The issue of drug abuse is increasing worldwide and has been associated with various social problems. Methamphetamine (MA) is a highly addictive psychostimulant that causes adverse biological effects, such as acute toxic effects on the cardiovascular system, acute renal failure, altered behavioral and cognitive functions, and permanent brain damage [1
]. Therefore, illicit use of MA is prohibited in many countries. An accurate and rapid detection system for MA is required for clinical diagnostics and criminal forensics, such as drug screening at the workplace and monitoring patients during drug rehabilitation.
One of the current methods for quantitating MA is the Duquenois-Levine colorimetric test, in which an indicator reacts with MA and is compared with standardized color charts visually [3
]. Such a colorimetric approach is economic and simple, and can be performed without complicated training. However, the visual interpretation affects the accuracy of results, which is occasionally presumptive and limits the quantification. Immunochromatography, which uses a colloidal gold-labeled antibody or antigen as a detection agent provides a simple analytical procedure for drug screening in biological specimens [4
]. However, as the test result is typically interpreted by the observation of a color band on the membrane, the determination of a positive or negative result is also subjective. Another method, thin-layer chromatography (TLC) has been used to detect MA. However, TLC has limited accuracy when used on complex samples due to low resolution of separation; thus, the method is typically used for preliminary identification [3
]. Chromatographic methods, including gas chromatography and liquid chromatography, can be used in combination with mass spectrometry to provide high precision results [5
]. However, these methods involve long sample preparation times, complicated experimental procedures that require a high-level of scientific knowledge and advanced skills and expensive instruments. Enzyme-linked immunosorbent assay (ELISA) has been used as a primary tool for the quantification of MA [7
]. However, ELISA involves multiple steps, including several washing and incubating steps, which require one or two days to complete the entire procedure. Thus, a highly accurate assay with easy sample treatment is needed for rapid, onsite MA detection.
Antibody-based reagentless fluorescence immunoassays offer the advantages of only taking minutes to complete by eliminating washing steps as well as the use of secondary antibodies. However, these types of assays offer a few challenges in the developmental stage, such as different dye conjugation efficiencies to individual antibodies and non-specific binding to nontarget molecules. For example, labeling an N-hydroxysuccinimide ester-conjugated dye to the lysine (Lys) R-group amines is advantageous due to the numerous Lys residues on the antibody surface. However, this method has the disadvantage such that a good number of Lys residues are distributed all over the antibody, making it difficult to exactly quantify a target antigen due to the random labeling of the fluorescent dye, which is not consistently producing a conjugate product with a known dye:antibody conjugation ratio. Another potential issue is that the Lys residues in the antigen binding region might be conjugated to the dye, which could hamper the antigen-binding activity. To increase the site-specific dye-antibody conjugation efficiency, we developed a new antibody-labeling method using a cysteine (Cys)-containing peptide tag (Cys-tag; MSKQIEVNCSNET [8
], a mimic of ProX-tag (MSKQIEVN*SNET, * = amber codon [9
]) that was developed for the incorporation of unnatural amino acids), and applied it to the generation of various Quenchbodies (Q-bodies). A Q-body is an innovative immunosensor that fluoresces upon antigen-dependent removal of the fluorophore quenching [9
]. In the absence of an antigen, the fluorescence of the dye is quenched by a photo-induced electron transfer from the tryptophan (Trp) residues in the nearby antigen-binding sites of antibody. When an antigen binds to an antibody, the binding stabilizes the conformation of the antibody variable region and the dye associated with the antigen-binding site is sterically hindered from interacting with the Trp, which leads to de-quenching (Figure 1
In addition to the dye-Trp interaction, a quenching effect can be achieved by dimerization of two neighboring dyes called H-dimer [10
]. For example, a double-labeled antigen-binding fragment (Fab)-type Q-body, in which the same dye was incorporated in both the H and L chains, showed a higher fluorescent response in the presence of antigen than single-labeled one, which might be due to dye-dye interaction-mediated deeper quenching [8
]. The advantage of Q-body-based assays over conventional immunoassays is its simplicity and rapid quantification of various antigens. Q-body assays function in a one-step, one-pot manner. The Q-body reagent is added to the sample and fluorescence is measured after a short incubation time, without any other experimental steps, which is inherent to the conventional immunoassay that needs several incubation and washing steps with blocking agent or additional antibody. Since the fluorescence of the Q-body is quenched in the absence of antigen, its signal-to-background ratio is higher than that of other conventional dye-conjugated antibodies. We have utilized Q-bodies as biosensors for the rapid detection of various targets in a solution, and in some cases on cells, without the need for washing steps [13
]. Moreover, Q-body assay for detecting BGP peptide was successfully performed not only in the biochemical buffer-based samples but also in 50% plasma with no apparent loss of response [8
]. Therefore, Q-body has potential as a universal reagent for monitoring MA in biological fluid such as blood sample. The anti-MA Q-body was previously generated using an amber-codon-based cell-free transcription and translation system. This Q-body showed a dose-dependent response to the MA derivative concentration up to 7.2-fold [8
]. However, as the Q-body is produced via an in vitro translation system, the high cost of reagents including unnatural aminoacyl-tRNA-conjugated dye and the low yield remain as obstacles in practical applications.
In this paper, we detail the synthesis of a Q-body using a combination of Escherichia coli (E. coli) expression for high yields and Cys-tagging for site-specific fluorescence dye conjugation. The conjugating involves the mild reduction of the sulfhydryl group of a Cys-tag followed by labeling with a maleimide-conjugated dye. To develop a one-pot MA detection method, we generated a Q-body that recognizes 3-[(2S)-2-(methylamino)propyl] phenol, a MA derivative with high structural similarity. A major focus was the position and number of the fluorophore, as well as the length of the spacer between the maleimide and dye to synthesize a Q-body with a high response to the antigen. The results indicate the potential use of high yield E. coli-based recombinant antibody production and thiol-based antibody labeling with fluorescent dyes against various biomarkers, including MA.
4. Results and Discussion
The anti-MA scFv gene M9 used in this study was originally cloned and affinity-matured by G. Georgiou’s group [17
]. Since this anti-MA antibody had the same number of Trp residues in both the heavy chain variable (VH) and light chain variable (VL) domains (three Trp residues in each domain), we constructed three different DNA genes with different sites for fluorophore-labeling: close to the H chain, to the L chain, or to both the chains. We inserted a Cys-tag for conjugating a dye at the N-terminal regions of the H and L chains (Figure 2
). The anti-MA Fab gene with a single Cys-tag at the N-terminal region of the H/L chain was cloned into the pUQ1H/pUQ1L vector. Similarly, we generated a gene for a double-dye-labeled Q-body with two Cys-tags at the N-terminal regions of both the H and L chains.
Previously, the VH and VL genes of clone M9 had been codon-optimized and subcloned into the pROX vector to generate a cell-free transcription-translation system-based anti-MA Q-body with a fluorophore located at the N-terminal region of the H chain [pROX1H(MeM9)] [9
]. In this study, the DNA was subcloned into pUQ1H, pUQ1L, and pUQ2 to express each Fab in the cytoplasm of E. coli
]. The anti-MA Fabs were expressed in soluble form and then purified by immobilized metal affinity chromatography (IMAC) via the His-tags. After a mild reduction of the cysteine thiol group of the Cys-tag, a maleimide-conjugated fluorescent dye was conjugated to the reduced thiol group utilizing the maleimide-thiol reaction (Figure 3
). Next, the Q-bodies were tandemly purified by IMAC and anti-Flag affinity beads to remove the excess free dyes. The Q-bodies were then used for fluorescence measurements in the presence of denaturant or antigen.
Our previous study utilizing a Fab-type Q-body against bone Gla protein (BGP) showed that the differences in linker length between the maleimide and Cys-tag affected the fluorescent response of the Q-body in the presence of antigen [8
]. Based on these results, we varied the spacer length between the dye and maleimide: TAMRA-CO-maleimide (C0), TAMRA-C2-maleimide (C2), or TAMRA-C5-maleimide (C5) (Figure 4
a). We evaluated the maximum degree of quenching (quenching capacity) of each Q-body by measuring the fluorescence intensities in the presence of a denaturant (7 M guanidine hydrochloride and 100 mM dithiothreitol) and normalizing the values against those obtained under the non-denatured condition (PBST added instead of the denaturant). The responses of the denatured Q-bodies were 1.56 ± 0.03-, 1.19 ± 0.15-, 0.73 ± 0.38-, 2.48 ± 0.26-, 2.80 ± 0.24-, 5.13 ± 0.79-, 2.35 ± 0.36-, 3.67 ± 0.68-, and 2.31 ± 0.33-fold for the C0-labeled H, C0-labeled L, C0-labeled HL, C2-labeled H, C2-labeled L, C2-labeled HL, C5-labeled H, C5-labeled L, and C5-labeled HL, respectively (Figure 4
b,c). For the C2-linker, the responses of two single dye-labeled Q-bodies (H and L) were similar, and these values were approximately two times lower than for the double dye-labeled Q-body (HL), as estimated based on the same number of Trp residues in the VH and VL domains. The C0/C5-linker Q-bodies did not show this trend. Although the three-dimensional structures of these Q-bodies have not been determined, we can state that the optimal position of dyes around the Trp residues affects the degree of quenching. We estimated that the C0 linker was too short to stay close to the Trp residues in VH as well as in VL. However, the C2 and C5 linkers were a suitable length to increase the quenching capacity.
The double TAMRA-C2-maleimide labeled Q-body had the highest quenching capacity and was used for testing the antigen dose-dependency. Since MA is an illicit drug, it was not generally available. Therefore, we used 3-[(2S)-2-(methylamino)propyl]phenol as a surrogate since the only structural difference was the addition of a hydroxyl group on the benzene ring (Figure 5
a). The fluorescence of the double TAMRA C2-labeled Q-body was measured at different concentrations (0.1 to 104
µg/mL) of 3-[(2S)-2-(methylamino)propyl]phenol. The fluorescence intensity increased to 4.40 ± 0.12- fold at 104
µg/mL of 3-[(2S)-2-(methylamino)propyl]phenol, similar to the fluorescence intensity in the presence of denaturant (Figure 4
c and Figure 5
b). This indicated that the dye was almost completely de-quenched by the antigen. This data suggested that the fluorescence dye was successfully incorporated into the antigen-binding domain of the Fab fragment and displaced by antigen, and that quenching was removed in an antigen-concentration-dependent manner.
In our previous study, a single labeled Fab-type Q-body was generated through a cell-free translation and transcription system. The resultant Ultra Q-body showed MA-dependent fluorescence increases up to 7.2-fold [8
]. The lower response of the Q-bodies generated in this study may reflect the differences in the dye conjugation method. The previously published cell-free based Q-body was generated with a TAMRA-tRNA conjugated to the amber-tag with the (GlySer)2
linker on the N-terminus of the H chain. Therefore, the distance between the dye and Trp was not the same as that of the TAMRA-C2-maleimide-labeled Q-body. As a primary reason for the higher EC50
of this double-labeled Q-body than the value of the single-labeled Q-body [8
], we think that the dye-dye stacking occurred between the two dyes, resulting in the need of high concentration of antigen for de-quenching [10
]. Additionally, we removed the cysteine residues at the C-terminal of the Fd and L chains, which are used for the disulfide bond between them, to increase the efficiency of site-specific labeling at the cysteine of the Cys-tag. This could have caused the Q-body to have somewhat lower antigen affinity and resultant lower sensitivity. Although the fluorescence response and sensitivity were lower than the cell-free based one, the E. coli
-based approach for constructing Q-bodies have several advantages. First, the yield of Q-body produced by E. coli
is higher than the cell-free system. Better yield increases cost efficiency, which is one of the major obstacles for the large-scale production of Q-bodies for practical applications. The expression of recombinant antibody fragments in E. coli
has a higher yield than hybridoma-based production of full-sized antibodies, and such an inexpensive bacterial production may result in lower product costs. Thus, this concept holds promise as a general strategy for the development of a commercially available biosensor. Second, the maleimide-conjugated dye is less expensive to synthesize than the tRNA-conjugated dye, and has a wider range of colors. The wider range of multi-color labels for Q-bodies provides greater flexibility in developing multi-plex drug screening kits. Finally, to test the cross-reactivity of anti-MA Q-body against other MA derivatives, we used phenethylamine and methoxyphenamine. These chemicals have fewer structural similarities to those of MA than 3-[(2S)-2-(methylamino)propyl]phenol. The anti-MA Q-body exhibited a negligible response in the presence of phenethylamine or methoxyphenamine. The lack of response indicates that there is no cross-reactivity to other MA derivatives, which suggests that the anti-MA Q-body has a high MA selectivity (Figure 5
We produced an E. coli-based Q-body against a hydroxyl MA derivative with the goal of developing a fast and convenient on-site MA screening kit. In particular, we focused on the number and position of fluorescent dyes with different linker lengths to produce a Q-body with an optimized response range. In addition, the Q-body reported here is highly selective to 3-[(2S)-2-(methylamino)propyl]phenol. The major advantage of the Q-body is that the test takes a few minutes of incubation followed by the fluorescence measurement. It is a single step test with no need for multiple incubation and washing steps, like those required for other conventional immunoassays such as ELISA. Therefore, this anti-MA Q-body has potential as a reagent in a drugs-of-abuse analysis platform when used in conjunction with a portable fluorophotometer. This type of rapid and simple assay tool may be useful in a range of areas, such as drug screening at the workplace, multiplexed point-of-care testing for simultaneous on-site detection of various stimulant drugs including cocaine, morphine, and suspicious substance identification, and monitoring patients during drug rehabilitation. Additionally, the Cys-tag-based Q-body-synthesis method reported in this study can be applied to the generation of Q-bodies of interest by fusing fluorescence dye to antibodies in a number of different antibody formats.