title: Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy.
abstract: Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics. To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein. OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle. An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli. The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library. This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis. Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease.
authors.affiliation: Center for Human and Clinical Genetics, Leiden University Medical Center, 2333 AL, Leiden, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: van Koningsbruggen
authors.forename: H
authors.initials: H
authors.lastname: de Haard
authors.forename: P
authors.initials: P
authors.lastname: de Kievit
authors.forename: R W
authors.initials: RW
authors.lastname: Dirks
authors.forename: A
authors.initials: A
authors.lastname: van Remoortere
authors.forename: A J
authors.initials: AJ
authors.lastname: Groot
authors.forename: B G M
authors.initials: BG
authors.lastname: van Engelen
authors.forename: J T
authors.initials: JT
authors.lastname: den Dunnen
authors.forename: C T
authors.initials: CT
authors.lastname: Verrips
authors.forename: R R
authors.initials: RR
authors.lastname: Frants
authors.forename: S M
authors.initials: SM
authors.lastname: van der Maarel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002161:Camelids, New World / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D039141:Muscular Dystrophy, Oculopharyngeal / Q000276:immunology*; D019151:Peptide Library
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library