A simplified method to assess affinity of insulin autoantibodies
Introduction
Insulin autoantibodies (IAA) are often the first autoantibodies detected during the natural history of childhood type 1 diabetes (T1D) [1], [2], [3], [4], [5]. Many IAA-positive children also develop autoantibodies to other islet autoantigens, such as glutamate decarboxylase (GADA), insulinoma-associated protein 2 (IA-2A), or zinc transporter 8 (ZnT8A), and eventually progress to clinical diabetes [6], [7], [8], [9]. In contrast, children who remain positive only for IAA rarely develop the disease [9], [10], [11]. The affinity of IAA is fixed relatively early in the autoimmune response; those children with high-affinity IAA are much more likely to develop multiple islet autoantibodies [12], [13], [14], whereas those with low-affinity IAA usually show no progression of islet autoimmunity [12], [13]. Affinity can therefore discriminate between IAA-associated diabetes risks at an early stage in pathogenesis. The utility of testing IAA affinity was confirmed in T1D and control samples by an international workshop organized by the Diabetes Autoantibody Standardization Program (DASP) [15]. However, wide scale application of IAA affinity measurements is limited by the amount of serum as well as the cost and time required for performing the assay. Current IAA affinity assays measure antibody binding to labeled insulin in the presence of up to eight concentrations of unlabeled competitor [12], [13], [14], [15]. In this study we define a single concentration of unlabeled insulin competitor that discriminates between IAA of high and low-affinity, markedly simplifying the method, thereby making it feasible for wider application.
Section snippets
Subjects and samples
To establish the methodology for simplified assessment of IAA affinity we used 122 serum samples from young non-diabetic first degree relatives of T1D patients from Germany who were prospectively followed for the development of islet autoimmunity and diabetes in the BABYDIAB study [2], the BABYDIET study [16], or the Munich family study [17]. All sera were pre-screened for islet autoantibodies and selected for the presence of IAA and included 47 that were also positive for GADA, IA-2A and/or
Determining IAA affinity and relative-binding
IAA affinity in the 122 IAA-positive sera from Germany ranged between 7.5 × 104 and > 2.0 × 1011 L/mol (median affinity 1.4 × 109 L/mol; IQR 3.5 × 107 to 1.4 × 1010). For six competitor concentrations of unlabeled insulin used in the affinity measurement (5.5 × 10− 11 to 2.8 × 10− 7 mol/L), cpm-binding was expressed as percentage of the cpm-binding in the absence of competition and referred to as IAA relative-binding (Fig. 1). In this way, high relative-binding corresponds to low competition and theoretically,
Discussion
The development of autoantibodies to multiple islet autoantigens signals an important event in the pathogenesis of T1D, which is strongly associated with progression to clinical diabetes [20]. IAA affinity measurement provides an early immune marker that can distinguish between single IAA-positive children who are likely to progress in islet autoimmunity and those in whom progression is rather unlikely [12], [13]. This study aimed to simplify IAA affinity measurement. We show that inclusion of
Conclusions
In conclusion, the use of one additional concentration of unlabeled insulin competitor in the micro-IAA radiobinding assay allowed discrimination between high-affinity and low-affinity IAA. We believe this approach is highly feasible and increases the prognostic information obtained from predictive testing using IAA. The method is relatively simple and robust and will be useful for risk assessment in prospective studies in infants as well as early selection of children with high diabetes risk
Role of the funding source
None of the funding sources have been involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.
Acknowledgments
This study was supported in part by grants from the German Federal Ministry of Education and Research (BMBF) to the German Center for Diabetes Research (DZD e.V.) and to the Competence Network for Diabetes mellitus (FKZ 01GI0805-07), and from the European Union (EP7-HEALTH-2007, DIAPREPP N202013). Peter Achenbach was supported by the Juvenile Diabetes Research Foundation (11-2005-1117), Kerstin Adler by the NIH/DFG Research Career Transition Award Program (KO 3418/1-1), and Ezio Bonifacio by
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