 1 diabetes, and determined how these can be used to stratify the likelihood of progression to clinical diabetes. The findings are consistent with the concept that high-titer multitarget responses signal late or aggressive preclinical diabetes and allow staging of diabetes risk on the basis of antibody measurements. RESEARCH DESIGN AND METHODSSera of all first-degree relatives of patients with type 1 diabetes from the Bart's Oxford (BOX) and the Munich family studies (13,17) were tested for IAA, GADA, and IA-2A. A total of 180 nondiabetic relatives (76 from BOX study and 104 from Munich family study) were selected on the basis of positivity for at least one of these antibodies on two or more occasions and whether a sufficient volume of the first positive sample was available for complete testing...

Children at risk for type 1 diabetes can develop early insulin autoantibodies (IAAs). Many, but not all, of these children subsequently develop multiple islet autoantibodies and diabetes. To determine whether disease progression is reflected by autoantibody maturity, IAA affinity was measured by competitive radiobinding assay in first and subsequent IAA-positive samples from children followed from birth in the BABYDIAB cohort. IAA affinity in first positive samples ranged from less than 10 6 l/mol to more than 10 11 l/mol. High affinity was associated with HLA DRB1*04, young age of IAA appearance, and subsequent progression to multiple islet autoantibodies or type 1 diabetes. IAA affinity in multiple antibody-positive children was on average 100-fold higher than in children who remained single IAA positive or became autoantibody negative. All high-affinity IAAs required conservation of human insulin A chain residues 8-13 and were reactive with proinsulin. In contrast, most lower-affinity IAAs were dependent on COOH-terminal B chain residues and did not bind proinsulin. These data are consistent with the concept that type 1 diabetes is associated with sustained early exposure to (pro)insulin in the context of HLA DR4 and show that high-affinity proinsulin-reactive IAAs identify children with the highest diabetes risk.

Children at risk for type 1 diabetes can develop early insulin autoantibodies (IAAs). Many, but not all, of these children subsequently develop multiple islet autoantibodies and diabetes. To determine whether disease progression is reflected by autoantibody maturity, IAA affinity was measured by competitive radiobinding assay in first and subsequent IAA-positive samples from children followed from birth in the BABYDIAB cohort. IAA affinity in first positive samples ranged from less than 10 6 l/mol to more than 10 11 l/mol. High affinity was associated with HLA DRB1*04, young age of IAA appearance, and subsequent progression to multiple islet autoantibodies or type 1 diabetes. IAA affinity in multiple antibody-positive children was on average 100-fold higher than in children who remained single IAA positive or became autoantibody negative. All high-affinity IAAs required conservation of human insulin A chain residues 8-13 and were reactive with proinsulin. In contrast, most lower-affinity IAAs were dependent on COOH-terminal B chain residues and did not bind proinsulin. These data are consistent with the concept that type 1 diabetes is associated with sustained early exposure to (pro)insulin in the context of HLA DR4 and show that high-affinity proinsulin-reactive IAAs identify children with the highest diabetes risk.

Insulin autoantibodies (IAA) are markers of the preclinical phase of insulin-dependent diabetes mellitus (IDDM) [1]. Their measurement, together with that of islet cell antibodies (ICA), antibodies to glutamic acid decarboxylase (GAD), and the protein tyrosine phosphatase-like antigen IA-2, forms the basis of current strategies for predicting future onset of IDDM [2]. IAA are particularly important when determining IDDM risk since their prevalence is significantly elevated in subjects developing the disease in childhood [3], and moreover, they are often the first autoantibodies to be detected [4].Methodologies for the detection of IAA are based upon the original radiobinding assay (RBA) of Kurtz et al. [5], the two established and validated assays being that described by Palmer [6] and that of Soeldner [7]. These methods are sensitive but require relatively large serum volumes of up to 600 ml. This limits the use of IAA in screening when capillary blood samples are obtained and when repeat testing is necessary or desired. Recently, Williams et al. described a novel RBA based on protein-A binding, rather than polyethylene glycol (PEG) precipitation of antibody bound insulin, which requires only 20 ml of serum [8]. In this study we compared results and performances of the novel micro RBA of Williams with those of the conventional RBA of Soeldner for the measurement of IAA in a large series of samples from new onset IDDM patients, control subjects and first degree relatives of IDDM patients. We report that the novel micro RBA has similar sensitivity and specificity to the Soeldner assay and should, therefore, facilitate high throughput screening, especially in young children. Diabetologia (1998) Summary Measurement of insulin autoantibodies (IAA) with a novel micro radiobinding assay which requires only 20 ml of serum was compared with that in a conventional radiobinding assay which uses 600 ml of serum. IAA were measured with both assays in samples from 94 new onset insulin-dependent diabetes mellitus (IDDM) patients, 97 control subjects, and 48 first degree relatives of IDDM patients selected for having IAA in the conventional radiobinding assay. Overall, 227 (95 %) of 239 samples tested were concordant, and IAA levels correlated well (r 2 = 0.7) between the two assays. Discordant results were obtained in 7 new onset patients, 4 control subjects, and 1 first degree relative, and these had low IAA levels in the respective assays. Sensitivity and specificity in the new onset IDDM patients and control subjects were 69 % and 98 % for the micro radiobinding assay and 72 % and 98 % for the conventional radiobinding assay. The use of the micro radiobinding assay should greatly facilitate islet related antibody screening, particularly in children. [Diabetologia (1998) 41: 681±683]

The related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2β are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2β to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2β and IA-2Δ 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611–620 (epitope JM1) and 621–630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules.

OBJECTIVE -Removal of the dietary wheat protein gluten protects against autoimmune diabetes in animal models. Furthermore, elimination of dietary gluten reduces the frequency of type 1 diabetes in patients with celiac disease. Herein we test the hypothesis that gluten is the driving antigen for type 1 diabetes-associated islet autoimmunity.RESEARCH DESIGN AND METHODS -Seven autoantibody-positive, first-degree relatives of patients with type 1 diabetes were placed on a gluten-free diet for 12 months followed by gluten reexposure for 12 months. Gliadin antibodies as well as the diabetes-related antibodies insulin autoantibody (IAA), GAD antibody (GADA), and tyrosin phosphatase IA2 antibody (IA-2A) were measured every 3 months; oral glucose tolerance tests were performed every 6 months. Changes in autoantibody titers were compared with those observed in a matched historical cohort.RESULTS -A reduction in IgG gliadin antibody titers was observed during the gluten-free period, but titers of diabetes-associated autoantibodies changed independently of gluten exposure. Type 1 diabetes-associated islet autoantibody levels at the end of the gluten-free diet period were not significantly different from those before commencement of the diet (P ϭ 0.2) or at the end of the gluten reexposure period (P ϭ 0.4). Changes in individual subjects were identified, but no differences were noted between the gluten-free and the gluten re-exposure periods, and the changes were similar to those observed in the historical control cohort (P ϭ 1.0). Major titer reductions (Ͼ50%) in the gluten-free period were observed in only one subject for all antibodies. Type 1 diabetes developed in this subject and in a second subject during the gluten reexposure period.CONCLUSIONS -The findings do not support the hypothesis that gluten is a driving antigen in type 1 diabetes. Diabetes Care 25:1111-1116, 2002D ietary gluten is the etiologic agent of celiac disease (CD) (1). CDassociated autoimmunity and disease activity are correlated with gluten exposure, and removal of gluten from the diet reduces titers of circulating CDassociated autoantibodies and restores intestinal mucosa histology and function, indicating that CD autoimmunity is driven by gluten (2). Dietary gluten has also been postulated as an etiologic agent in other autoimmune diseases, particularly type 1 diabetes (3-5). In animal models, autoimmune diabetes incidence is significantly reduced if nonobese diabetic mice are never exposed to gluten (4). In humans with CD, the frequency of type 1 diabetes, other autoimmune diseases, and associated autoantibodies is directly related to age at diagnosis of CD, suggesting that early elimination of gluten may prevent the manifestation of other autoimmune diseases (5). In accordance with this, it was recently reported that 11 patients with untreated CD and at least one diabetes-related autoantibody experienced complete loss of these autoantibodies within a 12-month period of glutenfree therapy (6).Furthermore, there are anecdotal reports of the disapp...

Insulin autoantibodies (IAA) are early sensitive markers of pre-diabetes in the young. The aim of this study was to assess whether, using IgG-specific measurement with a protein A/G assay, IAA are already present at birth, and whether this assay is suitable for early autoantibody screening. Cord blood and follow-up samples from offspring of parents with type 1 diabetes included in the BABYDIAB study were analyzed. Although insulin antibodies in cord blood from children of mothers with type 1 diabetes were readily detected and correlated well with levels in the maternal circulation, no insulin binding was detected in 247 cord blood samples from children of father probands. IgG IAA were detected at 2 yr in all 21 children who had multiple islet autoantibodies or who later developed type 1 diabetes, but were confirmed in only 6 of 58 with IAA by the conventional IAA assay in the absence of other islet autoantibodies. False positive IAAs in the conventional assay were often attributable to hemolysis. Hemolysis did not affect protein A/G IAA measurement, and results in whole capillary blood samples were comparable to those in corresponding serum samples (r2 = 0.99). These data show that IgG IAA appear early and after birth, and that the protein A/G IAA assay is sufficiently sensitive for early screening. The specificity of this assay requires further evaluation.

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