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Åke Lernmark

Principal investigator

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Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands

Author

  • Alberto Falorni
  • Eva Örtqvist
  • Bengt Persson
  • Åke Lernmark

Summary, in English

Autoantibodies are an important marker of human autoimmune diseases and the development of simple, precise and reproducible immunoassays to detect autoantibodies is important to our understanding of human autoimmunity. GAD65 autoantibodies occur frequently in insulin-dependent diabetic patients and is a useful marker for IDDM. A RIA to detect immunoreactive GAD65 has not been described. In the present study we describe a semi-automated fluid-phase immunoassay for the rapid detection of GAD65 autoantibodies in human serum. We also developed a sensitive RIA to determine immunoreactive human GAD65 in biological fluids and in vitro cell systems. Using in vitro translated recombinant human GAD65 in a multiwell-adapted procedure, our GAD65Ab RIA combines high specificity and sensitivity with a high capacity to analyze a large number of samples. In this report the three critical steps in the GAD65Ab RIA, DNA preparation, in vitro translation and immunoprecipitation, have been optimized. In our RIA, GAD65Ab were detected in 116 155 (75%) new onset Swedish IDDM children and in 1 85 (1.2%) healthy controls. In an immunoassay to detect autoantibodies against the proinsulin converting enzyme 2 (PC-2) no such antibodies were detected in IDDM patients. In the GAD65 RIA the lower detection limit was 2 ng/ml (31 fmol/ml). Our data demonstrate that autoantigen radioligands produced by in vitro translation are useful in RIA for autoantibodies and autoantigens in studies of human autoimmunity.

Publishing year

1995-10-12

Language

English

Pages

89-99

Publication/Series

Journal of Immunological Methods

Volume

186

Issue

1

Document type

Journal article

Publisher

Elsevier

Keywords

  • Autoantibody
  • Glutamic acid decarboxylase
  • In vitro translation
  • Insulin-dependent diabetes mellitus
  • Proinsulin converting enzyme-2
  • Radioimmunoassay

Status

Published

ISBN/ISSN/Other

  • ISSN: 0022-1759