Isolation and Characterization of Phage-Displayed Single Chain Antibodies Recognizing Nonreducing Terminal Mannose Residues. 1. A New Strategy for Generation of Anti-Carbohydrate Antibodies
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- 作者：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
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：January 9, 2007
- 年：2007
- 卷：46
- 期：1
- 页码：253 - 262
- 全文大小：252K
- 年卷期：v.46,no.1(January 9, 2007)
- ISSN：1520-4995
文摘
Phage-display technology is probably the best available strategy to produce antibodies directedagainst various carbohydrate moieties since conventional hybridoma technologies have yielded mostlylow-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties inimmobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for proteinantigens cannot be readily applied. We adapted phage-display technology to generate human single chainantibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterizationof phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. Wefirst constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shufflingmethods with unique vector constructs. The library was subjected to four rounds of panning againstneoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE)by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) usingMan3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences wereisolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmonresonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residuesat nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose typeoligosaccharides 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 affinityand specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness ofthis strategy in constructing human scFv against various carbohydrate antigens. Further studies on thepurification and characterization of these scFvs are presented in an accompanying paper in this issue.