Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display

Christiane Mühle; Stefan Schulz-Drost; Alexey V. Khrenov; Evgueni L. Saenko; Jens Klinge; Holm Schneider

doi:10.1160/TH03-07-0473

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 91(03): 619-625
DOI: 10.1160/TH03-07-0473

New Technologies and Diagnostic Tools

Schattauer GmbH

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display

Authors

Christiane Mühle

¹Children’s Hospital and Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nuernberg, Germany
Stefan Schulz-Drost

¹Children’s Hospital and Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nuernberg, Germany
Alexey V. Khrenov

²American Red Cross Holland Laboratory, Rockville, USA
Evgueni L. Saenko

²American Red Cross Holland Laboratory, Rockville, USA
Jens Klinge

¹Children’s Hospital and Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nuernberg, Germany
Holm Schneider

¹Children’s Hospital and Nikolaus Fiebiger Center of Molecular Medicine, University of Erlangen-Nuernberg, Germany

Abstract

Summary

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patient’s plasma by affinity chromatography. FVIIIbinding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.

Keywords

Haemophilia - inhibitor - polyclonal antibodies - epitope mapping - phage display

Full Text

References

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