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Characterization of a linear epitope within the human pancreatic 64‐kDa glutamic acid decarboxylase and its autoimmune recognition by sera from insulin‐dependent diabetes mellitus patients

1993; Wiley; Volume: 212; Issue: 2 Linguagem: Inglês

10.1111/j.1432-1033.1993.tb17698.x

1432-1033

Ludwig Mauch, Charles C. Abney, H. Marijke van den Berg, Werner A. Scherbaum, Bodo Liedvogel, Wolfgang Northemann,

Immune Cell Function and Interaction

A 2.0‐kb cDNA coding for the full‐length 64‐kDa human glutamic acid decarboxylase (GAD 64 ) was isolated from a pancreatic carcinoma cDNA library by oligonucleotide screening, polymerase‐chain‐reaction amplification and subsequently characterized by sequence analysis. Five overlapping fragments of GAD 64 cDNA were constructed into the vector pH6EX3, allowing the highly efficient expression of corresponding fusion proteins with a histidine hexapeptide as an affinity ligand at their N‐termini in Escherichia coli . The recombinant GAD 64 fragments were analysed by Western blotting using sera from patients with early onset of insulin‐dependent diabetes mellitus (IDDM). We found that at least 20% of the patients with an onset of IDDM have developed autoantibodies which can specifically recognize a linear antigenic epitope within the GAD 64 . With a selected IDDM serum, an antigenic epitope was localized in a region of 31 amino acids located at the C‐terminus of GAD 64 , using epitope mapping techniques, and it was characterized. The possibility of using recombinant GAD 64 for the development of an immunoassay for a predictive diagnosis of IDDM is discussed.