Quantifying Autoantibodies to Zinc Transporter 8

Serum samples were analyzed for ZnT8 autoantibodies (ZnT8A)—arginine (ZnT8-RA), tryptophan (ZnT8-WA), and glutamine (ZnT8-QA)—using the radioligand binding assay as previously described (25 (link)). Briefly, the COOH-terminal constructs of ZnT8 were prepared using a Phusion site-directed mutagenesis kit (Finnzymes Oy, Espoo, Finland). The [³⁵S]methionine-labeled antigens were incubated overnight at 4°C with duplicate serum samples followed by precipitation of immune complexes with protein A-Sepharose (PAS; Amersham Biosciences, Uppsala, Sweden). The antibody-bound radioactivity was counted in a β-counter (1450 MicroBeta TriLux Microplate Scintillation-Luminescence Counter; PerkinElmer, Boston, MA), and concentrations of antibodies were estimated from a known standard curve and analyzed in GraphPad Prism 4.0 software (GraphPad). Our assays showed comparable precision (intra-assay coefficient of variation [CV] was 5.5% for ZnT8-RA, 5.3% for ZnT8-WA, and 4.9% for ZnT8-QA) and reproducibility (interassay CV was 13.8% for ZnT8-RA, 6.7% for ZnT8-WA, and 11.0% for ZnT8-QA). In the Diabetes Autoantibody Standardization Program (DASP) 2010 workshop (26 (link)), our laboratory was among the top-ranking laboratories in assays performance with workshop sensitivity of 52% and specificity of 100% for ZnT8-RA, 50 and 100% for ZnT8-WA, and 38 and 100% for ZnT8-QA, respectively.