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Q‑Bodies from Recombinant Single-Chain Fv Fragment with Better Yield and Expanded Palette of Fluorophores Hee-Jin Jeong,† Takuya Kawamura,‡ Jinhua Dong,† and Hiroshi Ueda*,† †

Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18, Nagatsuta-cho, Midori-ku, Yokoyama, Kanagawa 226-8503, Japan ‡ Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *

ABSTRACT: 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. KEYWORDS: immunoassay, fluorescent biosensor, Quenchbody, fluorescence quenching, multicolored probes, single chain Fv n past decades, the field of immunoassay has made a considerable impact on biological and biotechnological research and clinical and environmental applications. The first immunoassay approach was established using radioactive isotopes to assess the concentration of insulin in human plasma.1 Various radioimmunoassay methods have been demonstrated and applied to extensive areas by taking advantage of their high sensitivity and specificity.2 However, the hazardous effects of radioactive compounds for users and the environment and their limited half-life period frequently prevent their application in long-term assays.3 To address these problems, a fluoroimmunoassay based on the variation in a fluorescent property when an antigen binds to an antibody have been extensively provided as an alternative for the classic radioimmunoassay.4,5 A common approach is Förster resonance energy transfer (FRET), in which energy created by fluorescence excitation of a fluorophore is transferred to an adjacent fluorophore. However, a weakness of the method is that FRET requires a pair of probes, which limits the range of fluorophore pairs to prevent their overlapped emission spectra. The change in a FRET signal is usually small and sometimes smaller than the background fluorescence. Thus, the measurement almost always requires control experiments, which are ratiometrically calculated, to obtain data.6 Another approach, which involves labeling with environmentally sensitive fluo-

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rophores to make reagentless biosensors, has recently extended.7 Because it is based on the coupling of fluorophore to mutated cysteine (Cys) residue near the antigen-binding site of an antibody, the conjugation site should be carefully designed not to hinder antigen-binding activity;8 the variation in a suitable and environmentally sensitive dye is limited. To address this issue, our group has recently developed an innovative fluoroimmunosensor named Quenchbody (Qbody), which operates based on the principle of the antigendependent removal of the quenching effect on a fluorophore that had been quenched by tryptophan (Trp) residues in the antibody fragment.9−13 The use of Q-body-based immunoassays has increased due to its high sensitivity and applicability to an extensive range of antigens. An advantage of the Q-body method is its simplicity, which is straightforwardly performed by mixing a Q-body with an antigen subsequent to fluorescence measurement. It is complementary to traditional methods, which require several incubation and washing steps, including laborious manipulations. In the primary stage of development, we made Q-bodies using a cell-free transcription-translation system.10−12 We also Received: September 4, 2015 Accepted: October 26, 2015

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ATTO-TEC (Siegen, Germany). Rhodamine6G (R6G)-C5-mal and 5(6)-TAMRA-C0-mal were obtained from Setareh biotech LLC (Eugene, OR). TAMRA-C2-mal was obtained from Anaspec (Fremont, CA). TAMRA-C5-mal was obtained from Biotium (Hayward, CA). Rhodamine red (Rho)-C2-mal was obtained from Life Technologies (Carlsbad, CA). His Mag Sepharose Ni was obtained from GE healthcare (Piscataway, NJ). Anti DYKDDDDK tag antibody magnetic beads, DYKDDDDK Peptide, Supersep PAGE gel, and a silver staining kit were obtained from Wako pure chemicals (Osaka, Japan). The Nanosep Centrifugal-3 k Ultrafiltration Device was obtained from Pall Corporation (Ann Arbor, MI). The Costar 3590 microplate was obtained from Corning Japan (Tokyo, Japan). Immunoblock was obtained from DS Pharma (Osaka, Japan). PentaHis-HRP conjugate was obtained from Qiagen (Hilden, Germany). 3,3′,5,5′-Tetramethylbenzidine (TMBZ) was obtained from Sigma (St. Louis, MO). C-terminal peptides from BGP (BGP-C7, NH2RRFYGPV-COOH, MW = 894) and biotinylated C-terminal peptides from BGP (BGP-C11, bio-NH2-QEAYRRFYGPV-COOH) were obtained from Lifetein (South Plainfield, NJ). Other chemicals and reagents, unless otherwise indicated, were obtained from Wako pure chemicals. Construction of scFv-type Q-Body Gene. A Fab-type Q-body expression vector pUQ1H(KTM219)9 was digested by EcoRV and BamHI and blunted with T4 DNA polymerase and ligation to remove L chain fragments. It was digested by AgeI and EagI to remove H chain fragments and was ligated using ligation high with AgeI- and EagIdigested scFv fragments of KTM219, which were amplified by the primers KTMAgeBack (5′-GGAATTCACCGGTCAAGTAAAGCTGCAGCAGTC-3′) and T7term (5′-TGCTAGTTATTGCTCAGCGG3′), pROX-FL92.1amber(KTM219) as a template, and KOD-plus DNA polymerase. To insert Flag-tag, the ligated gene was amplified by the primers KTMAgeBack and pROXHis6Bamfor (5′-GTCGGATCCGCCATGATGATGATGATGATGATAAC-3′) and digested using AgeI and BamHI, followed by ligation to AgeI- and BamHI-digested pUQ1H(KTM219), which produced pSQ(KTM219). The obtained plasmid was prepared with the PureYield plasmid miniprep system, and the entire coding region sequence was confirmed. Synthesis and Purification of Protein. SHuffle T7 Express lysY cells were transformed with pSQ(KTM219) and cultured at 30 °C for 16 h in LBA medium (LB medium containing 100 μg/mL ampicillin) and 1.5% agar. A single colony was picked and grown at 30 °C in 4 mL of LBA medium until a OD600 of 0.9 was attained; 1.6 mL were utilized to inoculate 100 mL of LBA medium. The cells were cultured at 30 °C until an OD600 of 0.6 was attained, when 0.4 mM isopropylthio-βgalactopyranoside was added. The solution was incubated for an additional 16 h at 16 °C and centrifuged (8000 × g, 20 min, 4 °C). The pellet was resuspended in 10 mL of Talon wash buffer (50 mM phosphate, 0.3 M sodium chloride (NaCl), 5 mM imidazole, pH 7.4) and sonicated. After centrifugation (8000 × g, 20 min, 4 °C), the supernatant was incubated with 0.2 mL of Talon metal resin on a rotating wheel for 30 min at 25 °C. The beads were washed three times with 8 mL Talon wash buffer. After the addition of 4 mL Talon elution buffer (50 mM phosphate, 0.3 M NaCl, 0.5 M imidazole, pH 7.4) and incubation at 25 °C for 30 min, the eluent was collected using a Talon disposable gravity column. The eluent was subjected to an ultrafiltration device and equilibrated with PBST (10 mM phosphate, 137 mM NaCl, 2.7 mM potassium chloride, 0.05% Tween 20, pH 7.4) and concentrated to 250 μL. The expression and purification of the protein were confirmed using a SDS-PAGE analysis, and the concentration of the protein was determined using ImageJ software (National Institutes of Health, Bethesda, MD) with the varied concentration of bovine serum albumin (BSA) as a standard. Fluorescence Labeling and Purification. A volume of immobilized TCEP disulfide reducing gel slurry that was equivalent to the volume of 450 μg of purified protein was added to a microtube and centrifuged (100 × g, 1 min, 4 °C). After removing the supernatant, 450 μg of purified protein was added and incubated for 1 h at room temperature on a rotating wheel. After centrifugation at 100 g for 1 min, the supernatant was recovered and divided into 50 μg samples. Each sample was reacted with 20 × mol of the fluorescence

developed a method for its large-scale production, which combines Escherichia coli (E. coli) expression of recombinant Fab fragment and postfluorescence chemical labeling.9 Both cell-free and E. coli-based methods have respective advantages: the E. coli-based method enables improvement of the production yield and cost-efficiency. The Q-body made from a cell-free system showed a higher fluorescent response than the response of a Q-body made from E. coli. Subtle differences in the Q-body’s structure have been inferred, namely, the chemical structures of the linker between the dye and the protein differ due to distinct labeling principles. In our previous study, in which we made a tetramethylrhodamine (TAMRA)labeled Fab-type antiosteocalcin (bone gla protein, BGP) Qbody, p-(TAMRA-aminocaproyl)-aminophenylalanine was employed for labeling via the aminoacyl-tRNA that was utilized during the cell-free translation reaction, whereas TAMRA-C5maleimide was employed for thiol-maleimide reaction-based chemical labeling.9 Because the spacer length of a fluorophore significantly affects the accessibility of dye to Trp residues, we assumed that adjusting the fluorophore and spacer length would be effective solutions for making a superior Q-body. We considered that an increased variation of usable fluorophores would improve the performance of the Q-body toward prospective and simultaneous fluorescent imaging by a series of different colors. Therefore, to expand the applicability of a Q-body, we labeled the Q-body using several types of fluorescent dyes. When we previously constructed a vector for making an E. coli-based Fab-type Q-body, we eliminated the “tail” Cys residues of H (Fd) and L chains to prevent fluorescent labeling in these positions. Because the affinity between the folded H chain and the folded L chain in their constant regions is usually sufficient, the two chains could be noncovalently bonded even without a disulfide bond. Although many Fabs exhibited antigen-binding activity and an antigen-dependent fluorescent response, the expression yields of the two chains were dependent on their sequences and the final ratio of the two chains was not always one-to-one. Although the majority of the free H and L chains can be washed by tandem purification using two C-terminal tag sequences, this procedure frequently produces a considerable loss of target protein; thus, additional enhancement of the production yield has been desired.14−16 To circumvent these “developable” problems, we considered that a single-chain Fv (scFv) in two variable regions of an antibody VH and VLare linked by a flexible amino acid linker, which enables improvement in the yield of Q-body without any additional purification steps for isolating the Fab complex.17,18 To extend the functionality of the Q-body and attain its full potential, we made multicolored Q-bodies by postlabeling a recombinant scFv fragment using several dyes and examined their properties.

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EXPERIMENTAL SECTION

Materials. KOD-plus, T4 DNA polymerases, and the Ligation-high ligation kit were obtained from Toyobo (Osaka, Japan). Restriction enzymes and E. coli Shuffle T7 Express lysY were obtained from New England Biolabs Japan (Tokyo, Japan). Oligonucleotides were obtained from Operon-Eurofins (Tokyo, Japan). A PureYield plasmid miniprep kit was obtained from Promega Japan (Tokyo, Japan). Talon metal affinity resin and a Talon disposable gravity column were obtained from Takara Bio (Otsu, Japan). Immobilized TCEP disulfide reducing gel and the Zeba spin desalting column (MWCO 7 k) were obtained from Thermo Pierce (Rockford, IL). ATTO495-C2maleimide (mal) and ATTO520-C2-mal were obtained from B

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Figure 1. Scheme for the construction of scFv type Q-body from E. coli. Anti-BGP scFv fragment was expressed in E. coli cytoplasm and purified by IMAC via His-tag. After mild reduction of the exposed SH group of N-terminal Cys-tag, the fluorophore was labeled and the Q-body was purified by IMAC and anti-Flag affinity beads. dyes ATTO495-C2-mal, ATTO520-C2-mal, R6G-C5-mal, Rho-C2mal, TAMRA-C0-mal, TAMRA-C2-mal, and TAMRA-C5-mal in 2 μL of DMSO or with 2 μL of DMSO in the dark for 2 h at 25 °C. Each reaction mixture was incubated with 10 μL of His Mag Sepharose Ni beads on a rotating wheel at room temperature for 30 min. The beads were washed three times with 1 mL of His wash buffer (20 mM phosphate, 0.5 M NaCl, 60 mM imidazole, 0.1% polyoxyethylene(23)lauryl ether, pH 7.4) on a magnetic rack. After adding 500 μL of His elution buffer (20 mM phosphate, 0.5 M NaCl, 0.5 M imidazole, 0.1% polyoxyethylene(23)lauryl ether, pH 7.4) and incubating at 25 °C for 15 min, the eluent was collected on a magnetic rack and applied to Nanosep Centrifugal-3 k ultrafiltration. After equilibration twice with 500 μL of PBST by centrifuge (14 000 × g, 20 min, 4 °C), the supernatant was concentrated to 200 μL and subsequently purified using Flag-tag as follows: Anti-DYKDDDDK tag antibody magnetic beads (10 μL) were added to the supernatant. After incubation at 25 °C for 1 h, the beads were washed three times with 1 mL of Flag wash buffer (20 mM phosphate, 0.5 M NaCl, 5 mM imidazole, pH 7.4) on a magnetic rack and incubated with 100 μL of wash buffer that contained 50 μg of Flag peptide at 25 °C. After 1 h, the eluent was collected on a magnet, and 100 μL of PBST was added twice to wash the beads and mixed with the eluent; the total eluent mix (300 μL) was stored at 4 °C. An aliquot (3 μL) of each mixture was mixed with 3 μL of SDS loading buffer (0.125 M Tris-HCl, 4% (w/v) SDS, 20% (w/v) glycerol, 0.01% (w/v) BPB, 100 mM DTT, pH 6.8), boiled at 95 °C for 5 min, and loaded to 12.5% PAGE. A fluorescence image was obtained using a transilluminator with excitation at 500 nm (Gelmiére, Wako), and the protein concentration was determined after silver staining by comparing with varied concentrations of BSA standard using ImageJ software. Enzyme-Linked Immunosorbent Assay. After 100 μL of streptavidin (100 μg/mL in PBS) was immobilized on the Costar 3590 microplate for 16 h at 4 °C, the well was filled with 200 μL of PBS that contains 20% Immunoblock for 2 h at 25 °C, and washed three times with PBST. Subsequently, 100 ng/mL of biotinylated BGP peptide in 100 μL of PBST was added and incubated for 1 h at 25 °C. After washing three times with PBST, 10 ng of Q-body or unlabeled protein in 100 μL of PBST was added and incubated for 2 h at 25 °C. The well was washed three times with PBST and bound protein was probed with 4000-fold diluted anti-His-HRP antibody in PBST for 1 h at 25 °C. The well was washed three times with PBST and developed with 100 μL of substrate solution (100 μg/mL TMBZ, 0.04 μg/mL hydrogen peroxide in 100 mM sodium acetate, pH 6.0). After incubation for 15 min, the reaction was stopped with 50 μL of 10% sulfuric acid, and the absorbance was read at 450 nm with a reference at 655 nm using a microplate reader Model 680 (Bio-Rad). As a control, PBS was employed instead of biotinylated BGP-peptide, and the same procedure was performed. Fluorescence Measurements. First, 10 ng of Q-body in 250 μL of PBST was added in a 5 × 5 mm2 quartz cell (Starna Scientific, Hainault, UK), and 10 μM of BGP-C7 peptide was subsequently added. After incubation at 25 °C for 3 min, the spectral measurement was performed at 25 °C using the fluorescence spectrophotometer Model FP-8500 (JASCO, Tokyo, Japan). To denature the protein structure, 250 μL of 7 M guanidium hydrochloride (GdnHCl) and 100 mM of dithiothreitol (DTT) were added instead of PBST in a cell and

the same procedure was performed. Second, various concentrations of BGP-C7 were added in titration at 3 min intervals, after each fluorescence spectrum was measured. Both the excitation and emission slit widths were set to 5.0 nm. The excitation wavelength was 495, 520, 530, 546, and 565 nm for ATTO495-, ATTO520-, R6G-, TAMRA-, and Rho-labeled Q-body, respectively. Dose−response curves were constructed by fitting the intensities at the maximum emission wavelength of each Q-body using the Kaleida Graph 4.1 (Synergy Software, Reading, PA). The EC50 value was calculated from the curve fitting to a 4-parameter logistic equation. Kinetic Analysis Using Biolayer Interferometry. To evaluate the antigen binding activity of Q-bodies, biolayer interferometry measurements were performed using a BLItz instrument (Pall ForteBio, Menlo Park, CA). Streptavidin-conjugated biosensor (ForteBio) was presoaked in water for 10 min. Subsequently, biotinylated BGP-C11 peptide (Lifetein) (4 μL, 10 μg/mL in PBS) was immobilized onto the biosensor for 5 min and allowed to equilibrate in PBS prior to analysis. A binding assay using 4 μL of PBS that contains R6G-labeled Q-body in various concentrations was performed according to the following cycle conditions: oscillation at 1000 rpm, baseline measurement in PBS for 30 s, association measurement in sample for 5 min, and dissociation measurement in PBS for 5 min. The biosensor was regenerated by immersion in glycine-HCl (pH 2.0) for 1 min and immersion in PBS for 1 min prior to the next cycle. The data were prepared for analysis by subtracting the background signal (using PBS without Q-body) and importing into Blitz software for calculating the kinetic constants. Fluorescence-Based Thermal Shift Assay. The fluorescence intensity of 50 ng of R6G-labeled Q-body in 100 μL PBST was measured using a MiniOpticon real-time thermal cycler (Bio-Rad) with HEX filter. The temperature was increased from 25 to 98 °C at 1 °C intervals over the course of 1 min, and the fluorescence intensity was measured at each interval. The fluorescence data were plotted, and the first derivative of the temperature was calculated to obtain the melting temperature (Tm).

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RESULTS AND DISCUSSION Novel Expression Vector for Making scFv Q-Bodies. We constructed a new vector named pSQ to express the recombinant scFv for making a scFv-type Q-body. In this vector, a Cys-tag encoding NH2-MAQIEVNCSNETG-COOH for introducing a cysteine residue for the labeling of thiolreactive fluorescent dye and His-tag followed by Flag-tag for tandem affinity purification was incorporated to the 5′terminus and 3′-terminus, respectively, of the scFv sequence (Figure 1). The sequence of the anti-BGP scFv fragment KTM21919 was introduced to the vector to compare the performances of scFv BGP Q-bodies made by this system and the performances of scFv BGP Q-bodies made by a cell-free system.11 We expressed a protein from pSQ(KTM219) in E. coli and purified the scFv protein in the cytosol. From the SDSPAGE analysis, we confirmed that highly purified scFv fragment was obtained as a soluble form with the amount of approximately 400−600 μg from the 100 mL shake-flask culture, which exceeded more than the Fab fragment that was C

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Figure 2. (A) Chemical structure of the fluorophores in this study. (B) Fluorescence image of SDS-PAGE for Q-bodies. The estimated molecular mass of scFv, including tags, is 29.4 kDa.

previously obtained from E. coli (approximately 100−200 μg from the 100 mL culture) (Figure S-1 in Supporting Information). This was probably due to the multidomain structure of Fab, which was more complex than the structure of scFv.20 The fluorescence measurement was performed using 10 ng of Q-body; the amount of Q-body obtained from 50 μg of purified scFv was approximately 1 μg, which was sufficient for conducting ∼100 measurements. Note that the pSQ vector is designed to enable simple insertion of the scFv fragment: First, AgeI and BamHI sites can be incorporated at the 5′ and 3′ termini of the majority of scFv fragment sequences by PCR using primers that are designed by modifying specific primer sets, such as the “Primer set for generation of highly diversified mouse phage display libraries” (Thomas Grunwald and Greg Winter, MRC Centre for Protein Engineering, Cambridge, UK, 2000). Second, the amplified fragment can be double-digested by AgeI and BamHI and inserted to the AgeI- and BamHI-digested pSQ vector by ligation. If other AgeI and/or BamHI sites exist in the scFv, cloning by the In-fusion method21 enables insertion of the amplified scFv fragment without restriction of PCR fragments. Q-Bodies with High Purity and Antigen Binding Activity. To determine the best fluorescent dye for making a Q-body, we examined the labeling of purified protein with a range of fluorophores that span distinct molecular and spectral properties (Figure 2A). To investigate the effect of distance between the dye and the protein, we employed three commercially available types of TAMRA-maleimide dyes with different spacer lengths (i.e., TAMRA-C0-mal, TAMRA-C2mal, and TAMRA-C5-mal, which indicates the number of carbon atoms between the dye and maleimide). We performed labeling with eight types of fluorophore to the reduced Cys residue of Cys-tag by maleimide−thiol chemistry and purified them by tandem purification with immobilized metal and antiFlag affinity chromatographies. From the result of the SDSPAGE analysis on a transilluminator, we confirmed the efficient fluorescence labeling of scFv and the efficient elimination of unbound fluorophores (Figure 2B). We examined the antigen-binding activity of Q-body by an enzyme-linked immunosorbent assay (ELISA). The wells with immobilized antigens showed strong signals, whereas negligible signals were observed for the wells without antigens, which indicates that each Q-body retains sufficient antigen-binding activity (Figure 3). The signals of eight labeled scFvs and the unlabeled scFv were almost identical, which suggests the

Figure 3. Specific binding of the Q-body to antigens probed by ELISA. The colored bars represent the signal in the presence of antigens, and the white bars represent the signal in the absence of antigen. Error bars represent ±1 standard deviation (SD) (n = 3).

negligible effect of the introduced dye on the antigen binding activity of scFv. Despite similar antigen binding activity, the fluorescence responses of these labeled scFvs remarkably differed, as described later. Antigen Dependency of the Fluorescence Spectra. We measured the fluorescence spectra of the Q-bodies in both the absence of antigen BGP-C7 and the presence of antigen BGPC7. Because the fluorescence from unreacted excess dye significantly affects measurements, such as background intensity, thorough washing was performed to attain high signal-to-background ratios. To investigate the optimal method to eliminate unreacted dyes, we purified the Q-bodies with three different approaches(i) using IMAC, (ii) using IMAC followed by desalting column, and (iii) using IMAC followed by anti-Flag beadsand compared the responses after the addition of the antigen. By purifying using not only IMAC but also desalting or anti-Flag column, the response of R6G-labeled Q-body was noticeably increased (Figure S-2). Because R6G is hydrophobic and easily forms aggregates,22−25 the excess R6G had been nonspecifically adsorbed to the IMAC resin or the IMAC-protein complex even after washing and was subsequently detached by the elution. Thus, the remaining excess dye after the first purification was successfully eliminated by the second purification. Based on these results, the importance of thorough purification to remove unbound dyes was confirmed to be vital to obtain adequate responses especially when hydrophobic dyes or characteristically unknown dyes are employed for the labeling. D

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Figure 4. (A) Fluorescence spectra of Q-bodies in the presence and absence of 10 μM of antigen. The black shaded area represents ±1 SD (n = 3). (B) Schematic images of quenched, dequenched, and denatured Q-bodies (left), and normalized fluorescence intensities of Q-bodies in the presence of 10 μM of antigen and in the presence of denaturant (right). Error bars represent ±1 SD (n = 3).

Figure 5. Antigen concentration-dependent fluorescent response of the Q-bodies. Error bars represent ±1 SD (n = 3).

The fluorescence intensities of the Q-body labeled with ATTO495-C2-mal, ATTO520-C2-mal, R6G-C5-mal, Rho-C2mal, TAMRA-C0-mal, TAMRA-C2-mal, and TAMRA-C5-mal after purification with IMAC and anti-Flag beads was increased 1.1-, 2.7-, 5.0-, 1.7-, 2.9-, 2.0-, and 4.0-fold, respectively, in the presence of an antigen (Figure 4A). We compared these values with the fluorescent responses for a denaturing condition,

which shows the available maximum dequenching status (Figure 4B and Figure S-3). As a result, the changes in fluorescence after the denaturation correlated with the changes in fluorescence after the addition of antigen, which indicates the quenching of the dye by scFv in its native state and its almost complete release after the addition of antigen. The results collectively suggest that these dyes are suitable for making a QE

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ACS Sensors Table 1. Binding Constants of the Proteins to Immobilized BGP-C11 Peptidea

a

protein

ka (×105 M−1 s−1)

kd (×10−2 s−1)

KA (×107 M−1)

KD (×10−8 M)

Unlabeled scFv R6G-labeled Q-body

3.42 ± 1.19 3.01 ± 1.07

1.16 ± 0.268 1.25 ± 0.413

3.21 ± 1.86 2.85 ± 2.10

3.78 ± 1.75 4.82 ± 2.80

An average and a standard error are shown (n = 3).

and R6G-labeled Q-body, which indicates that the binding affinity of scFv for the antigen was preserved after the incorporation of fluorescent dye into the N-terminal region of scFv (Figure S-4 and Table 1). Second, we performed a fluorescence-based thermal shift assay using a real-time thermal cycler to investigate the thermal denaturation property. According to the increase in incubation temperature, the Q-body began to unfold and bound the dye in its hydrophobic area, which produced a higher fluorescent signal (Figure S-5A). The first derivative of the raw fluorescence curve (Figure S-5B) confirmed that this Q-body has a thermal stability with a melting temperature of 61 °C, which is equivalent to the thermal stability of the anti-BGP scFv Q-body that was prepared by a cell-free system.10

body. In particular, Rhodamine-red (Rho) was newly revealed in this study as a suitable dye for making a Q-body. Because this dye has longer excitation and emission wavelengths, the Rholabeled Q-body is expected to be applied to fluorescence imaging of various biological phenomena in vivo, for instance, the imaging of calcium ion-related molecular events.26 The observed fluorescent response was dependent on the fluorophore, which was primarily attributed to the variable extent of fluorescence quenching. Although the reasons for the observed variety cannot be precisely described, the different properties among the fluorophores, including spacer length, hydrophobicity, and molecular size, are assumed to be probable reasons. One of the most important factors is the spacer length, despite the difficulty of precisely calculating the distances between the fluorophore and the internal Trp residues of the Q-body. The response of the R6G-C5-mal and TAMRA-C5mal-labeled Q-bodies were optimal. When the three TAMRACn-mal (n = 0, 2, 5)-labeled Q-bodies were compared, the higher quenching effect was observed for the Q-body with the longer spacer. If the spacer is short (C0, C2), the dye may be too far away to interact with the internal Trp residues and the resulting low quenching subsequently produces a lower response than the response with a longer spacer (C5). The R6G-C5-mal-labeled Q-body showed a response that is superior to the response of TAMRA-C5-mal, which shares the same spacer. Because R6G is more hydrophobic than TAMRA,25 it was easily quenched around the hydrophobic VH/ VL interface. When the increasing concentrations of antigen were added to each Q-body, the fluorescence intensity of the majority of Qbodies showed an antigen dose-dependent increase with a broad detection range (Figure 5). The EC50 value of ATTO520, R6G, TAMRA-C5, and the Rho-labeled Q-body was 6.3, 11.4, 4.4, and 70.9 nM, respectively, which indicates a high sensitivity and utility as a practical sensor for BGP.27 Note that the maximal fluorescence responses and the EC50 values of ATTO520-, R6G-, and TAMRA C5-labeled Q-bodies were almost comparable with the maximal fluorescence responses and the EC50 values of cell-free based scFv type Q-bodies (i.e., the ATTO520-, R6G-, and TAMRA-labeled Q-body showed 2.9-, 7.5-, and 5.7-fold responses, respectively, in the presence of an antigen, with an EC50 of 17.7, 84.6, and 38.0 nM, respectively),11 which indicates the usefulness of this E. colibased approach for constructing a Q-body in terms of the response and the sensitivity. Characterization of R6G-Labeled Q-Body. The antigen binding affinity and thermal stability are critical factors of a recombinant antibody. To evaluate both properties of an R6Glabeled Q-body, which showed the largest response in this study, we assessed the kinetic constants and melting temperature. First, the binding kinetics of the Q-body to the varied concentration of antigen was examined by a biolayer interferometry (BLI) assay. The biotinylated antigen was immobilized on a streptavidin-coated BLI sensor, and association/dissociation constants were analyzed. As a result, almost similar KD values were obtained from the unlabeled scFv

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CONCLUSION In this study, we successfully constructed an E. coli-based scFv type Q-body with a high yield and response and improved color variation. The variation in the fluorescent response was dependent on the fluorophore and the spacer length between the dye and maleimide, which reflects the differences in their quenching behavior. Due to its simplicity and versatility, these multicolored Q-bodies may become one of the most attractive biosensors for a range of applications, such as for imaging of multiple molecular targets in situ, with the ability to be simultaneously excited and monitored at several wavelengths.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.5b00089. Figures for protein purity, effect of purifications, fluorescence spectra, affinity measurements, and thermal shift assay (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81-45-924-5248. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was partly supported by a Grant-in-Aid for Scientific Research (No. 15H04191 to HU and No. 26889028 to HJJ) from the Japan Society for the Promotion of Science and partly supported by Ushio Inc.

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REFERENCES

(1) Yalow, R. S.; Berson, S. A. Assay of plasma insulin in human subjects by immunological methods. Nature 1959, 184 (Suppl 21), 1648−9. (2) Ekins, R. P. New perspectives in radioimmunoassay. Nucl. Med. Commun. 1993, 14, 721−35.

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DOI: 10.1021/acssensors.5b00089 ACS Sens. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acssensors.5b00089 ACS Sens. XXXX, XXX, XXX−XXX