High affinity antibodies against Lex and sialyl Lex from a phage display library.
Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.
Dinh
Q
Q
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Weng
N P
NP
Kiso
M
M
Ishida
H
H
Hasegawa
A
A
Marcus
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000915:Antibody Affinity; D001435:Bacteriophages / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002999:Clone Cells / Q000378:metabolism; D015723:Gene Library; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D016256:Lewis X Antigen / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000235:genetics / Q000276:immunology*; D000080506:Sialyl Lewis X Antigen
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D016256:Lewis X Antigen; D009844:Oligosaccharides; D000080506:Sialyl Lewis X Antigen