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

Jönsson, Ida LU ; Lynch, Kristian LU ; Hallmans, Göran; Lernmark, Åke LU and Rolandsson, Olov (2011) In Autoimmunity 44. p.129-136
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 (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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Autoimmunity
volume
44
pages
129 - 136
publisher
Taylor & Francis
external identifiers
  • WOS:000288236200007
  • PMID:20670115
  • Scopus:79551519263
ISSN
0891-6934
DOI
10.3109/08916934.2010.482117
language
English
LU publication?
yes
id
babf6f91-fad4-4ecd-9559-9d1080215e8f (old id 1644443)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/20670115?dopt=Abstract
date added to LUP
2010-08-05 10:50:26
date last changed
2016-10-13 04:30:53
@misc{babf6f91-fad4-4ecd-9559-9d1080215e8f,
  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 (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.},
  author       = {Jönsson, Ida and Lynch, Kristian and Hallmans, Göran and Lernmark, Åke and Rolandsson, Olov},
  issn         = {0891-6934},
  language     = {eng},
  pages        = {129--136},
  publisher    = {ARRAY(0x89b1ba0)},
  series       = {Autoimmunity},
  title        = {High-titer GAD65 autoantibodies detected in adult diabetes patients using a high efficiency expression vector and cold GAD65 displacement.},
  url          = {http://dx.doi.org/10.3109/08916934.2010.482117},
  volume       = {44},
  year         = {2011},
}