title: An improved phage display antibody cloning system using newly designed PCR primers optimized for Pfu DNA polymerase.
abstract: An improved combinatorial library system to raise mouse monoclonal antibodies was constructed. PCR primers have been newly designed to optimize the reaction for Pfu DNA polymerase, which has proofreading activity. The phagemid vector (pPDS) is designed to accommodate VH and Vk cDNAs, which had previously been assembled by PCR either in single chain fragment of variable regions (scFv) or Fab form. Antibody cloned in scFv form can be converted to Fab form by substituting the scFv linker of (Gly4Ser)3 with a fragment containing murine CH1 cDNA. This vector will produce soluble Fab in non-amber suppressor cells and allow the shuffling of light chains against a heavy chain. Hybridoma cell lines producing anti-human procollagenase monoclonal antibodies were used as the source of antibody mRNA. Antigen-binding ability of both scFv- and Fab-displaying phage was confirmed by ELISA against human procollagenase. They were also analyzed by DNA sequencing to verify the fidelity of Pfu DNA polymerase and to identify the primer incorporated. The mutation rate was considerably reduced compared to the mutation rate achieved by Taq DNA polymerase. Primers are incorporated into target sequences in most cases.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D017364:Collagenases / Q000276:immunology*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D004792:Enzyme Precursors / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D003062:Codon; D017931:DNA Primers; D004792:Enzyme Precursors; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C072146:Pfu DNA polymerase; D004259:DNA-Directed DNA Polymerase; D017364:Collagenases; C022440:procollagenase