Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis. 2003

Claus Bornemann, and Tilman Burggraef, and Günter Heimbüchel, and Franz-Georg Hanisch, and Sandra Winkels
Analytis Gesellschaft für Laboruntersuchungen mbh, Ludwigshafener Str. 1, 50389 Wesseling, Germany. bornemann@analytis.de

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.

UI MeSH Term Description Entries
D007140 Immunoglobulin Fab Fragments Univalent antigen-binding fragments composed of one entire IMMUNOGLOBULIN LIGHT CHAIN and the amino terminal end of one of the IMMUNOGLOBULIN HEAVY CHAINS from the hinge region, linked to each other by disulfide bonds. Fab contains the IMMUNOGLOBULIN VARIABLE REGIONS, which are part of the antigen-binding site, and the first IMMUNOGLOBULIN CONSTANT REGIONS. This fragment can be obtained by digestion of immunoglobulins with the proteolytic enzyme PAPAIN. Fab Fragment,Fab Fragments,Ig Fab Fragments,Immunoglobulins, Fab Fragment,Fab Immunoglobulin Fragments,Immunoglobulin Fab Fragment,Immunoglobulins, Fab,Fab Fragment Immunoglobulins,Fab Fragment, Immunoglobulin,Fab Fragments, Immunoglobulin,Fragment Immunoglobulins, Fab,Fragment, Fab,Immunoglobulin Fragments, Fab
D007525 Isoelectric Focusing Electrophoresis in which a pH gradient is established in a gel medium and proteins migrate until they reach the site (or focus) at which the pH is equal to their isoelectric point. Electrofocusing,Focusing, Isoelectric
D011786 Quality Control A system for verifying and maintaining a desired level of quality in a product or process by careful planning, use of proper equipment, continued inspection, and corrective action as required. (Random House Unabridged Dictionary, 2d ed) Control, Quality,Controls, Quality,Quality Controls
D001826 Body Fluids Liquid components of living organisms. Body Fluid,Fluid, Body,Fluids, Body
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D002852 Chromatography, Ion Exchange Separation technique in which the stationary phase consists of ion exchange resins. The resins contain loosely held small ions that easily exchange places with other small ions of like charge present in solutions washed over the resins. Chromatography, Ion-Exchange,Ion-Exchange Chromatography,Chromatographies, Ion Exchange,Chromatographies, Ion-Exchange,Ion Exchange Chromatographies,Ion Exchange Chromatography,Ion-Exchange Chromatographies
D004921 Erythropoietin Glycoprotein hormone, secreted chiefly by the KIDNEY in the adult and the LIVER in the FETUS, that acts on erythroid stem cells of the BONE MARROW to stimulate proliferation and differentiation.
D005453 Fluorescence The property of emitting radiation while being irradiated. The radiation emitted is usually of longer wavelength than that incident or absorbed, e.g., a substance can be irradiated with invisible radiation and emit visible light. X-ray fluorescence is used in diagnosis.
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic

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