Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies.

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies. Research Abstract Details 

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Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies. Abstract Text:

Wei Zhang,Ayano Matsumoto-Takasaki,Yu Kusada,Hiroyuki Sakaue,Keiko Sakai,Munehiro Nakata,Yoko Fujita-Yamaguchi,

Since phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties, we employed phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as carbohydrate antigens. An accompanying paper in this issue describes how phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues were isolated and characterized. In this study, four independent scFv genes, isolated by a mannotriose (Man3)-bearing lipid as an antigen as previously described, were used to construct expression vectors to produce soluble scFv proteins in quantity. Both bacterial and mammalian expression systems were used to produce glutathione S-transferase-scFv fusion proteins and scFv-human IgG1 Fc conjugates, respectively. The expressed scFv fusion proteins were purified to apparent homogeneity with yields of approximately 1 and 48 mg, from 1 L of bacterial culture and myeloma cell media, respectively. Surface plasmon resonance and ELISA analyses confirmed that purified scFv proteins showed Man3 specificity. The humanized antibody in scFv-Fc form, derived from clone 5A3, was a disulfide-liked dimer with a molecular mass of 108 kDa. According to a bivalent model, the kinetics parameters of its binding to Man3 were determined to be ka = 4.03 x 104 M-1 s-1, kd = 5.77 x 10-4 s-1, KA = 6.98 x 107 M-1, and KD = 1.43 x 10-8 M. This study thus established the foundation for isolation of carbohydrate-specific scFv genes and eventual production of humanized scFv-Fc type antibodies.

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies. Publishing Authors By Initials

W Zhang,A Matsumoto-Takasaki,Y Kusada,H Sakaue,K Sakai,M Nakata,Y Fujita-Yamaguchi,

For similar investigative techniques: chemistry, analytical: surface plasmon resonance research abstracts see: investigative techniques: chemistry, analytical: surface plasmon resonance research

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Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies. Journal Published:

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

Journal: Biochemistry

VOLUME: 46

Page Numbers: 263-70

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. 2. Expression, purification, and characterization of recombinant single chain antibodies. Information

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LANGUAGE: eng

NlmUniqueID: 370623

Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain antibodies. Keywords Mesh Terms:

KEYWORDS: Surface Plasmon Resonance

MESH TERMS: isolation & purification

Chemical & Substance for Abstract: Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 2. Expression, purification, and characterization of recombinant single chain 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. 2. Expression, purification, and characterization of recombinant single chain 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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