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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1644443
- author
- Jönsson, Ida
LU
; Lynch, Kristian
LU
; Hallmans, Göran
; Lernmark, Åke
LU
and Rolandsson, Olov
- organization
- publishing date
- 2011
- 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
- pmid:20670115
- 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
- 2016-04-04 09:27:23
- date last changed
- 2022-01-29 17:54:46
@article{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 = {{Taylor & Francis}},
series = {{Autoimmunity}},
title = {{High-titer GAD65 autoantibodies detected in adult diabetes patients using a high efficiency expression vector and cold GAD65 displacement.}},
url = {{https://lup.lub.lu.se/search/files/5329327/1671807.pdf}},
doi = {{10.3109/08916934.2010.482117}},
volume = {{44}},
year = {{2011}},
}