Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of Autoreactive T-cells in type 1 Diabetes report of phase II of the second international Immunology of Diabetes Society workshop for standardization of T-cell assays in type 1 Diabetes

• Published in: Diabetes (New York, N.Y.) Vol. 50; no. 8; pp. 1749 - 1754
• Main Authors: PEAKMAN, Mark, TREE, Timothy I, ENDL, Josef, VAN ENDERT, Peter, ATKINSON, Mark A, ROEP, Bart O
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 01-08-2001
• Subjects: Animals; Antigens; Autoantigens - immunology; Baculoviridae; Biological and medical sciences; Cell Line; Clone Cells; Cloning; Diabetes; Diabetes Mellitus, Type 1 - diagnosis; Diabetes Mellitus, Type 1 - immunology; Diabetes research; Diabetes. Impaired glucose tolerance; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Endotoxins - immunology; Glutamate Decarboxylase - immunology; Humans; Immunology; Insulin; Insulin - immunology; Insulin - pharmacology; Islets of Langerhans - immunology; Isoenzymes - immunology; Lymphocyte Activation - drug effects; Management. Various non-drug treatments. Langerhans islet grafts; Medical sciences; Phosphatase; Proinsulin - immunology; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases - immunology; Receptor-Like Protein Tyrosine Phosphatases, Class 8; Recombinant Proteins - immunology; Risk Factors; Sensitivity and Specificity; Spodoptera; T-Lymphocytes - drug effects; T-Lymphocytes - immunology; Tetanus Toxoid - immunology; Third party; Transfection; United States; Endocrinopathy; Proinsulin; Enzyme; Toxicity; Langerhans islet; Standardization; Phosphoric monoester hydrolases; Autoantibody; Esterases; Lyases; Glutamate decarboxylase; Carbon-carbon lyases; Carboxy-lyases; Aminoacid; T-Lymphocyte; Insulin dependent diabetes; Hydrolases; Islet cell antibody; Detection; Endocrine pancreas; Protein-tyrosine-phosphatase
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/diabetes.50.8.1749

The identification, quantification, and characterization of T-cells reactive with the islet autoantigens GAD65, proinsulin (PI), and tyrosine phosphatase-like molecules IA-2 and phogrin are major research goals in type 1 diabetes. 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 of the inconsistency in published studies of peripheral blood T-cell reactivity to islet autoantigens. Poor antigen quality has also hampered the development of novel technologies for the detection of islet-reactive T-cells. For these reasons, in the present study, several preparations of GAD65, PI, and IA-2 were collected and evaluated for endotoxin content, ability to stimulate a panel of relevant T-cell clones, and inhibitory effects on proliferation to unrelated third-party antigens. Through this process, we have been able to identify preparations of GAD65 and IA-2, generated in insect cells using the baculovirus expression system, that stimulate relevant clones and display low inhibitory effects on third-party antigens. In addition, we characterized a PI preparation generated in bacteria as being free of effects on proliferation to third-party antigens and low in endotoxin content. These preparations are important to promote the development of robust and sensitive assays of islet-reactive T-cells in patients with type 1 diabetes or patients at high risk for developing the disease.