title: Immunohistochemical differentiation of monoclonal GAD antibodies recognizing linear or conformational epitope regions.
abstract: GAD65 is targeted by different patterns of autoantibodies [glutamic acid decarboxylase (GAD)-AAbs] in insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). To study differentiation of the GAD-AAb pattern by immunohistochemistry, we examined the immunostaining of 15 monoclonal GAD antibodies (mc-GAD-Abs), which recognized different epitope regions of the antigen, on human pancreatic sections that were unfixed or fixed with different fixatives. By a competitive sandwich enzyme-linked immunosorbent assay (ELISA), three binding patterns of mc-GAD-Abs were identified: 5 of 15 mc-GAD-Abs recognized a linear N-terminal epitope (p1), 5 of 15 were reactive with a conformational GAD65 epitope region (p2), and 5 of 15 were cross-reactive with GAD67 (p3). These patterns of mc-GAD-Abs were tested for islet cell binding by indirect immunofluorescence on pancreatic sections treated with either (1) Bouin's solution, (2) Zamboni's solution, or (3) phosphate-buffered formaldehyde for 0.5, 1, 2, and 18 h at 4 degrees C. After fixation for up to 2 h no differentiation of immunoreactivity of patterns was observed using the three fixatives. mc-GAD-Abs recognizing conformational epitope regions (p2) revealed a marked reduced immunoreactivity on pancreatic sections fixed for 18 h with 4% formaldehyde, while mc-GAD-Abs reactive with linear epitopes (p1, p3) were detectable with strong binding. This fixation procedure was used to compare the immunoreactivity of GAD-AAb+ or GAD-AAb- islet cell cytoplasmic antibody-positive (ICA+) sera of IDDM (n = 27) and SMS patients (n = 3). The three SMS sera were reactive with GAD on fixed islets but showed a reduced titer, whereas the majority of IDDM sera (22/27; 81.5%) were not detectable; 70.6% (12/ 17) of GAD-AAb+ IDDM sera were not detectable on fixed islets. Furthermore, all 10 GAD-AAb- IDDM sera tested failed to react with fixed pancreas, which also suggested an alteration of non-GAD-ICA antigens. In conclusion, the fixation of human pancreatic sections with formaldehyde for 18 h allows the differentiation of GAD-AAbs recognizing linear and conformational epitope regions.
authors.affiliation: Department of Immunochemistry, Institute of Diabetes Gerhardt Katsch, University of Greifswald, Karlsburg, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Augstein
authors.forename: B
authors.initials: B
authors.lastname: Ziegler
authors.forename: M
authors.initials: M
authors.lastname: Schlosser
authors.forename: S
authors.initials: S
authors.lastname: Flassig
authors.forename: M
authors.initials: M
authors.lastname: Strebelow
authors.forename: M
authors.initials: M
authors.lastname: Ziegler
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D001667:Binding, Competitive; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D019084:Fluorescent Antibody Technique, Indirect; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D011994:Recombinant Proteins
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase