Measurements of colour constancy by using a forced-choice matching technique

In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies 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 strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera.