Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence

Abstract

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.

Figure 1. Synthesis of the Fab-containing TAMRA at the N-terminal region of the H chain.

(a) Scheme of the incorporation of TAMRA-C6-AF into the H chain (Fd, V_H-C_H1) of Fab in response to a UAG codon in a cell-free translation system. (b) SDS-PAGE fluorescence image of the expressed anti-BGP Fab containing the TAMRA-C6-AF in reducing and non-reducing conditions. TAMRA fluorescence was detected with an excitation at 532 nm and emission at 580 nm. The fluorescent marker shown in green was detected with an excitation at 488 nm and emission at 520 nm. (c) Western blotting analysis of the same gel using anti-Flag and anti-His antibodies.

Figure 2. Antigen-dependent fluorescence enhancement of TAMRA-labeled anti-BGP Fab.

(a) Fluorescence spectra of TAMRA-scFv with an excitation at 550 nm in the absence and presence of BGP-C7 peptide. (b) Standard curve of the fluorescence intensity at 580 nm. The intensities are the relative values in the absence of BGP-C7 peptide. (c) Normalized standard curves. (d) Fluorescence spectra of double TAMRA-Fab with an excitation at 550 nm in the presence of BGP-C7 peptide as shown. (e) Standard curve of the fluorescence intensity at 580 nm. The intensities are the relative values to that in the absence of BGP-C7 peptide. (f) Normalized dose-response curves.

Figure 3. Antigen-dependent fluorescence enhancement of TAMRA/ATTO655-labeled Fab.

(a) Scheme of the incorporation of TAMRA-C6-AF and ATTO655-C6-AF into the H and L chains of Fab in response to a UAG and 4-base codons in a cell-free translation system, respectively. (b) Fluorescence spectra of the Fab labeled at the N-terminal region of the H chain with TAMRA and the L chain with ATTO655 (HTLA), with excitation at 530 nm in the presence of BGP-C7 peptide as indicated. (c) The same with Fab labeled with ATTO655 at the H chain and with TAMRA at the L chain (HALT). (d) Standard curves of the fluorescence intensity at 580 nm. The intensities are the relative values to that in the absence of BGP-C7 peptide. (e–g) The results of the anti-SA UQ-bodies for detecting human serum albumin. The conditions were the same as in (b–d), except that HSA was used as an antigen.

Figure 4. Titration curves of TAMRA-labeled anti-narcotics UQ-bodies.

Anti-Morphine double-labeled Fab (a), anti-methamphetamine H chain-labeled Fab (b), and anti-cocaine double-labeled Fab (c) were used. Standard curves of fluorescence intensity at 580 nm with excitation at 530 nm are shown. Normalized fluorescence intensities show relative values with respect to that in the absence of antigen. An average with 1 SD of the eight samples is shown.

Figure 5. Preparation of UQ-bodies from E. coli cytoplasm.

(a) Scheme of constructing UQ-bodies. Cys-tagged anti-BGP Fab fragment was expressed in E. coli SHuffle cells, and purified via the His₆ tag at the C-terminus of the H (Fd) chain. After mild reduction of the exposed SH group, dye-maleimide was reacted and purified via the Flag tag at the C-terminus of the L chain. (b–c) Dose-response relationship of TAMRA-labeled (b) and ATTO520-labeled (c) UQ-bodies. An average with 1 SD of the three samples is shown. (d) Microscopic observation of anti-Flag M2 beads with and without double ATTO520-labeled UQ-body. The transmission and fluorescence images are shown. (e) Transmission and (f) fluorescence images of U2OS cells at the indicated conditions.