Epitope spreading and a varying but not disease-specific GAD65 antibody response in Type I diabetes

Aims/hypothesis. The aim of this study was to analyse the conformational an d linear epitope profiles of glutamic acid decarboxylase antibody (GAD65-ab )-positive sera to find disease-specific epitope profiles and to study, whe ther GAD65-ab epitope recognition changes or spreads during the prediabetic period and, thus, can provide markers to differentiate early from later st ages of progression to diabetes. Methods. Sera from subjects before (n = 21), at onset (n = 44), or at incre ased risk of Type I (insulin-dependent) diabetes mellitus (n = 20) and from patients with stiff-man syndrome (SMS, n = 18) or polyendocrine autoimmune syndrome (PAS, n = 21) were analysed for conformational and linear GAD65 e pitope recognition by an immunohistochemical blocking test based on human m onoclonal GAD65-ab (MICA 1-10) and western blotting of a GAD65 epitope-cDNA -library. Results. A redundant reactivity of many GAD65-ab positive sera to three maj or conformational (EP-1, EP-2, EP-3) and two dominant linear epitope cluste rs (amino acid 1-124 and 535-585) was observed in diabetes, polyendocrine a utoimmune syndrome and stiff-man syndrome and no disease-specific epitopes or epitope-profiles were detected. Epitope recognition broadened with highe r titres and with the vulnerability of patients to acquire additional autoi mmune diseases apart from diabetes. Low GAD65-ab serum titres (<1200 arbitr ary units) and EP-1 recognition in the absence of EP-2 binding characterise d the early immune response. Changing epitope profiles combined stable reco gnition of EP-1 with gain or loss of reactivity to C-terminal epitopes duri ng follow-up. Conclusion/interpretation. A maturing autoantibody response, which could sp read from EP-1-recognition to other regions of GAD65, resulted in titre-rel ated rather than disease-specific epitope profiles which were not sufficien t to predict whether GAD65-ab positive subjects will progress to Type I dia betes, autoimmune polyendocrine syndrome or stiff-man syndrome.