Detection of Antibodies Directed to the N-Terminal Region of GAD Is Dependent on Assay Format and Contributes to Differences in the Specificity of GAD Autoantibody Assays for Type 1 Diabetes

Abstract

GAD autoantibodies (GADAs) are sensitive markers of islet autoimmunity and type 1 diabetes. They form the basis of robust prediction models and are widely used for the recruitment of subjects at high risk of type 1 diabetes to prevention trials. However, GADAs are also found in many individuals at low risk of diabetes progression. To identify the sources of diabetes-irrelevant GADA reactivity, we analyzed data from the 2009 and 2010 Diabetes Autoantibody Standardization Program GADA workshop and found that binding of healthy control sera varied according to assay type. The characterization of control sera found positive by radiobinding assay (RBA), but negative by ELISA, showed that many of these sera reacted to epitopes in the N-terminal region of the molecule. This finding prompted development of an N-terminally truncated GAD65 radiolabel, (35)S-GAD65(96-585), which improved the performance of most GADA RBAs participating in an Islet Autoantibody Standardization Program GADA substudy. These detailed workshop comparisons have identified a source of disease-irrelevant signals in GADA RBAs and suggest that N-terminally truncated GAD labels will enable more specific measurement of GADAs in type 1 diabetes.

Figure 1

The AS95 plotted against the AUC from receiver operator characteristic analysis of GADA assays participating in the DASP 2010 workshop. Using these threshold-independent measures, better performance is demonstrated by those assays located toward the top right-hand corner (circled), a cluster that includes all the commercial ELISAs.

Figure 2

Heatmaps of laboratory-defined positive-negative calls for the 10 healthy control sera found positive most often in the DASP 2010 (A) and the same sera in the DASP 2009 (B) for those assays with a laboratory-defined specificity of >90%, sorted according to assay type. Positive-negative calls were found to cluster according to assay type; sera shaded in blue were found positive most commonly by commercial ELISAs, those in yellow by RBAs, and those in orange by RBAs using ¹²⁵I-labeled GAD₆₅. ID, identification.

Figure 3

Epitope specificity of eight healthy control sera from the DASP 2010 workshop that showed assay-related differences in reactivity. The left panel shows the different GAD constructs used to assess epitope specificity with regions derived from GAD₆₅ shown in black, and those from GAD₆₇ shown in white. The right panel shows the reactivity of the control sera with these GAD constructs and the epitope reactivity ascribed to those sera based on the pattern of binding with the different constructs. Four sera (S8531, N53371, TS23727, and LQ21235) showed reactivity with GAD₆₅ N-terminal epitopes that was abolished by deletion of the first 95 amino acids (aa). Three control sera (N56575, N51532, and N59932) showed weak reactivity with the middle (MID) region of GAD₆₅ (amino acids 235–444), and this binding was not reduced by use of the N-terminally truncated labels.

Figure 4

AS95 for 10 RBAs using ³⁵S or ¹²⁵I-GAD₆₅(1–585) and ³⁵S-GAD₆₅(96–585) to measure workshop samples in the IASP 2012 GADA substudy. The improved performance of assays using the N-terminally truncated GAD is shown by the eight laboratories that lie above the line of equivalence (hatched line).