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