title: Immunological applications of single-domain llama recombinant antibodies isolated from a naïve library.
abstract: We describe the rapid isolation of single-domain recombinant antibodies in VHH format from a pre-immune llama library created in our laboratory. Such naïve library has demonstrated to be a versatile tool and enabled the isolation of several different antibodies for any of the six proteins panned in parallel. The binders specific for human fibroblast growth factor receptor 1 (FGFR1) were successively analyzed in more detail and resulted suitable for both western blot and immunofluorescence analyses. Several milligrams per liter of antibodies were purified by affinity chromatography and used for kinetic and thermodynamic characterization. Their K(D) was in the nanomolar range and they apparently bound a FGF receptor 1 domain not overlapping the region recognized by its physiological ligand FGF. Altogether, the collected data indicate that the new library can enable the recovery of binders of high affinity, specificity and functionality in the conventional immunological tests, avoiding the necessity of further maturation steps. Such results confirmed recent reports of high affinity pre-immune IgNARs and supported the choice of using large single-domain recombinant antibody naïve libraries as an alternative to the preparation of immune libraries for selecting monoclonal antibodies, at convenient cost and time conditions.
authors.affiliation: Consortium for Genomic Technologies, Biochemistry Unit, IFOM-IEO Campus, Via Adamello 16, 20139 Milano, Italy.
authors.forename: Ana
authors.initials: A
authors.lastname: Monegal
authors.forename: Diletta
authors.initials: D
authors.lastname: Ami
authors.forename: Chiara
authors.initials: C
authors.lastname: Martinelli
authors.forename: He
authors.initials: H
authors.lastname: Huang
authors.forename: Marisa
authors.initials: M
authors.lastname: Aliprandi
authors.forename: Paola
authors.initials: P
authors.lastname: Capasso
authors.forename: Chiara
authors.initials: C
authors.lastname: Francavilla
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ossolengo
authors.forename: Ario
authors.initials: A
authors.lastname: de Marco
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D001483:Base Sequence; D002161:Camelids, New World / Q000276:immunology*; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D005346:Fibroblast Growth Factors; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1