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.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BDA-F (50))
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Stewart, Randall R
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Sanger Institute
United Kingdom
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Staudt, Nicole; Müller-Sienerth, Nicole; Fane-Dremucheva, Alla et al. (2015) A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining. Biochem Biophys Res Commun 456:527-33
Staudt, Nicole; Müller-Sienerth, Nicole; Wright, Gavin J (2014) Development of an antigen microarray for high throughput monoclonal antibody selection. Biochem Biophys Res Commun 445:785-90
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