Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1diabetes - Report of phase II of the second international immunology of diabetes society workshop for standardization of T-cell assays in type 1 diabetes

The identification, quantification, and characterization of T-cells reactiv e with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphat ase-like molecules IA-2 and phogrin are major research goals in type 1 diab etes. In the Immunology of Diabetes Society First Workshop on Autoreactive T-Cells, the quality of recombinant preparations of these autoantigens was identified as a significant weakness, a finding that may account for much o f the inconsistency in published studies of peripheral blood T-cell reactiv ity to islet autoantigens. Poor antigen quality has also hampered the devel opment of novel technologies for the detection of islet-reactive T-cells. F or these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to sti mulate a panel of relevant T-cell clones, and inhibitory effects on prolife ration to unrelated third-party antigens. Through this process, we have bee n able to identify preparations of GAD65 and IA-2, generated in insect cell s using the baculovirus expression system, that stimulate relevant clones a nd display low inhibitory effects on third-party antigens. In addition, we characterized a PI preparation generated in bacteria as being free of effec ts on proliferation to third-party antigens and low in endotoxin content. T hese preparations are important to promote the development of robust and se nsitive assays of islet-reactive T-cells in patients with type 1 diabetes o r patients at high risk for developing the disease.