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Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays.

Rui, M; Hampe, CS; Wang, C; Ling, Z; Gorus, FK; Lernmark, Åke LU ; Pipeleers, DG and De Pauw, PE (2007) In Journal of Immunological Methods 319(1-2). p.133-143
Abstract
The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively... (More)
The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to > 100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
GAD65, Immunoassay, Type 1 diabetes, Pancreatic beta cells
in
Journal of Immunological Methods
volume
319
issue
1-2
pages
133 - 143
publisher
Elsevier
external identifiers
  • scopus:33846238106
ISSN
1872-7905
DOI
10.1016/j.jim.2006.11.007
language
English
LU publication?
yes
id
c880ed6e-3137-4513-8664-b536a3d4017e (old id 1140981)
date added to LUP
2008-08-14 09:33:16
date last changed
2017-01-01 08:11:58
@article{c880ed6e-3137-4513-8664-b536a3d4017e,
  abstract     = {The 65 kDa isoform of human glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (T1D). In the present study, we have developed a sensitive sandwich time-resolved fluorescence immunoassay (TRFIA) for the quantification of GAD65 in cell extracts, cell media and serum. The monoclonal antibody GAD-6 is used to selectively capture GAD65 but not the slightly larger isoform GAD67, and the utilization of different detecting antibodies with distinct GAD65 epitope specificity allows modulating the specificity of the assay. To this effect we have biotinylated a recombinant antigen-binding fragment (rFab) with epitope specificity for the N-terminal region of rat and human GAD65 (rFab N-GAD65) and another rFab that selectively binds to the middle part of human GAD65 (rFab b96.11). In the assay the biotinylated rFabs are recognized by Europium labeled streptavidin. The obtained time-resolved fluorescence (TRF) is directly proportional to the concentration of GAD65 over a large measuring range (0.1 to > 100 ng/mL). Based on total error estimation including both bias and imprecision, the lower limit of quantitation (LLOQ) of GAD65 in cell extracts is 0.33 ng/mL with the N-GAD65 TRFIA, and 0.10 ng/mL with the b96.11 TRFIA, but the latter is suitable for human GAD65 only, whereas the N-GAD65 TRFIA has equal sensitivity with rat and human GAD65. Specificity was further checked with GAD65/67 fusion proteins, confirming that the presence of intact capture as well as detection epitope on the analyte is a prerequisite for recognition in both assays. We show that the beta cell-specific marker GAD65 can be quantified in pancreatic cell extracts and in serum, allowing studies on discharge during cell death in vitro as well as in vivo.},
  author       = {Rui, M and Hampe, CS and Wang, C and Ling, Z and Gorus, FK and Lernmark, Åke and Pipeleers, DG and De Pauw, PE},
  issn         = {1872-7905},
  keyword      = {GAD65,Immunoassay,Type 1 diabetes,Pancreatic beta cells},
  language     = {eng},
  number       = {1-2},
  pages        = {133--143},
  publisher    = {Elsevier},
  series       = {Journal of Immunological Methods},
  title        = {Species and epitope specificity of two 65 kDa glutamate decarboxylase time-resolved fluorometric immunoassays.},
  url          = {http://dx.doi.org/10.1016/j.jim.2006.11.007},
  volume       = {319},
  year         = {2007},
}