This proposal outlines how a unique resource of zebrafish cell surface and secreted proteins will be used in conjunction with a novel method of antibody selection to create a resource of 200 new zebrafish monoclonal antibody reagents. The antibody targets have been carefully selected primarily from their known in situ hybridization patterns so as to be of maximum benefit to the zebrafish community. Targets include: new cellular markers and proteins whose subcellular localization is important for function such as morphogens, polarity proteins and neural cell surface proteins. The throughput required to select this large number of antibodies will be achieved by using a novel pooling immunization strategy, antibody selection using protein microarrays and the recombinant cloning of antibody V-regions. The antibody selection process will identify antibodies that are non-crossreactive with proteins from the same family and recognize formalin-resistant epitopes. Finally, each antibody will be tested on whole mount embryos and cryosections and will be freely and widely distributed. These antibodies, when used in conjunction with the large genetic resources available to zebrafish researchers, will allow new mechanistic insights into protein function which may eventually be used to design novel strategies for therapeutic intervention.

Public Health Relevance

The zebrafish is an increasingly popular laboratory organism with many experimental advantages that biologists exploit to discover the detailed processes that underlie vertebrate development and disease. Zebrafish research, however, is limited by a paucity of good antibody reagents that are essential tools for determining the precise details of biological processes. This research proposal is aimed at providing the zebrafish community with a major resource of new antibody reagents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS063400-05
Application #
8304115
Study Section
Special Emphasis Panel (ZRG1-BDA-F (50))
Program Officer
Stewart, Randall R
Project Start
2008-07-01
Project End
2013-12-30
Budget Start
2012-07-01
Budget End
2013-12-30
Support Year
5
Fiscal Year
2012
Total Cost
$184,024
Indirect Cost
$13,172
Name
Sanger Institute
Department
Type
DUNS #
346013253
City
Cambridge
State
Country
United Kingdom
Zip Code
Wright, Gavin J; Washbourne, Philip (2011) Neurexins, neuroligins and LRRTMs: synaptic adhesion getting fishy. J Neurochem 117:765-78
Crosnier, Cecile; Staudt, Nicole; Wright, Gavin J (2010) A rapid and scalable method for selecting recombinant mouse monoclonal antibodies. BMC Biol 8:76