Zinc transporter 8 ( Z n T 8) autoantibody epitope specificity and affinity examined with recombinant Z n T 8 variant proteins in specific Z n T 8 R and Z n T 8 W autoantibody‐positive type 1 diabetes patients

Summary Variant‐specific zinc transporter 8 autoantibodies ( Z n T 8 A ) against either arginine ( R ) or tryptophan ( W ) at amino acid (aa) position 325 of the zinc transporter 8 ( Z n T 8) has been identified in type 1 diabetes ( T 1 D) patients. Reciprocal cross‐over tests revealed differences in half‐maximal binding to indicate variable affinity of patient Z n T 8 autoantibodies. Insufficient recombinant Z n T 8 variant proteins have precluded detailed analyses of Z n T 8 autoantibody affinity. The aims in the present study were to (i) generate recombinant Z n T 8 R ‐ and Z n T 8 W ‐aa275‐369 proteins; (ii) test the Z n T 8 R ‐ and Z n T 8 W ‐aa275‐369 proteins in reciprocal competitive radiobinding assays ( RBA ) against Z n T 8 R ‐ and Z n T 8 W ‐aa268‐369 labelled with 35 S ‐methionine; and (iii) determine the specificity and affinity of sera specific for either Z n T 8 arginine ( R ) or Z n T 8 tryptophan ( W ) autoantibodies in newly diagnosed T 1 D patients. The results demonstrate, first, that it was possible to produce recombinant human MBP – Z n T 8‐aa275‐369 protein purified to homogeneity for RBA reciprocal competition experiments. Secondly, high‐titre Z n T 8 WA sera diluted to half maximal binding showed significant specificity for respective variants of either Z n T 8 R or Z n T 8 W . Thirdly, Z n T 8 WA ‐positive sera showed high affinity for Z n T 8 W compared to Z n T 8 RA for Z n T 8 R . These data demonstrate that T 1 D patients may have single amino acid‐specific autoantibodies directed against either Z n T 8 R or Z n T 8 W and that the autoantibody affinity to the respective variant may be different. Further studies are needed to assess the mechanisms by which variant‐specific Z n T 8 A of variable affinity develop and their possible role in the pathogenic process leading to the clinical onset of T 1 D .