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NDT Advance Access first published online on January 23, 2006
This version published online on January 25, 2006
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfk101
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received October 25, 2005
Accepted January 5, 2006

Technical Report

Characteristics of polyclonal anti-human nephrin antibodies induced by genetic immunization using nephrin cDNA

Togo Aoyama 1 *, Kouju Kamata 1, Nozomu Yamanaka 1, Yasuo Takeuchi 1, Masaaki Higashihara 1, and Seishi Kato 2

1 Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa, Japan
2 Department of Rehabilitation Engineering, Research Institute, National Rehabilitation Center for the Disabled, Tokorozawa, Saitama, Japan

* To whom correspondence should be addressed.
Togo Aoyama, E-mail: dm02001x{at}st.kitasato-u.ac.jp



  Abstract

Background. Nephrin is an essential protein for maintaining the normal structure of the podocyte foot process and the glomerular filtration barrier. To analyse the mechanism of proteinuria and treatment of nephrotic syndrome, we produced and characterized polyclonal anti-human nephrin antibodies using a newly developed genetic immunization technique.

Methods. An expression vector with full-length or fragmented cDNA of human nephrin protein was administered to female Lewis rats once a week for 12 weeks using a gene-gun method. Antibody production against different fragments of nephrin protein was then analysed by ELISA, Western blot analysis, immunoprecipitation and FACS analysis. We also analysed recognition of native nephrin protein using immunohistochemistry and electron microscopy, and conducted a functional analysis of in vitro clustering of nephrin protein.

Results. Serum anti-nephrin antibody titers reached a maximum level at 8 or 12 weeks. Four of five antibodies induced with cDNA of five different Ig-like motif fragments showed antigen-specific binding to Escherichia coli and produced recombinant human nephrin protein. Only two of these four antibodies, plus one antibody induced by full-length human nephrin protein cDNA, bound to solubilized native nephrin protein. These three IgG antibodies, subclasses IgG1, IgG2a and IgG2b, showed fine granular staining along the glomerular basement membrane of normal human glomeruli and clustering of human nephrin protein on plasma membranes of HEK293 cells.

Conclusions. We successfully produced polyclonal anti-human nephrin antibodies by genetic immunization using nephrin cDNA. These new antigen-specific polyclonal antibodies will be useful for functional analysis and tissue staining of native nephrin protein.

Keywords: anti-nephrin antibody; clustering; DNA immunization; gene-gun; nephrin; proteinuria.
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