Novel fusion proteins in the analysis of diabetes-associated autoantibodies to GAD65 and IA-2

Assays to detect autoantibodies to glutamic acid decarboxylase (GAD65) and the protein tyrosine phosphatase-like molecule IA-2, which are both present in pancreatic islets, have been used in the diagnosis and prediction of ty pe 1 diabetes. In this study a novel fusion protein combining the entire GA D65 molecule with the 40 kDa intracellular domain of IA-2 (GAD-IA-2) was co nstructed to detect autoantibodies to both antigens by one single assay. Fo r the same purpose a truncated version of this fusion protein which contain ed the entire GAD65 linked to the 203 carboxy-terminal amino acids of IA-2 (GAD-dIA-2) was made. A panel of 34 diabetic sera which represented unequiv ocally positive or negative antibody responses to GAD65 and/or IA-2 as well as 20 serum samples from healthy controls were tested in a radioligand bin ding assay with the constructed fusion proteins as antigens. Nine of the sa mples from patients with type 1 diabetes reacted with GAD65 while being neg ative for IA-2. Six sera were positive for IA-2 only, 11 were double positi ve, and 8 negative for both antibodies using the standard in vitro transcri ption translation assay with single antigens. The full-length, as well as t he truncated fusion protein detected all samples positive for antibodies ei ther to GAD65 or IA-2 or both, except for one GAD65 antibody positive sampl e. All samples from healthy controls tested negative in all assays. We conc lude that the principle of a combinatorial molecule where a fusion protein expresses both GAD65 and IA-2 epitopes is feasible, and such a fusion prote in can be used instead of the single antigens to reduce time and costs of l arge-scale screening for clinical purposes. (C) 2000 Elsevier Science B.V. All rights reserved.