Poster Presentation The 13th International Congress of the Immunology of Diabetes Society 2013

Epitope specificity and affinity determined with recombinant ZnT8-aa275-369 variant proteins in specific ZnT8R and ZnT8W autoantibody positive type 1 diabetes patients  (#121)

Hanna Skärstrand 1 , Eva Krupinska 2 , Tatu Haataja 2 , Fariba Vaziri-Sani 1 , Jens O Lagerstedt 2 , Åke Lernmark 1
  1. Clinical Sciences, Lund University, Skåne University Hospital SUS, Malmö, Skåne, Sweden
  2. Experimental Medical Science, Lund University, Lund, Sweden

Background: Autoantibodies against Zinc Transporter 8 (ZnT8) are important predictive markers of type 1 diabetes (T1D) progression in islet autoantibody positive subjects. While it has been suggested that the specificity of the ZnT8A is restricted to the 325-epitope the specificity and affinity are poorly understood. We aimed to produce recombinant ZnT8-aa275-369 for competitive Radiobinding assays (RBA), and to determine the specificity and affinity of T1D sera specific for either ZnT8 Arginine (R) or ZnT8 Tryptophan (W) autoantibodies.

Methods: cDNA encoding ZnT8R C-terminal (aa275-369) was subcloned into pETMBP_1 for bacterial expression of a fusion protein between maltose-binding protein (MBP) and ZnT8R-aa275-369. MBP-ZnT8W-aa275-369 was created using ChickChange lightning mutagenesis. Affinity-chromatography yielded fusion proteins of high purity (>95%). In parallel, 35S-methionine-radiolabeled ZnT8R-aa268-369 and ZnT8W-aa268-369 proteins were generated by in vitro transcription and translation. Newly diagnosed T1D patients (1.5-14.9 years) with high-risk HLA (DQ2, DQ8 or DQ2/8) and either ZnT8R-specific (n=12) or ZnT8W-specific (n=12) autoantibodies were selected for competitive RBA wherein non-radiolabeled ZnT8R/W-aa275-369 at 0.001-100 µg/mL were tested against 35S-methionine-radiolabeled ZnT8R/W-aa268-369.

Results: Median titers were significantly different for R- and W-specific sera (4466 U/mL range; 2986-8306 and 6751 U/mL range; 3905-14454 U/mL; p<0.02). ZnT8RA-specific sera were effectively displaced by 100 µg/mL ZnT8R-protein (98%; range 95-99% displacement) but not by ZnT8W (34%; range 10-89% displacement). ZnT8WA-specific sera were displaced by 100 µg/mL ZnT8W-protein (98%: range 97-99% displacement) but not by ZnT8R (8%; range 0-39% displacement). Kd for the ZnT8R-protein in ZnT8RA-specific sera was 87 ng/mL and undeterminate in ZnT8WA. Kd for the ZnT8W-protein in ZnT8WA-specific sera was 70 ng/mL and essentially undeterminate in the ZnT8RA. The end-point titers did not correlate to the Kd-value.

Conclusions: The affinity of ZnT8WA was higher compared to the ZnT8RA in the newly diagnosed T1D patients. Also, the affinity was independent of the autoantibody titers in the patients.