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Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies

Li, L. ; Jiang, J. ; Hagopian, W. A. ; Karlsen, A. E. LU ; Skelly, M. ; Baskin, D. G. and Lernmark, A. LU orcid (1995) In Journal of Histochemistry and Cytochemistry 43(1). p.53-59
Abstract

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet β-cells,... (More)

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet β-cells, whereas α-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in α-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet α- and β-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

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author
; ; ; ; ; and
publishing date
type
Contribution to journal
publication status
published
keywords
γ-aminobutyric acid, GABA, Immunostaining, Insulin-dependent diabetes, IDDM, Stiff-man syndrome, SMS
in
Journal of Histochemistry and Cytochemistry
volume
43
issue
1
pages
53 - 59
publisher
Histochemical Society
external identifiers
  • scopus:0028855244
  • pmid:7822765
ISSN
0022-1554
DOI
10.1177/43.1.7822765
language
English
LU publication?
no
id
66313220-2106-4b91-8a5b-1e99c986a9d5
date added to LUP
2019-09-11 09:01:48
date last changed
2024-03-13 08:26:57
@article{66313220-2106-4b91-8a5b-1e99c986a9d5,
  abstract     = {{<p>We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet β-cells, whereas α-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in α-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet α- and β-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.</p>}},
  author       = {{Li, L. and Jiang, J. and Hagopian, W. A. and Karlsen, A. E. and Skelly, M. and Baskin, D. G. and Lernmark, A.}},
  issn         = {{0022-1554}},
  keywords     = {{γ-aminobutyric acid, GABA; Immunostaining; Insulin-dependent diabetes, IDDM; Stiff-man syndrome, SMS}},
  language     = {{eng}},
  month        = {{01}},
  number       = {{1}},
  pages        = {{53--59}},
  publisher    = {{Histochemical Society}},
  series       = {{Journal of Histochemistry and Cytochemistry}},
  title        = {{Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies}},
  url          = {{http://dx.doi.org/10.1177/43.1.7822765}},
  doi          = {{10.1177/43.1.7822765}},
  volume       = {{43}},
  year         = {{1995}},
}