title: Selection of single chain variable fragments specific for the human-inducible costimulator using ribosome display.
abstract: We applied a ribosome display technique to a mouse single chain variable fragment (scFv) library to select scFvs specific for the inducible costimulator (ICOS). mRNA was isolated from the spleens of BALB/c mice immunized with ICOS protein. Heavy and κ chain genes (VH and κ) were amplified separately by reverse transcriptase polymerase chain reaction, and the anti-ICOS VH/κ chain ribosome display library was constructed with a special flexible linker by overlap extension PCR. The VH/κ chain library was transcribed and translated in vitro using a rabbit reticulocyte lysate system. Then, antibody-ribosome-mRNA complexes were produced and panned against ICOS protein under appropriate conditions. However, in order to isolate specific scFvs for ICOS, negative selection using CD28 was carried out before three rounds of positive selection on ICOS. After three rounds of panning, the selected scFv DNAs were cloned into pET43.1a and detected by SDS-PAGE. Then, enzyme-linked immunosorbent assay showed that we successfully constructed a native ribosome display library, and among seven clones, clone 5 had the highest affinity for the ICOS and low for the CD28. Anti-ICOS scFvs are assessed for binding specificity and affinity and may provide the potential for development of the humanized and acute and chronic allograft rejection.
authors.affiliation: Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, People's Republic of China.
authors.forename: Yangbin
authors.initials: Y
authors.lastname: Pan
authors.forename: Weiping
authors.initials: W
authors.lastname: Mao
authors.forename: Xuanxuan
authors.initials: X
authors.lastname: Liu
authors.forename: Chong
authors.initials: C
authors.lastname: Xu
authors.forename: Zhijuan
authors.initials: Z
authors.lastname: He
authors.forename: Wenqian
authors.initials: W
authors.lastname: Wang
authors.forename: Hao
authors.initials: H
authors.lastname: Yan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000235:genetics; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D060889:Inducible T-Cell Co-Stimulator Protein / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology; D012270:Ribosomes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology
chemical_list: D018106:CD28 Antigens; C556692:ICOS protein, human; D007135:Immunoglobulin Variable Region; D060889:Inducible T-Cell Co-Stimulator Protein; D019151:Peptide Library; D012333:RNA, Messenger; D057127:Single-Chain Antibodies