title: A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.
abstract: A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark. Electronic address: rac@sund.ku.dk.
authors.forename: Rasmus P
authors.initials: RP
authors.lastname: Clausen
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Andreas Ø
authors.initials: AØ
authors.lastname: Mohr
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Erik
authors.initials: E
authors.lastname: Riise
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Anders A
authors.initials: AA
authors.lastname: Jensen
authors.affiliation: Department of Biochemistry & Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.
authors.forename: Avinash
authors.initials: A
authors.lastname: Gill
authors.affiliation: Department of Biochemistry & Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.
authors.forename: Dean R
authors.initials: DR
authors.lastname: Madden
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
authors.forename: Jette S
authors.initials: JS
authors.lastname: Kastrup
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark; Department of Clinical Biochemistry, Copenhagen University Hospital, Hvidovre, DK-2650, Denmark. Electronic address: peter.durand.skottrup@regionh.dk.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Skottrup
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000276:immunology / Q000378:metabolism; D001923:Brain Chemistry; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D005260:Female; D007114:Immunization; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000302:isolation & purification*; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D000072417:Protein Domains; D055503:Protein Multimerization; D051381:Rats; D018091:Receptors, AMPA / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment
chemical_list: D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D018091:Receptors, AMPA; D011994:Recombinant Proteins; C104724:glutamate receptor ionotropic, AMPA 4
keywords: Fab; GluA4; Phage