title: Expression cloning of camelid nanobodies specific for Xenopus embryonic antigens.
abstract: Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.
authors.affiliation: Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Keiji
authors.initials: K
authors.lastname: Itoh
authors.affiliation: Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
authors.forename: Sergei Y
authors.initials: SY
authors.lastname: Sokol
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology*; D000941:Antigens / Q000276:immunology*; D002162:Camelus / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000276:immunology*; D005260:Female; D005775:Gastrula / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D014981:Xenopus / Q000196:embryology* / Q000276:immunology*; D029867:Xenopus Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D061905:Single-Domain Antibodies; D029867:Xenopus Proteins