Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence.
Recently, we described a novel reagentless fluorescent biosensor strategy named Quenchbody, which functions via the antigen-dependent removal of the quenching effect on a fluorophore that is attached to a single-chain antibody variable region. To explore the practical utility of Quenchbodies, we prepared antibody Fab fragments that were fluorolabeled at either one or two of the N-terminal regions, using a cell-free translation-mediated position-specific protein labeling system. Unexpectedly, the Fab fragment labeled at the heavy chain N-terminal region demonstrated a deeper quenching and antigen-dependent release compared to that observed using scFv. Moreover, when the Fab was fluorolabeled at the two N-termini with either the same dye or with two different dyes, an improved response due to enhanced quenching via dye-dye interactions was observed. On the basis of this approach, several targets, including peptides, proteins, and haptens, as well as narcotics, were quantified with a higher response up to 50-fold. In addition, differentiation of osteosarcoma to osteoblasts was successfully imaged using a similarly fluorolabeled recombinant Fab protein prepared from E. coli. Due to its versatility, this "Ultra-Quenchbody" is expected to exhibit a range of applications from in vitro diagnostics to the live imaging of various targets in situ.
Ushio Inc., 6409 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa 225-0004, Japan.
Ryoji
R
Abe
1] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan [2] Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
Hee-Jin
HJ
Jeong
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Dai
D
Arakawa
1] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan [2] Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
Jinhua
J
Dong
1] Ushio Inc., 6409 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa 225-0004, Japan [2] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Hiroyuki
H
Ohashi
Ushio Inc., 6409 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa 225-0004, Japan.
Rena
R
Kaigome
Ushio Inc., 6409 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa 225-0004, Japan.
Fujio
F
Saiki
1] Ushio Inc., 6409 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa 225-0004, Japan [2] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Kyosuke
K
Yamane
Ushio Inc., 6409 Moto-Ishikawa-cho, Aoba-ku, Yokohama, Kanagawa 225-0004, Japan.
Hiroaki
H
Takagi
1] Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan [2] Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan.
Hiroshi
H
Ueda
D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015675:Osteocalcin / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D000906:Antibodies; D000941:Antigens; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D012235:Rhodamines; D015675:Osteocalcin