Diabetes Antibody Standardization Program: Evaluation of assays for autoantibodies to glutamic acid decarboxylase and islet antigen-2

Unit for Diabetes and Coeliac Disease, Institution of Clinical Sciences, Clinical Research Centre, University Hospital MAS, Malmö, Sweden.
Diabetologia (Impact Factor: 6.67). 05/2008; 51(5):846-52. DOI: 10.1007/s00125-008-0967-2
Source: PubMed


Islet autoantibodies are important in diabetes classification and risk assessment, and as endpoints in observational studies. The Diabetes Autoantibody Standardization Program (DASP) aims to improve and standardise measurement of autoantibodies associated with type 1 diabetes. We report results for glutamic acid decarboxylase autoantibodies (GADA) and islet antigen-2 autoantibodies (IA-2A) from three DASP workshops (2002--2005).
Up to 60 laboratories in 18 countries participated in each workshop. Participants received coded serum aliquots from 50 patients with newly diagnosed type 1 diabetes (median age 18 years, range 9-35 years) and 100 blood donor controls. Results were analysed using receiver operator characteristic (ROC) curves with sensitivity adjusted to 95% specificity in workshop controls.
GADA assays performed well in all three workshops (median area under the ROC curve [AUC] 0.94; interquartile range 0.91-0.95) and performance was similar to DASP 2000. Performance of IA-2A assays improved over the workshop programme. Median AUC was 0.81 (interquartile range 0.79-0.83) in DASP 2002, 0.82 (interquartile range 0.78-0.84) in 2003, and 0.85 (interquartile range 0.82-0.87) in 2005 (p < 0.0001). Performance of GADA ELISA improved between 2002 and 2005, and, in DASP 2005, achieved higher median AUC and adjusted sensitivity than RIA. IA-2A ELISA improved and, in DASP 2005, achieved AUCs equivalent to in-house RIA. Assays using IA-2ic or full length IA-2 clones were more sensitive than those using IA-2bdc, with higher AUC (p = 0.004).
GADA and IA-2A assays perform well in discriminating health and disease. The workshop format highlights systematic differences related to assay method and allows full evaluation of novel methods. The programme of autoantibody workshops in type 1 diabetes provides a model for other autoimmune diseases.

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    • "One of the current methods for the detection of IA-2As is radioimmunoassay (RIA), which is based on immunoprecipitation of 125I- or 35S-methionine-labeled recombinant IA-2 (intracellular portion) [9], [10]. Although several IA-2 RIAs have been reported to achieve high levels of sensitivity and specificity [11], they are expensive and usually take more than 24 h to carry out. In addition, they have the disadvantage of requiring special precautions and licensing because radioactive isotopes are used. "
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    ABSTRACT: Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.
    PLoS ONE 07/2014; 9(7):e103088. DOI:10.1371/journal.pone.0103088 · 3.23 Impact Factor
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    • "Human leukocyte antigen (HLA) genotyping is performed by high-resolution sequencing-based typing of exons 2 and 3 of HLA-DRB1 and HLA-DQB1, including heterozygous ambiguity resolution (Conexio Genomics, Fremantle, Western Australia). Blood samples are used to determine autoantibodies to insulin, glutamic acid decarboxylase, insulinoma-associated protein 2, and zinc transporter 8, as previously described [14,15], with the upper limit of normal for each assay corresponding to the 99th percentile of control subjects [16–18]. "
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    ABSTRACT: The diabetes mellitus Incidence Cohort Registry (DiMelli) aims to characterize diabetes phenotypes by immunologic, metabolic, and genetic markers. We classified patients into three groups according to islet autoantibody status and examined whether patients with multiple diabetes-associated autoantibodies, one autoantibody, or without autoantibodies differed with respect to clinical, metabolic, and genetic parameters, including an insulin sensitivity (IS) score based on waist, HbA1c, and triglycerides. We also assessed whether metabolic markers predicted the immune status. As of June 2012, 630 patients in Bavaria, Germany, aged <20 years diagnosed with any type of diabetes within the preceding 6 months were registered in DiMelli. We compared the clinical and laboratory parameters between islet autoantibody status defined patient groups. Parameters showing the strongest associations were included in principal component analysis. Receiver operating characteristic curves were used to assess the ability of the IS Score to predict islet autoantibody status. Patients with multiple islet autoantibodies, one autoantibody, or without autoantibodies were significantly different in terms of BMI percentile, weight loss before diagnosis, fasting C-peptide (all, P<0.001), and IS Score (P=0.034). However, principal component analysis revealed no distinct patterns according to autoantibody status. At the optimal IS Score cut-off for predicting islet autoantibody positivity (single compared to none), the specificity was 52.0% and the sensitivity was 86.8%. With respect to prediction of multiple autoantibodies (compared to none), specificity and sensitivity were slightly lower and in combination inferior to those obtained using the BMI percentile and fasting C-peptide. The DiMelli study indicated that patients with and without islet autoantibodies differed with respect to metabolic and genetic markers but there was considerable overlap of phenotypes, and autoantibody status could not be predicted by these parameters. Thus, our results suggest that refined diabetes classification may require both immune and metabolic phenotyping.
    PLoS ONE 09/2013; 8(9):e74339. DOI:10.1371/journal.pone.0074339 · 3.23 Impact Factor
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    • "Insulin autoantibodies (IAAs) are usually the first to appear before T1D development and they are most frequently found in young children, as their level and prevalence at diagnosis inversely correlate with age [2]. One of the current methods for the detection of T1D autoantibodies is enzyme-linked immunosorbent assay (ELISA), in which the immobilized antigen captures autoantibodies from the sample and detection is achieved using labeled antigen [2], [3]. However, this method cannot be applicable when measuring IAAs, because it appears that human IAAs cannot react with insulin directly bound to plates [4], [5]. "
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    ABSTRACT: Type 1 diabetes (T1D) is an autoimmune disease which results from the destruction of pancreatic beta cells. Autoantibodies directed against islet antigens are valuable diagnostic tools. Insulin autoantibodies (IAAs) are usually the first to appear and also the most difficult to detect amongst the four major islet autoantibodies. A non-radioactive IAA bridging ELISA was developed to this end. In this assay, one site of the IAAs from serum samples is bound to a hapten-labeled insulin (GC300-insulin), which is subsequently captured on anti-GC300 antibody-coated 96-well plates. The other site of the IAAs is bound to biotinylated insulin, allowing the complex to be detected by an enzyme-streptavidin conjugate. In the present study, 50 serum samples from patients with newly diagnosed T1D and 100 control sera from non-diabetic individuals were analyzed with our new assay and the results were correlated with an IAA radioimmunoassay (RIA). Using IAA bridging ELISA, IAAs were detected in 32 out of 50 T1D children, whereas with IAA RIA, 41 out of 50 children with newly diagnosed T1D were scored as positive. In conclusion, the IAA bridging ELISA could serve as an attractive approach for rapid and automated detection of IAAs in T1D patients for diagnostic purposes.
    PLoS ONE 07/2013; 8(7):e69021. DOI:10.1371/journal.pone.0069021 · 3.23 Impact Factor
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