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Application of phage display peptide library to autoimmune diabetes: identification of IA‐2/ICA512bdc dominant autoantigenic epitopes

Autoantigenic epitope mapping represents a critical issue in autoimmune diseases. The islet tyrosine phosphatase‐like protein IA‐2/ICA512bdc is a major autoantigen in type 1 diabetes (IDDM), but the epitopes responsible for autoantibody binding have been only partially defined. The aim of our study was to identify ICA512bdc epitopes, and in particular mini‐epitopes, utilizing a novel strategy for autoimmune diseases. The study was performed in three sequential steps: (1) construction of a λ‐phage surface‐displayed ICA512bdc cDNA library with the methodology of tagged random priming with peptides displayed as a fusion to the C terminus of the capsid protein D; (2) affinity selection of the resulting library, followed by immunoscreening, enzyme‐linked immunosorbent assay and sequence analysis of positive clones, and (3) radioimmunoprecipitation to detect autoantibodies to the selected clones. This strategy resulted in the identification of two epitopes (IA‐2 residues 761 – 964 and 929 – 979), which were recognized by 100 % and 62.9 % ICA512bdc‐positive IDDM patients, respectively. Interestingly, the larger clone was detected also by a proportion (16.7 %) of new onset ICA512bdc‐negative patients, thus suggesting that this region contains not only the main autoantigenic repertoire of ICA512bdc molecule, but is able to detect IA‐2 autoantibodies in even higher percentages of patients. In addition, this study showed the existence of multiple epitopes located in the C‐terminal domain of the IA‐2 protein, one of which is formed by the 50 C‐terminal amino acids, and provided evidence that the strategy used represents a valid tool for identification of epitopes within autoantigenic molecules.