Peptide specificity of high-titer anti-glutamic acid decarboxylase (GAD)65 autoantibodies

To study systematically the linear epitope specificity of anti-glutamic acid decarboxylase (GAD) autoantibodies associated with insulin-dependent diabetes mellitus (IDDM), we produced 93 overlapping 12-residue synthetic peptides derived from the sequence of the human GAD65 protein and covering the entire length of the protein. These peptides were used as antigens in an enzyme immunoassay to screen the sera from 10 IDDM patients, all of which contained at high level autoantibodies directed against GAD65. Three out of ten (30%) IDDM patients had antibodies that reacted with one or more of the synthetic peptides. Two of the peptide-reactive IDDM sera, which also bound denatured recombinant GAD65 on western blots, had the highest titers of anti-GAD antibodies in ELISA assay. Moreover, the anti-GAD antibodies-GAD complexes formed with these sera were characterized by low dissociation rates, indicative of their good stability. A fine specificity analysis, using analogs of antigen peptide 1 (residues 1-12), allowed us to identify the residues at positions 5-9 (GSGFW) as critical for antibody recognition.