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High-titer GAD65 autoantibodies detected in adult diabetes patients using a high efficiency expression vector and cold GAD65 displacement.
Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Nutritional Research.ORCID iD: 0000-0001-9581-3845
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2011 (English)In: Autoimmunity, ISSN 0891-6934, Vol. 44, no 2, 129-136 p.Article in journal (Refereed) Published
Abstract [en]

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 ³⁵S-GAD65 generated by the pEx9 plasmid, we tested the hypothesis that GADA in LADA patients preferentially react with ³⁵S-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 ³⁵S-GAD65 generated from both the pEx9 and pThGAD65 plasmids were used in displacement assays with an excess of recombinant human GAD65 (2 μg/mL) to correct for non-specific binding. ³⁵S-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 ³⁵S-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 ³⁵S-GAD65 and that displacement with recombinant GAD65 may detect very high-titer GADA with possible clinical relevance.

Place, publisher, year, edition, pages
Informa Healthcare , 2011. Vol. 44, no 2, 129-136 p.
Keyword [en]
Latent autoimmune diabetes in adults, radio-binding assay, stiff person syndrome, type 1 diabetes, type 2 diabetes
National Category
Medical and Health Sciences
URN: urn:nbn:se:umu:diva-41163DOI: 10.3109/08916934.2010.482117PubMedID: 20670115OAI: diva2:404832
Available from: 2011-03-18 Created: 2011-03-18 Last updated: 2015-04-22Bibliographically approved

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