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High-titer GAD65 autoantibodies detected in adult diabetes patients using a high efficiency expression vector and cold GAD65 displacement.

Author:
  • Ida Jönsson
  • Kristian Lynch
  • Göran Hallmans
  • Åke Lernmark
  • Olov Rolandsson
Publishing year: 2011
Language: English
Pages: 129-136
Publication/Series: Autoimmunity
Volume: 44
Document type: Journal article
Publisher: Taylor & Francis

Abstract english

Adult type 2 diabetes patients with GAD65 autoantibodies (GADA) are known as latent autoimmune diabetes in adults (LADA). It has been suggested that GADA in LADA patients preferentially bind to the N-terminal end of GAD65. Using the N-terminal end extension of (35)S-GAD65 generated by the pEx9 plasmid, we tested the hypothesis that GADA in LADA patients preferentially react with (35)S-GAD65 from the pEx9 plasmid compared to the normal length pThGAD65 plasmid. Healthy control subjects (n = 250) were compared with type 1 (n = 23), type 2 (n = 290), and unspecified (n = 57) diabetes patients. In addition, radio-binding assays for GADA with (35)S-GAD65 generated from both the pEx9 and pThGAD65 plasmids were used in displacement assays with an excess of recombinant human GAD65 (2 mug/mL) to correct for non-specific binding. (35)S-GAD65 produced by either pEx9 or pThGAD65 did not differ in binding among the healthy controls and among the type 1 diabetes patients. Among the type 2 and unspecified patients, there were 4/290 and 3/57 patients, respectively, with binding to the pEx9 but not to the pThGAD65 generated (35)S-GAD65. In the displacement assay, we discovered 14 patients with very high-titer GADA among the type 1 (n = 3, 12,272-29,915 U/mL), type 2 (n = 7; 12,398-334,288 U/mL), and unspecified (n = 4; 20,773-4,053,580 U/mL) patients. All samples were fully displaced following appropriate dilution. We conclude that pThGAD65 is preferred for the coupled in vitro transcription translation of (35)S-GAD65 and that displacement with recombinant GAD65 may detect very high-titer GADA with possible clinical relevance.

Keywords

  • Rheumatology and Autoimmunity

Other

Published
  • Diabetes and Celiac Unit
  • ISSN: 0891-6934
E-mail: ake [dot] lernmark [at] med [dot] lu [dot] se

Principal investigator

Diabetes and Celiac Unit

+46 40 39 19 01

+46 70 616 47 79

60:11:015

Jan Waldenströms gata 35, Malmö

33

Lund University Diabetes Centre, CRC, SUS Malmö, Jan Waldenströms gata 35, House 91:12. SE-214 28 Malmö. Telephone: +46 40 39 10 00