title: Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions.
abstract: We have constructed a large (3.6 x 10(8) clones) phage display library of human single chain Fv (scFv) antibody fragments by combining 49 germline VH genes with synthetic heavy chain CDR3 (HCDR3) regions and seven light chains. The HCDR3 regions varied in length between 6 and 15 residues and were designed to contain fully randomized stretches of amino acid residues flanked by regions of limited residue variability that were composed of amino acid residues that frequently occur in natural antibodies. We reasoned that this approach would increase the frequency of functional molecules in our library and, in addition, permit us to efficiently utilize available cloning space. By direct selection on solid phase-bound antigens we obtained phage antibodies with binding activities to 13 different antigens, including Von Willebrand factor, the DNA-binding HMG box of transcription factor TCF-1 and the tumor antigen EGP-2. In addition, we applied a competitive selection procedure to target phage antibodies to the desired portion of a recombinant fusion protein and to select phage antibodies capable of discriminating between the two highly homologous homeobox proteins PBX1a and PBX2. The functional capacity of monoclonal phage antibodies was assessed in immuno-histochemical staining of tissue specimens. Western blotting assays and immunofluorescent analysis of cells by flow cytometry. The results demonstrate that this large human phage antibody library contains a broad assortment of binding specificities that can be applied in a variety of biochemical assays.
authors.affiliation: Department of Immunology, University of Utrecht, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: de Kruif
authors.forename: E
authors.initials: E
authors.lastname: Boel
authors.forename: T
authors.initials: T
authors.lastname: Logtenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001435:Bacteriophages; D001483:Base Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D000882:Haplorhini; D004854:Herpesvirus 4, Human; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000276:immunology*; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000276:immunology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013961:Thyroid Gland / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004268:DNA-Binding Proteins; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D046911:Macromolecular Substances; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D018799:Intercellular Adhesion Molecule-1