title: Generation and validation of canine single chain variable fragment phage display libraries.
abstract: Single chain variable region fragments (scFvs) are composed of an immunoglobulin (Ig) variable heavy (VH) and variable light (VL) chain joined by a flexible serine-glycine linker. They represent the smallest antibody fragments that maintain antigen specificity and they hold significant potential for therapeutic antigen targeting in vivo. Here we report on the design and validation of a series of degenerate primers that amplify the recombined variable regions of canine Ig heavy and light chain genes from lymphocyte cDNA. We show that these VH and VL amplicons can be randomly combined by a flexible linker using splicing by overlap extension PCR to form scFv constructs that can be expressed on the surface of M13 bacteriophage. To demonstrate that scFvs with specificity for previously encountered antigens are contained within these scFv phage display libraries we used simple panning procedures to isolate canine parvovirus (CPV) specific scFvs from a library made from the splenocytes of a dog immunized against CPV. These studies reveal the feasibility of this approach for generating diverse canine scFv libraries and pave the way toward future studies to isolate canine antigen-specific scFv of interest that may be tested as targeting agents for the treatment of infectious, inflammatory and neoplastic diseases in the dog.
authors.affiliation: Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, 19106, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Braganza
authors.forename: Koranda
authors.initials: K
authors.lastname: Wallace
authors.forename: Laura
authors.initials: L
authors.lastname: Pell
authors.forename: Colin R
authors.initials: CR
authors.lastname: Parrish
authors.forename: Don L
authors.initials: DL
authors.lastname: Siegel
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Mason
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004285:Dogs / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005822:Genetic Vectors / Q000235:genetics; D017993:Parvovirus, Canine / Q000235:genetics; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D017931:DNA Primers; D019151:Peptide Library; D057127:Single-Chain Antibodies