Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies.

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Research Abstract Details 

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Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Abstract Text:

Keiko Sakai,Yoshitaka Shimizu,Tomoki Chiba,Ayano Matsumoto-Takasaki,Yu Kusada,Wei Zhang,Munehiro Nakata,Naoya Kojima,Kazunori Toma,Atsushi Takayanagi,Nobuyoshi Shimizu,Yoko Fujita-Yamaguchi,

Phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties since conventional hybridoma technologies have yielded mostly low-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties in immobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for protein antigens cannot be readily applied. We adapted phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterization of phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. We first constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) using Man3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences were isolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residues at nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Furthermore, first round characterization of scFvs expressed from respective phages indicated good affinity and specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness of this strategy in constructing human scFv against various carbohydrate antigens. Further studies on the purification and characterization of these scFvs are presented in an accompanying paper in this issue.

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Publishing Authors By Initials

K Sakai,Y Shimizu,T Chiba,A Matsumoto-Takasaki,Y Kusada,W Zhang,M Nakata,N Kojima,K Toma,A Takayanagi,N Shimizu,Y Fujita-Yamaguchi,

For similar peptides: peptide library research abstracts see: peptides: peptide library research

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Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 46

Page Numbers: 253-62

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 9

MONTH: Jan

YEAR: 2007

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Information

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LANGUAGE: eng

NlmUniqueID: 370623

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Keywords Mesh Terms:

KEYWORDS: Peptide Library

MESH TERMS: immunology

Chemical & Substance for Abstract: Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. Information

Substance Name: Mannose

Registry Number: 31103-86-3

Grant and Affiliation Information for Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies.

AFFILIATION: Institute of Glycotechnology, Tokai University, Kanagawa 259-1292, Japan.

Country: United States

AGENCY: United States NCI

GRANT: CA65767

ACRONYM: CA

MEDLINETA: Biochemistry

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