title: Platelet integrin alpha2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display.
abstract: 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 alpha2 integrin as a model. The alpha2beta1 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 glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 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 alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 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 splice-variant specific antibodies that recognise native protein.
authors.affiliation: Department of Haematology, University of Cambridge, UK.
authors.forename: Darren L
authors.initials: DL
authors.lastname: Hughes
authors.forename: Prachi
authors.initials: P
authors.lastname: Stafford
authors.forename: Samir W
authors.initials: SW
authors.lastname: Hamaia
authors.forename: lan J
authors.initials: lJ
authors.lastname: Harmer
authors.forename: Anne
authors.initials: A
authors.lastname: Schoolmeester
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.forename: Willem H
authors.initials: WH
authors.lastname: Ouwehand
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Watkins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001792:Blood Platelets / Q000276:immunology*; D003001:Cloning, Molecular; D018604:Epitope Mapping; D006801:Humans; D007114:Immunization / Q000379:methods; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D038982:Integrin alpha2beta1 / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D019151:Peptide Library; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D038982:Integrin alpha2beta1; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA