Cadherin-2 plays a fundamental role during zebrafish CNS and heart morphogenesis. Profiling zCdh2 expression via antibody staining is essential to achieving better understanding of its role during zebrafish development. Generation of recombinant human antibodies to zCdh2 by phage display was used to identify monoclonal antibodies with reduced unspecific binding patterns when compared to available commercial antibodies. Specificity was profiled using flow cytometry of wild type, zCdh2-defective mutant and zCdh2-GFP zebrafish cells. The epitopes recognized by the novel antibodies were mapped to peptides located in the first or second extracellular domains of zCdh2. These antibodies allowed improved observations of the spatial distribution of zCdh2 from imaging of whole mount zebrafish preparations. Since the generated antibodies are sequence defined, they can always be reconstituted from the information stored in the respective computer file, securing future reproducibility of respective experiments. The results further suggest that sequence defined antibodies with specificities thoroughly controlled by flow cytometry and genetic antigen-defective mutants or knockouts can substantially reduce the risk of misleading, false-positive results in whole mount imaging and other assays, and thus can improve the scientific value of such assays.
Keywords: Antibody engineering; Antibody specificity; Central nervous system; Epitope mapping; Flow cytometry; Immunohistochemistry; Phage display; Primary cell culture; Recombinant antibody; scFv; scFv-Fc.
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