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Research Article

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

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Pages 129-136 | Received 29 Jan 2010, Accepted 29 Mar 2010, Published online: 29 Jul 2010
 

Abstract

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.

Acknowledgments

This study was supported by Västerbotten County Council (OR), the Swedish Research Council (K2008-55X-15312-04-3) (ÅL), UMAS Funds, and the Skåne County Council of Research and Development. We kindly thank Diamyd Medical AB, Stockholm, for their generous donation of human recombinant GAD65. We thank ÅsaÅgren for technical assistance.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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