Zinc transporter 8 antibodies complement GAD and IA-2 antibodies in the identification and characterization of adult-onset autoimmune diabetes: Non Insulin Requiring Autoimmune Diabetes (NIRAD) 4.

OBJECTIVE: Zinc transporter 8 (ZnT8) is an islet beta-cell secretory granule membrane protein recently identified as an autoantibody antigen in type 1 diabetes. The aim of this study was to determine the prevalence and role of antibodies to ZnT8 (ZnT8As) in adult-onset diabetes. RESEARCH DESIGN AND METHODS: ZnT8As were measured by a radioimmunoprecipitation assay using recombinant ZnT8 COOH-terminal or NH(2)-terminal proteins in 193 patients with adult-onset autoimmune diabetes having antibodies to either GAD (GADAs) or IA-2 (IA-2As) and in 1,056 antibody-negative patients with type 2 diabetes from the Non Insulin Requiring Autoimmune Diabetes (NIRAD) study. RESULTS: ZnT8As-COOH were detected in 18.6% patients with autoimmune diabetes and 1.4% with type 2 diabetes. ZnT8As-NH(2) were rare. ZnT8As were associated with younger age and a high GADA titer. The use of GADAs, IA-2As, and ZnT8As in combination allowed a stratification of clinical phenotype, with younger age of onset of diabetes and characteristics of more severe insulin deficiency (higher fasting glucose and A1C, lower BMI, total cholesterol, and triglycerides) in patients with all three markers, with progressive attenuation in patients with two, one, and no antibodies (all P(trend) < 0.001). Autoantibody titers, association with high-risk HLA genotypes, and prevalence of thyroid peroxidase antibodies followed the same trend (all P < 0.001). CONCLUSIONS: ZnT8As are detectable in a proportion of patients with adult-onset autoimmune diabetes and seem to be a valuable marker to differentiate clinical phenotypes.

Radiobinding assay for detecting autoantibodies to single epitopes.

Autoantibodies, a hallmark of autoimmune disease, are directed against diverse antigens and epitopes. This diversity aids in characterising disease progression and identifying disease-associated autoantibodies. Here we aimed to develop a sensitive assay to detect and quantify epitope-specific autoantibodies. We generated constructs to integrate known peptide epitopes into the TrxA protein, and used these to in vitro synthesize radio-labelled proteins for a radiobinding assay (RBA). This assay was first validated with an animal model using mice immunized with the MOG(40-55) peptide. Using type 1 diabetes-associated protein tyrosine phosphatase-like autoantigen IA-2 as a human model, we expressed IA-2(609-621), IA-2(619-631), or IA-2(609-631) peptide in the TrxA construct and used the RBA to test sera from 113 patients with type 1 diabetes and 87 controls. Antibodies were detected to the IA-2(609-631) epitope in 37 of 113 patient sera and one control; 17 sera were reactive to the IA-2(609-621) (JM1) epitope, and 16 to the IA-2(619-631) (JM2) epitope. Interestingly, a novel third epitope (JM3) was identified in 7 sera that reacted to IA-2(609-631) but not the JM1 and JM2 sub-specificities. An ELISA using IA-2(609-631) was less sensitive than the RBA, as it detected only some of the JM1 and JM3 antibody positive sera and none of the JM2. Mutagenesis of single IA-2(609-631) amino acids in combination with the RBA showed that antibodies to JM2 and JM3 were highly diverse in their specific residue requirements. Our strategy using in vitro synthesized antigens and the RBA was thus a highly sensitive and versatile method to identify epitopes and quantify epitope-specific antibodies.