title: Production of a single chain anti-CEA antibody from the hybridoma cell line T84.66 using a modified colony-lift selection procedure to detect antigen-positive ScFv bacterial clones.
abstract: Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies (MAbs) such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The scFv antibody (designated RK10.2) was generated using anti-CEA T84.66 hybridoma cells as a source of genetic starting material and the Pharmacia Recombinant Phage Antibody System (RPAS). Escherichia coli clones expressing antigen-positive soluble scFv were identified using a modified colony-life selection procedure and antigen-coated filters. The resultant anti-CEA scFv (designated RK10.2) had a molecular weight of approximately 33.6 kDa and an isoelectric point of 5.2 at 15 degrees C. The RK10.2 scFv interacted with LS174 T cells bearing the CEA antigen and inhibited the anti-CEA MAb/CEA antigen interaction in ELISA and the anti-CEA MAb/LS174 T cell interaction in a RIA. The modified colony-lift approach circumvented the more time-consuming phage-display approach that is normally taken to affinity select for antigen-positive scFv clones.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham 35294-3300, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Rodenburg
authors.forename: R
authors.initials: R
authors.lastname: Mernaugh
authors.forename: G
authors.initials: G
authors.lastname: Bilbao
authors.forename: M B
authors.initials: MB
authors.lastname: Khazaeli
mesh_terms: D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012641:Selection, Genetic
chemical_list: D000912:Antibodies, Neoplasm; D002272:Carcinoembryonic Antigen; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins