Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Darren L. Hughes; Prachi Stafford; Samir W. Hamaia; Ian J. Harmer; Anne Schoolmeester; Hans Deckmyn; Richard W. Farndale; Willem H. Ouwehand; Nicholas A. Watkins

doi:10.1160/TH05-06-0410

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(06): 1318-1326
DOI: 10.1160/TH05-06-0410

New Technologies and Diagnostic Tools

Schattauer GmbH

Platelet integrin α2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Darren L. Hughes

¹Department of Haematology, University of Cambridge, UK

,

Prachi Stafford

¹Department of Haematology, University of Cambridge, UK

,

Samir W. Hamaia

¹Department of Haematology, University of Cambridge, UK

,

Ian J. Harmer^*

¹Department of Haematology, University of Cambridge, UK

,

Anne Schoolmeester

⁴Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium

,

Hans Deckmyn

⁴Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, Belgium

,

Richard W. Farndale

³Department of Biochemistry, University of Cambridge, UK

,

Willem H. Ouwehand

¹Department of Haematology, University of Cambridge, UK

²National Blood Service, Cambridge, UK

,

Nicholas A. Watkins

¹Department of Haematology, University of Cambridge, UK

› Author Affiliations

Abstract

Summary

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs).This system was validated using the I-domain of α2 integrin as a model. The α2β1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoproteinVI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunized with a DNA vaccine in which the α2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT).Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four α2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet α2β1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splicevariant specific antibodies that recognise native protein.

Keywords

Collagen - variable gene phage display - DNA immunisation - domain-specific - α2β1 integrin

Full Text

References

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