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

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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.

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Abbreviations

BGE:

background electrolyte

CE:

capillary electrophoresis

Epo:

Erythropoietin

Fab:

antigen-binding fragment

FITC:

fluorescein isothiocyanate

IEF:

isoelectric focusing

mAb:

monoclonal antibody

PBS:

phosphate-buffered saline

rHuEpo:

recombinant human erythropoietin

scFv:

(recombinant) single chain variable fragment

SDS-PAGE:

denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

ECL:

enzyme-coupled chemoluminescence

vH :

variable domain

cH1–3 :

constant domains of an antibody's heavy chain

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Acknowledgements

This work was supported by a grant from the German Federal Government (BMBF Schwerpunktprogramm Glykobiotechnologie). We thank F. Hoffmann-La Roche AG for a generous gift of recombinant human erythropoietin and Dr Stefan Müller for inspiring discussion. Peptide synthesis was performed by the ZMMK service lab, University at Cologne. Michael Kolloch and Ulrich Kutscher analysed and purified APP20.

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Authors and Affiliations

  1. Analytis Gesellschaft für Laboruntersuchungen mbh, Ludwigshafener Str. 1, 50389, Wesseling, Germany

    Claus Bornemann, Tilman Burggraef, Günter Heimbüchel & Sandra Winkels

  2. Institut für Biochemie II, Medical Faculty of the University at Cologne, Joseph-Stelzmann-Str. 42, 50938, Köln, Germany

    Franz-Georg Hanisch

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  1. Claus Bornemann
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  2. Tilman Burggraef
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  3. Günter Heimbüchel
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  4. Franz-Georg Hanisch
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  5. Sandra Winkels
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Correspondence to Claus Bornemann.

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Bornemann, C., Burggraef, T., Heimbüchel, G. et al. Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis. Anal Bioanal Chem 376, 1074–1080 (2003). https://doi.org/10.1007/s00216-003-2038-3

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  • DOI: https://doi.org/10.1007/s00216-003-2038-3

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