AbstractBackgroundIdentification of the major humoral epitopes in zinc transporter 8 (ZnT8) will expand the range of biomarkers for human type 1 diabetes and may provide clues to the mechanisms governing disease progression. Our initial studies suggested that most ZnT8‐reactive sera recognize conformational epitopes in the final 100aa region of the molecule. Subsequently we identified residue 325 as a major determinant in two epitopes linked to a genetic polymorphism with high minor allele frequency (rs13266634). The goal of the current study was to extend this analysis to identify non‐polymorphic epitopes in ZnT8.MethodsAlthough the carboxy‐terminal domains of human and mouse ZnT8 are ∼80% identical, the mouse probe is not precipitated by the majority of human type 1 diabetes sera. Thus to identify key residues we systematically ‘humanized’ the mouse probe at each position that differs and evaluated the probes in radio‐immunoassays.ResultsAs previously reported, only the alteration of Q > R325 by itself showed any restoration of binding to human sera. However, when clusters of structurally adjacent variant residues were also changed an additional region of antigenicity was revealed that depended on residues R332, E333, K336, and K340. Using a panel of 112 sera from recent onset subjects tested with the hC325Q and m‐R325R332E333K336K340 probes, 39.3% of the subjects were ZnT8(Q)A+, of which 38.6% (17/44) also recognized the mouse probe.ConclusionsWe conclude that the mR‐REKK probe identifies a third major epitope in ZnT8 that may add to the diagnostic utility of measuring autoantibodies to this molecule. Copyright © 2011 John Wiley & Sons, Ltd.