COMPARISON OF THE ISLET-CELL ANTIBODY PATTERN OF MONOCLONAL GLUTAMIC-ACID DECARBOXYLASE ANTIBODIES RECOGNIZING LINEAR AND CONFORMATIONAL EPITOPES

In order to compare the reactivity of glutamic acid decarboxylase (GAD ) antibodies recognizing linear and conformational epitopes as islet c ell cytoplasmic antibodies (ICA), monoclonal antibodies were generated . An ELISA displacement test using two biotinylated monoclonals recogn izing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GA D antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of huma n, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the m onoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antib odies recognized a linear GAD epitope located at the N-terminus (patte rn 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to pat terns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals s pecific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodie s in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with stron g reactivity. Furthermore, results obtained with monoclonals of patter n 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cr yosections, which may result in an ICA-negative test of GAD65 autoanti body positive sera.