title: An engineered bivalent single-chain antibody fragment that increases antigen binding activity.
abstract: Bivalent single chain Fv (scFv) was constructed by fusing a polypeptide extension containing one or two cysteines to the COOH-terminus of an scFv antibody fragment. The scFv protein was expressed and secreted in a recombinant Pichia pastoris system as a dimer with a C-terminal disulfide bridge, as determined by Western blot analysis under non-reducing conditions. We found that the scFv construct with one cysteine in the C-extension (scFv-1Cys) exhibited a much higher dimer/monomer ratio than the two cysteine counterpart (scFv-2Cys). Binding activity measurements performed by means of a competitive radioimmunoassay showed that scFv-1Cys exhibited specific antigen binding activity, which was almost the same as that of the parental MAb, and approximately four- and fortyfold higher than those of the control scFv monomer and scFv-2Cys.
authors.affiliation: Biotechnology Research and Development, AltaRex Inc., University of Alberta, Edmonton, Canada.
authors.forename: D
authors.initials: D
authors.lastname: Luo
authors.forename: M
authors.initials: M
authors.lastname: Geng
authors.forename: A A
authors.initials: AA
authors.lastname: Noujaim
authors.forename: R
authors.initials: R
authors.lastname: Madiyalakan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000502:physiology; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D000939:Epitopes / Q000502:physiology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000276:immunology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*
chemical_list: D018033:Antibodies, Bispecific; D000939:Epitopes; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D003545:Cysteine