Characterization of monoclonal ZnT8-specific antibody
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1
Institute of Biomedicine, University of Tartu, Estonia
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2
Institute of Molecular and Cell Biology, University of Tartu, Estonia
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3
Department of Biomedicine, University of Tartu, Estonia
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4
Institute of Technology, University of Tartu, Estonia
Immune-mediated destruction of insulin-producing beta cells of pancreas leads to autoimmune type 1 diabetes mellitus (T1D). Recently, ZnT8 (zinc transporter 8) has been identified as an autoimmune target in T1D. ZnT8 is expressed in the pancreatic tissue and regulates Zn metabolism in beta cells. Zn is critical for normal storage of insulin in the secretory vesicles of islet beta cells. ZnT8 autoantibodies are directed mostly against the C terminal domain. Molecular biological tools for basic research of ZnT8 role in T1D are needed.
In this report, we characterize a new monoclonal antibody to the ZnT8 C-terminus. Mice were immunized with Semliki Forest virus-like particles expressing full-length ZnT8 and hybridomas were obtained and cloned. Hybridomas were tested using LIPS (Luciferase Immunoprecipitation system) and ELISA using purified recombinant C-terminal domain of ZnT8. One of the hybridomas giving a very high signal was selected for further analysis. Protein A purified Mab was used to characterize binding epitope using 18 overlapping peptides designed according to C-terminal sequence of ZnT8. Binding epitope was identified using four methods: LIPS assay, ELISA, biosensor binding test, and immunohistochemistry assay with monkey pancreatic iselets.
In conclusion, we have characterized a new monoclonal antibody usable as a molecular biological tool for ZnT8 research.
Keywords:
ZnT8,
Autoantibodies,
autoimmune diabetes,
Monoclonal antibody,
beta cells
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Immune-mediated disease pathogenesis
Citation:
Ustinova
J,
Belitskin
D,
Juronen
E,
Zusinaite
E,
Utt
M and
Uibo
R
(2013). Characterization of monoclonal ZnT8-specific antibody.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00326
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Received:
14 Mar 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Miss. Julia Ustinova, Institute of Biomedicine, University of Tartu, Tartu, Estonia, lia@ut.ee