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

  1. Mark Peakman1,
  2. Timothy I. Tree1,
  3. Josef Endl2,
  4. Peter van Endert3,
  5. Mark A. Atkinson4 and
  6. Bart O. Roep5
  1. 1Department of Immunology, Guy’s King’s and St. Thomas’ School of Medicine, London, United Kingdom
  2. 2Roche Diagnostics, Penzberg, Germany
  3. 3Institut National de la Santé et de la Recherche Médicale Unité 25, Paris, France
  4. 4Department of Pathology, University of Florida, Gainesville, Florida
  5. 5Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands


    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.


    • Address correspondence and reprint requests to Dr. Mark Peakman, Department of Immunology, Guy’s King’s and St. Thomas’ School of Medicine, King’s College, London, Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, U.K. E-mail: mark.peakman{at}

      Received for publication 12 March 2001 and accepted 16 May 2001.

      EU, endotoxin unit; PBMC, peripheral blood mononuclear cell; PI, proinsulin; TCL, T-cell line; TCC, T-cell clone; TT, tetanus toxoid.

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