Q-Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores

被引:31
作者
Jeong, Hee-Jin [1 ]
Kawamura, Takuya [2 ]
Dong, Jinhua [1 ]
Ueda, Hiroshi [1 ]
机构
[1] Tokyo Inst Technol, Chem Resources Lab, Midori Ku, 4259-R1-18 Nagatsuta cho, Yokohama, Kanagawa 2268503, Japan
[2] Univ Tokyo, Sch Engn, Dept Chem & Biotechnol, Bunkyo Ku, 7-3-1 Hongo, Tokyo 1138656, Japan
来源
ACS SENSORS | 2016年 / 1卷 / 01期
基金
日本学术振兴会;
关键词
immunoassay; fluorescent bicsensor; Quenchbody; fluorescence quenching; multicolored probes; single chain Fv; ANTIGEN-DEPENDENT FLUORESCENCE; REAGENTLESS BIOSENSORS; ESCHERICHIA-COLI; BINDING-PROTEINS; ANTIBODY PROBES; RHODAMINE DYES; RADIOIMMUNOASSAY; QUENCHBODIES; AGGREGATION; EXPRESSION;
D O I
10.1021/acssensors.5b00089
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Fluorescence-based immunosensors serve a vital role in biotechnology and diagnostic and therapeutic applications. Our group recently developed a unique fluoroimmunosensor named Quenchbody (Q-body) that operates based on the principle of quenching and the antigen-dependent release of fluorophore, which is incorporated to a recombinant antibody fragment, either the single-chain Fv (scFv) or the Fab fragment of an antibody, using a cell-free transcription-translation system. With the objective of extending the functionality and diversity of the Q-body, here we attempted to make Q-bodies by labeling the recombinant scFv, which was prepared from. E. coli using several commercially available dye-maleimides. As a result, we reproducibly obtained larger amounts of antiosteocalcin Q-bodies, with an improved yield and cost-efficiency compared with those obtained from a conventional cell-free system. The fluorescence intensity of each Q-body, including that labeled with newly tested rhodamine red, was significantly increased in the presence of an antigen with a low detection limit, although some differences in response were observed for the dye with different spacer lengths between dye and maleimide. The results indicate the Q-body's applicability as a powerful multicolored sensor, with a potential to simultaneously monitor multiple targets in a sample.
引用
收藏
页码:88 / 94
页数:7
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