title: Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library.
abstract: Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.
authors.affiliation: Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), Hönggerberg HPM, Zürich CH-8093, Switzerland. schlattn@cell.biol.ethz.ch
authors.forename: Uwe
authors.initials: U
authors.lastname: Schlattner
authors.forename: Christof
authors.initials: C
authors.lastname: Reinhart
authors.forename: Thorsten
authors.initials: T
authors.lastname: Hornemann
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Tokarska-Schlattner
authors.forename: Theo
authors.initials: T
authors.lastname: Wallimann
mesh_terms: D000595:Amino Acid Sequence; D000918:Antibody Specificity; D003402:Creatine Kinase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D052276:Creatine Kinase, BB Form; D052279:Creatine Kinase, MB Form; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
chemical_list: D007135:Immunoglobulin Variable Region; D007527:Isoenzymes; D019151:Peptide Library; D003402:Creatine Kinase; D052276:Creatine Kinase, BB Form; D052279:Creatine Kinase, MB Form