Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (close homolog of L1) enhance neurite outgrowth.
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.
Dong
Ling
L
Zentrum für Molekulare Neurobiologie, Universität Hamburg, Germany.
Chen
Suzhen
S
Richter
Melanie
M
Schachner
Melitta
M
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
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