title: Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis.
abstract: A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex.
authors.affiliation: Analytis Gesellschaft für Laboruntersuchungen mbh, Ludwigshafener Str. 1, 50389 Wesseling, Germany. bornemann@analytis.de
authors.forename: Claus
authors.initials: C
authors.lastname: Bornemann
authors.forename: Tilman
authors.initials: T
authors.lastname: Burggraef
authors.forename: Günter
authors.initials: G
authors.lastname: Heimbüchel
authors.forename: Franz-Georg
authors.initials: FG
authors.lastname: Hanisch
authors.forename: Sandra
authors.initials: S
authors.lastname: Winkels
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000737:chemistry; D001826:Body Fluids / Q000737:chemistry*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000032:analysis*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000032:analysis / Q000276:immunology; D007525:Isoelectric Focusing; D011786:Quality Control; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D004921:Erythropoietin