Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library.
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.
Schlattner
Uwe
U
Institute of Cell Biology, Swiss Federal Institute of Technology (ETH), Hönggerberg HPM, Zürich CH-8093, Switzerland. schlattn@cell.biol.ethz.ch
Reinhart
Christof
C
Hornemann
Thorsten
T
Tokarska-Schlattner
Malgorzata
M
Wallimann
Theo
T
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
D007135:Immunoglobulin Variable Region; D007527:Isoenzymes; D019151:Peptide Library; D003402:Creatine Kinase; D052276:Creatine Kinase, BB Form; D052279:Creatine Kinase, MB Form