title: Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (close homolog of L1) enhance neurite outgrowth.
abstract: The neural cell adhesion molecule CHL1 (close homolog of L1) plays important roles in neurite outgrowth and neuronal survival in vitro. Reproducible and functionally active CHL1 antibodies are critical for a better understanding of the functional properties of CHL1 in vitro and in vivo. We have isolated human single-chain variable fragment (scFv) antibodies against mouse CHL1 from a human synthetic phage display library. To improve the binding activity of such antibodies, a clone (C12) was selected for affinity maturation by combined random mutagenesis of the V(H) gene and site-directed cassette mutagenesis to introduce random mutations in the complementarity determining region 3 (CDR3) of the V(L) gene. From the mutant phage display library, we selected a clone (6C2) that gave the strongest signal as determined by ELISA. The dissociation constant of 6C2 (Kd 2.28 x 10(-8) M) was increased approximately 85-fold compared with the wild-type clone C12 (Kd 1.93 x 10(-6) M). 6C2 detected CHL1 by Western blot analysis in mouse brain homogenates and detected CHL1 in CHL1-transfected cells by immunofluorescence. Furthermore, the wild-type and affinity-matured antibodies promoted neurite outgrowth of hippocampal and cerebellar neurons in vitro. Our results suggest that the affinity-matured CHL1 scFv antibody will serve a range of applications in vitro and in vivo.
authors.affiliation: Zentrum für Molekulare Neurobiologie, Universität Hamburg, Germany.
authors.forename: Ling
authors.initials: L
authors.lastname: Dong
authors.forename: Suzhen
authors.initials: S
authors.lastname: Chen
authors.forename: Melanie
authors.initials: M
authors.lastname: Richter
authors.forename: Melitta
authors.initials: M
authors.lastname: Schachner
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D000918:Antibody Specificity / Q000276:immunology*; D001483:Base Sequence / Q000235:genetics; D015815:Cell Adhesion Molecules; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002490:Central Nervous System / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D002525:Cerebellar Cortex / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D018076:DNA, Complementary / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D008565:Membrane Proteins; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics / Q000276:immunology; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016501:Neurites / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D019151:Peptide Library; D017434:Protein Structure, Tertiary / Q000235:genetics; D011506:Proteins
chemical_list: D000906:Antibodies; C490650:CHL1 protein, human; D015815:Cell Adhesion Molecules; C490649:Chl1 protein, mouse; D018076:DNA, Complementary; D008565:Membrane Proteins; D039842:Neural Cell Adhesion Molecule L1; D019006:Neural Cell Adhesion Molecules; D010446:Peptide Fragments; D019151:Peptide Library; D011506:Proteins