"Quenchbodies": Quench-Based Antibody Probes That Show Antigen-Dependent Fluorescence

Abstract

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

Full text

“Quenchbodies” : quench-
based antibody probes that show
antigen-dependent fluorescence
Journal:
Journal of the American Chemical Society
Manuscript ID:
ja-2011-05925j.R1
Manuscript Type:
Article
Date Submitted by the
Author:
n/a
Complete List of Authors:
Abe, Ryoji; Protein Express Co. Ltd.; Japan Advanced Institute of
Science and Technology, School of Materials Science
Ohashi, Hiroyuki; The University of Tokyo, Department of Chemistry
and Biotechnology
Iijima, Issei; Japan Advanced Institute of Science and Technology,
School of Materials Science
Ihara, Masaki; The University of Tokyo, Department of
Bioengineering
Takagi, Hiroaki; Protein Express Co. Ltd.
Hohsaka, Takahiro; Japan Advanced Institute of Science and
Technology, School of Materials Science
Ueda, Hiroshi; The University of Tokyo, Dept of Chemistry and
Biotechnology; The University of Tokyo, Department of
Bioengineering
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“Quenchbodies” : quench-based antibody probes that show antigen-
dependent fluorescence
Authors: Ryoji Abe†, §, Hiroyuki Ohashi‡, Issei Iijima†, Masaki Ihara±,||, Hiroaki
Takagi§, Takahiro Hohsaka† and Hiroshi Ueda‡, ±,*
†School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai,
Nomi, Ishikawa 923-1292, Japan
§Protein Express Co. Ltd., 1-8-15 Inohana, Chiba 260-0856, Japan
‡Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo,
7-3-Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
±Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, Japan
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ABSTRACT
Here we describe a novel reagentless fluorescent biosensor strategy based on the antigen-
dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a
cell-free translation-mediated position-specific protein labeling system, we found that an
antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region
showed a significant antigen-dependent fluorescence enhancement. Investigation of the
enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled
anti-osteocalcin scFv showed that antigen-dependency was dependent on semi-conserved
tryptophan residues near the VH/VL interface. This suggested that the binding of the antigen led to
the interruption of a quenching effect caused by the proximity of tryptophan residues to the
linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and
haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to
those observed in competitive ELISA were obtained, even in human plasma. Due to its versatility,
this “quenchbody” is expected to have a range of applications, from in vitro diagnostics, to
imaging of various targets in situ.
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INTRODUCTION)
)
The development of innovative fluorescence-based techniques for probing molecular
recognition is of major interest in biophysical chemistry. The naturally occurring amino acid
tryptophan (Trp) is of particular interest in fluorescence-based work on peptides and proteins. For
example, Trp can serve as an efficient electron donor in photoinduced electron transfer (PET)
reactions with certain dye molecules, a property conferred by the Trp indole side chain which is
the most readily oxidized functional group among all naturally occurring amino acids.1
In further promising applications, PET-based biosensors have been developed that use
conformationally induced alterations in PET efficiency upon binding for the specific detection of
DNA or RNA sequences or antibodies at the single-molecule level.2,3 In contrast to Förster
resonance energy transfer (FRET)-based systems, in which long-range dipole-dipole interactions
are probed, the above sensors require contact formation between the fluorophore and the
guanosine or tryptophan residue. Depending on the reduction potential of the fluorophore used,
efficient fluorescence quenching via PET can then occur. With careful design of
conformationally flexible molecules and the use of appropriate fluorophores, efficient single-
molecule sensitive PET sensors can be produced. Therefore, PET-based molecules offer an
elegant alternative to conventional biosensors based on FRET processes.
As a possible application for PET-based biosensors, immunological detection of target
molecules (immunoassay) is considered as a very attractive area of research. It is an
indispensable technique for quantifying various molecules, and is utilized in a range of fields,
from basic biological research to clinical diagnostics. Fluorescence-based assays that do not
involve separation steps are considered particularly useful, due to advantages such as short assay
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time and ease of handling. The reagentless fluorescent biosensor approach, which is based on the
fluorolabeled antibody fragment whose fluorescence intensity is altered by binding to its target
(antigen), is probably the simplest example of such attempts.4,5 For example, Bedouelle et al 6
and Winter et al 7 used site-specifically labeled antibodies that demonstrated increased
fluorescence upon binding to their target protein, using the environment-sensitive dye 7-
nitrobenz-2-oxa-1,3-diazole (NBD). However, to construct such protein biosensors, the
fluorolabeling position on the protein must be optimized, most typically in or near
complementarity determining regions (CDR) of the antibody, and this often requires considerable
time and labor.
Here we shows a novel strategy of PET-based biosensor for various antigens, using a
position-specific protein labeling methodology based on fluorolabeled aminoacyl tRNA and a
cell-free translation system.8 During the attempts to make FRET-based biosensor by this
methodology, we fortuitously discovered an antigen-dependent fluorescence enhancement of a
single chain variable region fragment (scFv) of the antibody fluorolabeled at its N-terminal
region. After investigation of the mechanism, it was found that this enhancement was observed
due to the quenching of used dye by the semi-conserved Trp residues in scFv, probably by PET
mechanism. According to the proposed mechanism, the phenomenon was considered to have
generality to other antibodies, and it was experimentally confirmed to be indeed the case.
RESULTS
Position-specific incorporation of TAMRA-labeled amino acid into anti-BGP scFv
As the model antibody to apply position-specific fluorolabeling technology, we chose anti-
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human osteocalcin (bone gla protein, BGP) scFv as the recognition unit for the bone-related
disease marker, and as the model dye we took 5-carboxytetramethylrhodamine (TAMRA). This
combination was chosen because the variable region, Fv, of this antibody has an interesting
property that it is markedly stabilized by bound BGP or its C-terminal epitope peptides.9 Also,
we have found in other experiments (Abe et al, in preparation) that position-specifically
TAMRA-labeled anti-BGP Fv at the N-terminal region of heavy chain variable region VH
demonstrates a modest but significant (1.7-fold) antigen-dependent fluorescence intensity change.
To prepare this construct, the two variable region genes (VH and VL) of the anti-BGP antibody
obtained from murine hybridoma KTM-219 cells were linked via a gene encoding a (Gly4Ser)3
linker (Figure 1A). The incorporation of p-(TAMRA-aminocaproyl)-aminophenylalanine
(TAMRA-C6-AF) into the N-terminus of the scFv was carried out using the amber suppression
method with an N-terminal ProX tag sequence (NH2-MSKQIEVNXSNE-COOH). This tag is an
optimized 12 amino acid sequence containing an amber codon (X) at the 9th position to enhance
the incorporation of non-natural amino acids as well as protein expression.10 In addition, a His
tag was fused at the C-terminus of scFv to allow ready detection and purification. The amber
codon was decoded by a highly efficient amber suppressor tRNA derived from Mycoplasma
capricolum Trp1 tRNA,11 which had been aminoacylated with TAMRA-C6-AF. The TAMRA-
C6-AF-tRNA was added to an E. coli cell-free translation system, together with the scFv genes
fused with a ProX coding sequence. The translation products were resolved by SDS-PAGE and
detected by fluorescence imaging of the gel. A clear fluorescent band showing TAMRA
fluorescence was observed at the expected molecular weight (32 kDa) (Figure 1B). This result
indicated that scFv containing TAMRA at the N-terminal region had been successfully expressed.
The scFv was also analyzed by Western blotting using an anti-His tag antibody. The full-length
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protein was clearly observed in the presence of TAMRA-C6-AF-tRNA, while a negligible
amount of the protein was observed in the absence of the suppressor tRNA (Figure 1C). These
results indicated that the TAMRA-C6-AF-tRNA decoded the amber codon specifically, and that
anti-BGP scFv had been site-specifically and near-quantitatively labeled with TAMRA.
Antigen-dependent fluorescence of TAMRA-labeled anti-BGP scFv
The TAMRA-labeled anti-BGP scFv was purified using nickel-affinity chromatography and
its fluorescence spectrum was measured in the absence and presence of a cognate antigen BGP-
C7 peptide (NH2-RRFYGPV-COOH). To our surprise, the fluorescence spectrum showed a
remarkable dose-dependent increase (5.6-fold) in intensity upon addition of BGP-C7 (Figure 2A).
A titration curve of the fluorescence intensity at 580 nm suggested that fluorescence increases
with BGP-C7 peptide concentration (Figure 2B). Also, when the same measurement was
performed with full-length BGP, similar dose-dependent fluorescence increase (max 5.2-fold)
was observed. Using curve fitting, the ED50 values for the peptide and the protein were estimated
as 2.5 × 10-8 M and 1.1 × 10-7 M, respectively, which compare well with the ED50 value obtained
by competitive ELISA (8.8 × 10-8 M for BGP-C7).9 The result indicates that the BGP peptide-
binding activity of scFv had not been affected by the incorporation of TAMRA-C6-AF at the N-
terminal region. To test if the observed fluorescence change could be used for imaging purposes,
the fluorescence intensity of TAMRA-scFv was visualized in a 384-well microplate, in the
presence and absence of the BGP peptide. The fluorogram obtained confirmed that the TAMRA-
labeled scFv could potentially be used for quantitative imaging of the antigen in situ (Figure 2C).
To further evaluate the binding specificity of TAMRA-scFv, the same assay was performed
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with the somewhat longer peptides BGP-C10 (NH2-EAYRRFYGPV-COOH, MW=1257) and
BGP-C10dV (NH2-EAYRRFYGP-COOH, MW=1158). The former includes the C-terminal valine
that is an essential epitope of this antibody, while the latter lacks this residue.9 Titration curves
indicated while BGP-C10 showed a similar 5.8-fold increase to that observed for BGP-C7, no
apparent increase in fluorescence was observed in the presence of BGP-C10dV (Figure 2D).
This clearly suggests that the binding specificity as well as the affinity of scFv for BGP is
preserved, even in the presence of the N-terminal TAMRA dye.
Quenching of TAMRA fluorescence by Trp residues
Since the BGP peptides tested did not contain moieties capable of enhancing the TAMRA
fluorescence, and no other dyes that might cause FRET between them were present either in the
antigen or the antibody, we reasoned that the observed fluorescence enhancement mechanism
involved electron transfer, rather than FRET. It has been reported previously that a number of
organic fluorophores are quenched by Trp residues, either in solution or in a polypeptide. This
quenching is thought to occur via a PET mechanism.12-14 We therefore investigated the possibility
that the observed fluorescence intensity change was attributable to intramolecular quenching by
Trp residues in scFv. The VH fragment of this antibody has four Trp residues, namely, Trp33H,
Trp36H, Trp47H and Trp103H (using the Kabat numbering scheme 15), while the VL fragment has
a single residue, Trp35L. Almost all of these Trp residues, with the exception of Trp33H, are
located in the framework region, and are generally conserved in antibodies derived from various
origins. A three-dimensional model (Figure. 3A) suggested that Trp47H and Trp103H contribute
to the hydrophobic interaction with VL, and that Trp33H contributes to the interaction with the
BGP peptide. Trp36H and Trp35L are located close to the interacting surfaces of VH and VL,
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respectively.
To evaluate the potential role of the Trp residues on antigen-dependent fluorescence
quenching, five Trp-to-Phe point mutants containing TAMRA-C6-AF were prepared, and the
antigen-dependent fluorescence intensities were measured. All mutants showed significantly
attenuated enhancements in fluorescence intensity upon addition of the BGP-C7 peptide, when
compared to the wild-type scFv. Both the magnitude and the antigen-dependency of fluorescence
were diminished, depending on the mutation, although different behaviors were observed for
different mutants (Figure 3, B and C). Compared with the basal intensity without antigen, the
fluorescence enhancements observed on addition of antigen were 3.7-, 4.7-, 2.5-, 3.5- and 3.1-
fold for the W33HF, W36HF, W47HF, W103HF and W35LF mutants, respectively. These values
are all considerably less than the value observed for the wild-type scFv (5.6-fold). It is worth
noting that a mutant of the Trp residue most distant to the TAMRA showed the most reduced
response, while fully maintaining its antigen dependency (i.e. binding affinity).
To further confirm the antigen-dependent removal of quenching mechanism proposed to
explain the observed fluorescence enhancement, the fluorescence of TAMRA-scFv proteins
under a denaturing condition was investigated. As summarized in Table 1, the fluorescence of
free TAMRA and the proteins were measured in a standard buffer (PBS containing 0.05% Tween
20, PBST) or that containing 7 M guanidine hydrochloride (GdnHCl) and 100 mM dithiothreitol
(DTT). While free TAMRA showed a modest (1.1-fold) fluorescence increase in the denaturant,
the wild-type TAMRA-scFv showed a 5.5-fold increment, with a slight red shift in emission
wavelength. The result is in accordance with our hypothesis that the quenching occurs in the
folded protein, and this was removed by the denaturation, allowing the fluorescence to be
revealed. On the other hand, the addition of BPG-C7 at saturating concentration to the wild-type
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TAMRA-scFv led to a 5.0-fold increment in fluorescence, supporting the “antigen-dependent
release from the quenched state” hypothesis. The mutant TAMRA-scFv proteins showed
diminished fluorescence increments upon denaturation, and also in the presence of saturated
antigen. The observed increments were similar to each other (shown as recovery ratio in Table 1),
indicating the active role of each Trp residue in the observed quenching. These results suggest
that in the absence of antigen, the TAMRA fluorophore is in close proximity to these Trp
residues. This includes the Trp35L residue near the VH/VL interface, which may be able to
interact with distant TAMRA due to the ProX tag sequence and the flexible aminohexyl linker
between the scFv and the fluorophore. We postulate that the addition of antigen promotes the
closure of the VH/VL interface, preventing the interaction of TAMRA with the Trp residues, and
therefore removing the quenching.
Fluorescence lifetime measurement
To verify the occurrence of quenching and its release, fluorescence lifetime measurement of
TAMRA-scFv was performed. Since multiple Trp residues participate in quenching, the observed
quenching was supposed to be dynamic rather than due to static interaction. In the case of
dynamic quenching, faster fluorescence decay will be observed in the presence of quenchers.13
When fluorescence lifetime measurement was performed for TAMRA-scFv in the presence or
absence of BGP-C7 peptide or denaturant, a significant difference in the amplitude of shorter
lifetime species (1.2-1.46 ns) was observed (Table 2, Figure S1). In the presence of antigen or
denaturant, longer lifetime species (3-4 ns, similar to reported values for free Trp16,17) dominated.
However, in the absence of these agents, the amplitude of shorter lifetime increased to almost
half, clearly suggesting the occurrence of dynamic quenching probably due to a PET mechanism.
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Fluorescence correlation spectroscopy analysis
Depending on its concentration, scFv molecules can form dimers or other higher order
species.18 Since TAMRA can form quenched dimer,19 there is a small possibility that the addition
of antigen induced dissociation of a TAMRA-scFv oligomer to monomers, thus resulted in
increased species that emit brighter fluorescence. To rule out this possibility, fluorescence
correlation spectroscopy (FCS) was utilized to measure the diffusion time of TAMRA-scFv, as a
measure of its average molecular size in solution. As shown in Figure S2, compared with
TAMRA-scFv alone, the addition of BGP-C7 or intact BGP resulted in increased diffusion time,
which means slower diffusion probably due to increased molecular weight and possibly due to
larger molecular size of scFv-antigen complex with exposed TAMRA dye. From this result and
low scFv concentration used throughout this study, it is highly unlikely that the observed
fluorescence increase was due to antigen-dependent increase of fluorescent monomers.
Effect of dye mobility
The observed fluorescence quenching may be resulted from the long, flexible linkage of the
VH N-terminus and the fluorophore. To confirm the effect of linker on quenching, flexible
peptide linkers of (Gly3Ser)n (n=1-3) were inserted between ProX tag and anti-BGP scFv to
increase the mobility of the fluorophore (Figure 4A). Fluorescence spectral measurements
showed that the antigen-dependent fluorescence increase was also observed for all the linker
lengths while the response was slightly decreased for longer linkers (Figure 4B).
To evaluate the effect of the linker length further, an anti-bisphenol A scFv20 was used as
another recognition unit. The anti-bisphenol A scFv has six Trp residues including four
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conserved ones, which were expected to contribute to the fluorescence quenching as in the case
of anti-BGP scFv (Table 3). The anti-bisphenol A scFv genes were constructed and expressed as
TAMRA-labeled proteins in a similar manner. Fluorescence spectra showed an increase in
fluorescence intensity upon the addition of bisphenol A, although the observed response was
much weaker (1.1-fold for the scFv without linker) than that of the anti-BGP scFv (Figure 4C).
Since this weak response may be due to suboptimal interaction between the fluorophore and Trp
residues, elongation of the peptide linker was attempted. The insertion of (Gly3Ser)n linkers, in
this case, markedly enhanced antigen-dependent fluorescence increase, and the scFv containing
the longest (Gly3Ser)5 linker showed 2.0-fold fluorescence. From the fitting of titration curves,
the EC50 was estimated as 2.0 × 10-8 M, which was approaching to the IC50 value determined by
competitive ELISA for the original scFv (1.4 × 10-9 M).
Taken together, the results suggest that the insertion of peptide linker between the
fluorophore and scFv affects the fluorescence quenching and its antigen-dependency. Although
the exact reason of antibody-specific difference in response is unclear, this may be resulted from
the difference in the optimal orientation of fluorophore to the differently positioned Trp residues
in each scFv.
Application of the quenchbody strategy to other scFvs
We propose to name this position-specifically fluorolabeled scFv as a “quenchbody” after
the quench phenomenon associated with it. Since many Trp residues are highly conserved in
antibodies of various origins (see below), we expected that this approach might be broadly
applicable to other antibody/antigen pairs. To further investigate this potential, we prepared
quenchbodies for other antigens. Firstly, hen egg lysozyme, estradiol and serum albumins (SAs)
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were used as model antigens. Examination of the primary sequence of these antibodies revealed
that a number of Trp residues were conserved among them (Trp36H, Trp47H, Trp103H and
Trp35L) (Table 3). While anti-hen egg lysozyme HyHEL-10 lacks Trp47H, and anti-SA 29IJ6
contains only the conserved Trp residues, the number of Trp residues present in each scFv made
us optimistic that antigen-dependent fluorescence would be observed.
To allow the performances of these antibodies to be compared under the same conditions,
TAMRA-labeled quenchbodies containing the (Gly3Ser)2 linker between the ProX tag and the
scFv were prepared as described above. The fluorescence intensity for the purified proteins in the
absence and presence of their respective antigen were evaluated by fluorescence spectral
measurements on titration with the antigens. As shown in Figure 5A-C, all the quenchbodies
showed antigen-dependent fluorescence enhancements. However, the extent of enhancement was
variable with the different scFvs. The anti-estradiol scFv showed the strongest antigen-dependent
increase in fluorescence (4.5-fold increase), while the anti-lysozyme and anti-SA scFvs showed
weaker responses (1.3- and 1.5-fold, respectively). However, it is worth noting that the calculated
ED50 for the lysozyme was 7.5 × 10-9 M, which is comparable to the reported Kd values of the Fv
and scFv (5.0 × 10-9 M and 1.2 × 10-8 M, respectively).21 The ED50 measured for human SA (~10-
5 M) was sufficiently lower than its reference range of 3.4 to 5.4 g/dL (0.5-0.8 mM) in serum
(MedlinePlus, http://www.nlm.nih.gov/medlineplus/ency/article/003480.htm) to indicate its
potential utility as a diagnostic reagent.
Measurement of BGP peptide in human plasma
To further demonstrate the utility of quenchbodies in clinical diagnostic applications, the
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performance of the quenchbody in a plasma sample was investigated using a fluorescence imager.
As shown in Figure 5D, the TAMRA-quenchbody for BGP showed an almost indistinguishable
dose-response for the BGP-C7 peptide in 50% human plasma to that in PBST. The maximum
fluorescence was as high as 7-fold of that observed in the absence of antigen. This result
compares well with our previous open sandwich enzyme-linked immunosorbent assay (OS-
ELISA) for BGP, which showed considerable signal reduction in serum-derived samples without
pretreatment to remove SAs.22
Application to morphine detection
Sensitive detection, identification and confirmation of opiates are considered highly
important in view of public health and crime prevention. However, most conventional analytical
methods for opiates such as morphine or heroin are laborious and time-consuming. As another
practical application of quenchbody technology, rapid detection of opiates was attempted. Based
on the published nucleotide sequence of an scFv (VL-VH) recognizing morphine-6-glucuronide
(M6G),23 a quenchbody for M6G was prepared and investigated for its fluorescence spectra upon
addition of morphine and related drugs (Figure 6B, Figure S3).
As shown in the dose-response curves in Figure 6A, the constructed quenchbody reacted
most strongly with codeine, and also heroin and morphine in this order with the calculated EC50
values of 1.9, 3.7, and 5.2 × 10-8 M, respectively. Although the maximum fluorescence increase
was about 1.5-fold over the background, 1-100 ng/mL of these compounds were detectable
within 3 min after reaction start. On the other hand, naloxone wherein the N-methyl group
observed in opiates is substituted with N-propenyl, as well as cocaine, ketamine and
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methamphetamine showed negligible fluorescence increase, probably due to the absence of this
N-methyl as the essential epitope.
To compare the antigen dose-dependency of quenchbody fluorescence with that of antigen
binding, we performed competitive ELISA using immobilized morphine-BSA and free morphine,
and the amount of bound quenchbody was evaluated using peroxidase-conjugated anti-His5
antibody. From the curve-fitting, the IC50 for morphine was obtained as 26 ± 12 nM (Figure S4),
which was a similar value to the EC50 of quenchbody fluorescence. These data suggest that the
anti-morphine quenchbody could detect as little as ppb range of opiates by just mixing the sample
and measuring its fluorescence, which obviates the needs of long and dangerous reaction/washing
steps inevitable in ELISA.
DISCUSSION
Our working model of the antigen-dependent quenchbody fluorescence observed with this
system is as follows. In the absence of antigen, TAMRA within the ProX tag sequence penetrates
between the VH/VL interface, and interacts with Trp residues by hydrophobic and/or!π−π!stacking
interactions (Figure 3A). This interaction leads to a quenching electron transfer from the Trp
residues to the dye due to transient contact between each Trp residue and the chromophore.13,14
Binding of an antigen, such as the BGP peptide, to the scFv induces tighter complexation of VH
and VL, and thus releases the TAMRA dye from interactions with the Trp residues near the
VH/VL interface.
Among the five tryptophans found to be responsible for the quenching of the TAMRA-
labeled anti-BGP quenchbody, four (Trp36H, Trp47H, Trp103H, and Trp35L) are located in the
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framework region, and are highly conserved among immunoglobulins of various origins.
According to a database of >35000 non-redundant immunoglobulin sequences (Abysis,
http://bioinf.org.uk/), the rate of conservation for the three H chain tryptophans are 99.0%, 93.3%,
and 97.0%, respectively, and that for Trp 35L is 98.0%, for 12483 chains including kappa and
lambda chains. These values, as well as the number of Trp residues involved, ensure that almost
all Fv could potentially be successfully incorporated into a quenchbody. It is interesting that
while the side chain of Trp35L is located in the protein core, and therefore is not exposed to
solvent in any antibody structure, it plays a considerable role in the quenching effect observed in
the anti-BGP quenchbody. Presumably, the dynamics of the scFv’s polypeptide backbone might
result in transient escape of this residue from the buried hydrophobic core, and/or penetration of
hydrophobic TAMRA dye into the vicinity of this residue.
In the anti-BGP quenchbody, additional quenching by Trp33H was observed. This CDR2
residue is likely to be in the antigen-binding site, and therefore the presence of TAMRA in close
proximity to this residue would be prohibited by antigen binding, which therefore inhibits
quenching and enhances fluorescence. It can be speculated that the different fluorescence
dynamic ranges observed for the quenchbodies based on different antibodies is due to differences
in their Trp residue content, both in number and in positions. As shown in Table 3, some scFvs
contain Trp residues in addition to the conserved ones. For example, anti-estradiol scFv, whose
quenchbody showed a large fluorescence increase, has two additional Trp residues in VL. These
may contribute to enhancing the quenching effect. On the other hand, the anti-SA scFv obtained
from a synthetic phage library, Tomlinson J,24 has no Trp other than the conserved ones, and its
quenchbody demonstrated a lower response. Nonetheless, the obtained results in overall suggest
that the conserved Trp residues are sufficient per se for an antigen-dependent fluorescence
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quenching effect to be observed. Therefore, the present strategy can be expected to be effective
for various scFvs, including those with minimal conserved Trp residues. An improved response is
likely for those scFvs containing additional residues, depending on their number and positions.
The putative working mechanism also suggests that antibodies that show a higher antigen-
dependent stabilization (larger signal change in OS-ELISA that detects the interaction between
VH and VL)25 are likely to show more fluorescence activation. This is because these antibodies
have a higher chance of having a solvent-exposed VH/VL interface. However, while fluorescence
activation was observed for the anti-SA scFv, the same Fv showed a minimal response to BSA in
a phage-based OS-ELISA.26 It is likely that a small antigen-dependent change in VH/VL
interaction strength is sufficient to obtain an observable change in fluorescence intensity. This
implies that quenchbodies can be applied not only to small molecule detection, but also to the
detection of various protein antigens, even if they contain several Trp residues.
PET is recognized as a useful mechanism to alter the fluorescence of organic dyes and their
derivatives using small molecules such as metal ions or reactive oxygen species.27,28 Only
recently, it has been found to be useful in the detection of natural biomolecules such as guanine 29
and tryptophan.13 Several organic fluorochromes have been shown quenched by tryptophans,
either by an intermolecular or intramolecular PET mechanism. For example, ATTO 655-labeled
streptavidin was efficiently quenched probably due to the spatial proximity of the fluorolabeled
Lys residues to Trp residues in streptavidin. The observed quenching effect was markedly
reduced in the presence of streptavidin’s ligand biotin 13. When compared with this system, the
quenchbody system has the benefit of a large range of detectable targets.
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CONCLUSION
We reported a discovery of novel biosensing principle utilizing fluorescence quenching of a
labeled dye by intrinsic Trp residues in antibody Fv region. Compared with conventional
fluoroimmunoassays, the quenchbody assay is simple, and requires no additional reagents to
perform the detection. In addition, compared with OS-ELISA using Fv molecules, scFv-based
quenchbodies generally show higher sensitivity due to the linkage of the two variable region
fragments. Another merit of this scFv-based system is their ready availability, as specific scFvs
can be directly obtained from phage-display libraries of various origins. Due to the simplicity of
the system, the application of quenchbodies is not likely to be limited to in vitro diagnostics, but
may also be applied to imaging in situ or in vivo. In future, the development of more robust
quenchbody production methods, such as in vivo incorporation of non-natural amino acids,30 will
further enlarge the scope of their applications.
)
EXPERIMENTAL METHODS
Materials. In-Fusion Advantage PCR Cloning Kit was from Takara Bio (Otsu, Japan). A
pIVEX2.3d vector and RTS 100 E. coli Disulfide Kit were from Roche Diagnostics (Basel,
Switzerland) or 5-Prime GmbH (Hamburg, Germany). C-terminal peptides for human osteocalcin
(bone gla protein, BGP) (BGP-C7: NH2-RRFYGPV-COOH, MW=894; BGP-C10: NH2-
EAYRRFYGPV-COOH, MW=1257; BGP-C10dV: NH2-EAYRRFYGP-COOH, MW=1158) were
obtained from Genscript (Piscaway, NJ, USA). Bovine serum albumin (BSA) was from Bovogen
(Essendon, Australia). Hen egg lysozyme (HEL) was from Wako (Tokyo, Japan). Human serum
albumin (HSA) and estradiol were from Sigma (Saint Louis, MO, USA). Pooled normal human
plasma was from Innovative Research (Northville, MI, USA).
Construction of scFv genes. An expression vector, pROX-BGP-scFv, harboring a T7
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promoter-controlled scFv gene of anti-BGP C-terminal fragment KTM-219 9 fused with an N-
terminal ProX tag containing an amber codon (ATG TCT AAA CAA ATC GAA GTA AAC
TAG TCT AAT GAG) and a C-terminal His tag, was constructed by overlap PCR. A 15-amino
acid linker with three Gly4-Ser repeats was inserted between the VH and VL. The VH chain was
amplified using the 5’-primer
(CTTTAAGAAGGAGATATACCATGTCTAAACAAATCGAAGTAAACTAGTCTAATGAG
ACCCAAGTAAAGCTGCAGCAGTC) and the 3’-primer
(CCAGAGCCACCTCCGCCTGAACCGCCTCCACCGCTCGAGACGGTGAC), and the VL
chain was amplified using the 5’-primer
(CAGGCGGAGGTGGCTCTGGCGGTGGCGGATCTGACATTGAGCTCACCC) and the 3’-
primer (TGATGATGAGAACCCCCCCCCCGTTTTATTTCCAG). The amplified VH and VL
genes were linked by overlap PCR using the VH 3’-primer and the VL 5’-primer, and the
construct was then cloned into NcoI- and SmaI-digested pIVEX2.3d using an In-Fusion PCR
cloning kit.
A wild-type (non-labeled) scFv was constructed by replacing the TAG codon with a TTT
codon in the ProX tag. For substitution of tryptophan (Trp) residues with phenylalanine (Phe),
TGG codons for Trp33, Trp36, Trp47 and Trp103 in the VH gene and Trp35 in the VL gene were
each replaced by a TTT codon. For scFv genes encoding anti-BGP and anti-bisphenol A20, the
corresponding genes were cloned in place of anti-BGP scFv, with an additional sequence
encoding N-terminal (G3S)0-5 linker. For scFv genes encoding anti-hen egg lysozyme,31 bovine
serum albumin (BSA),26 and estradiol (Fujioka et al., in preparation), the corresponding genes
were cloned in place of anti-BGP scFv, with an additional sequence encoding N-terminal (G3S)2
linker. The anti-morphine-3-glucuronide scFv gene was synthesized by Mr. Gene GmbH
(Regensburg, Germany) according to the published sequence for clone E3 23, assuming that the
undisclosed heavy chain FR4 sequence was derived of J4 segment. The gene for VL-VH type scFv
with N-terminal (G3S)2 and internal (G4S)4 linkers was inserted to pROX-BGP-scFv in place of
anti-BGP scFv as above.
Preparation of aminoacyl tRNA. TAMRA-C6-AF-pdCpA was synthesized as described
previously10. This was ligated to an amber suppressor tRNA, derived from Mycoplasma
capricolum Trp1 tRNA without the 3’ dinucleotide, by chemical ligation as described
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previously.11,32 The aminoacyl-tRNAs can be obtained as commercially available reagents
(CoverDirect tRNA reagents for site-directed protein labeling, Protein Express, Chiba, Japan).
Cell-free Transcription/Translation. The incorporation of TAMRA-C6-AF into the N-
terminal region of scFv was performed using an RTS 100 E. coli Disulfide kit. The reaction
mixture (50!µL) was comprised of 7!µL of an amino acid mix, 1!µL of methionine, 7!µL of the
reaction mixture, 25 µL of activated E. coli lysate, 5 µL of plasmid DNA (500 ng) and 5!µL of
TAMRA-C6-AF-tRNA (0.8 nmol). All the reagents used, with the exception of the plasmid and
the tRNA, were provided in the RTS 100 E. coli Disulfide kit. The reaction mixture was
incubated at 20°C with shaking on RTS ProteoMaster (Roche Diagnostics, Basel, Switzerland) at
600 rpm for 2 h, and subsequently at 4°C without shaking for 16 h. An aliquot of the reaction
mixture (0.5 µL) was applied to 15% SDS-PAGE33 and the gel was visualized using a
fluorescence scanner FMBIO-III (Hitachi, Tokyo, Japan). The gel was also analyzed by Western
blot analysis using anti-His tag (Novagen, La Jolla, CA, USA) and alkaline phosphatase-labeled
anti-mouse IgG (Promega, Madison, WI, USA).
To purify scFv, the reaction mixture (50 µL) was diluted in wash buffer (20 mM phosphate,
0.5 M NaCl, 60 mM imidazole, 0.1% polyoxyethylene(23)lauryl ether, pH 7.4) to a final volume
of 400 µL, and applied to a His Spin Trap Column (GE Healthcare, Piscataway, NJ, USA). After
incubation at room temperature for 15 min, the column was washed three times with wash buffer.
The labeled scFv proteins were eluted with two 200!µL volumes of wash buffer containing 0.5 M
imidazole. The eluate was passed through an UltraFree-0.5 centrifugal device (Millipore,
Billerica, MA) and equilibrated with phosphate buffered saline Tween-20 (PBST, 10 mM
phosphate, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween-20, pH 7.4) to allow buffer exchange and
concentration of the protein. The concentration of the labeled scFv protein was determined by
comparing the fluorescence intensities of a known concentration of TAMRA dye (Anaspec,
Fremont, CA, USA) and the sample under denaturing conditions in 7 M guanidine hydrochloride,
pH 7.4.
Fluorescence measurements of TAMRA-labeled scFvs. For fluorescence spectral
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measurements of anti-BGP scFv, the purified scFv (2 µg/mL, 25 µL) was diluted in 200!µL of
PBST containing 0.2% BSA, and BGP peptide was added by titration in a 3 × 3 mm quartz cell
(GL Sciences, Tokyo, Japan). After each addition of the antigen, the solution was incubated at
25°C, 70 min prior to the fluorescence spectral measurements.
Anti-lysozyme HyHEL-10, anti-estradiol, anti-M3G scFvs (2 µg/mL, 25 µL) were diluted
in PBST containing 1% BSA. Anti-SA scFv 29IJ6 (2!µg/mL, 25 µL) was diluted in PBST
containing 0.2% gelatin. The antigens were added by titration at 5 min intervals, except for anti-
M3G, to which antigens were added at 3 min intervals.
Fluorescence spectra were measured from 565 to 700 nm, with excitation at 550 nm at 25°C
on a FluoroMax-4 (Horiba Jobin-Yvon, Kyoto, Japan). Excitation and emission slit widths were
set to 5.0 nm. The ED50 values were calculated by curve fitting of the observed fluorescence
intensities at the maximum emission wavelength, using a sigmoidal dose-response model based
on ImageJ software (http://rsbweb.nih.gov/ij/).
For fluorescence imaging of anti-BGP scFv, the purified scFv (2 µg/mL, 6.25 µL) was
mixed with various concentrations of the antigen in PBST (50 µL) containing BSA (0.2%), or in
50% human plasma from which the endogenous antigen had been removed,22 in a 384-well
microplate (Olympus, Tokyo, Japan). The plate was visualized with excitation at 532 nm and
emission at 580 nm using a fluorescence scanner.
Fluorescence lifetime measurements were performed on a TemPro system (Horiba Jobin-
Yvon, Japan) using the time-correlated single-photon counting (TCSPC) technique. The
excitation source was a 495 nm SpectraLED with a pulse length of ~ 1 ns at a repetition rate of 1
MHz. Emission at > 600 nm was collected using a long-pass filter SCF-50S-60R (SIGMA
KOKI), and the decay parameters were determined by least-square deconvolution.
)
ASSOCIATED CONTENT
Supporting Information. Decay curves obtained in the fluorescence lifetime measurements,
FCS data and methods, fluorescence spectra for TAMRA-scFv specific to M3G, and competition
ELISA result for morphine. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
hueda@chembio.t.u-tokyo.ac.jp
Present Addresses
|| Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University,
Nagano, Japan
ACKNOWLEDGMENT
We indebt T. Ueda, N. Tomioka, T. Suganuma and S. Ebisu for valuable suggestions, T. Shinoda
for anti-BGP gene, H. Ohkawa for anti-BPA gene, I. Tomlinson for anti-SA gene, and Y. Fujioka
for anti-E2 gene. We also thank Y. Mizutani, N. Teshigawara and M. Nakamura in Central
Customs Lab., Ministry of Finance, Japan, for their help in morphine measurement. This project
was partly supported by City-area program from MEXT, Japan, a Grant-in-Aid for Scientific
Research (B20360368) from JSPS, Japan, a Grant-in-Aid for Scientific Research on Innovative
Areas (20107005) from MEXT, Japan, and by the Global COE Program for Chemistry
Innovation, MEXT, Japan.
)
ABBREVIATIONS
BGP, bone gla protein (osteocalcin); BPA, bisphenol A; CDR, complementarity determining
region; dNTPs, deoxyribonucleotide triphosphate; ELISA, enzyme-linked immunosorbent assay;
FCS, fluorescence correlation spectroscopy; Fv, antibody variable region fragments; PBS,
phosphate buffered saline; PBST, PBS containing 0.05% Tween 20; PCR, polymerase chain
reaction; scFv, single chain antibody variable region fragment;
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(22) Ihara, M.; Yoshikawa, A.; Wu, Y.; Takahashi, H.; Mawatari, K.; Shimura, K.; Sato, K.;
Kitamori, T.; Ueda, H. Lab. Chip 2010, 10, 92-100.
(23) Dillon, P. P.; Manning, B. M.; Daly, S. J.; Killard, A. J.; O'Kennedy, R. J Immunol
Methods 2003, 276, 151-161.
(24) de Wildt, R. M.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000,
18, 989-994.
(25) Ueda, H.; Tsumoto, K.; Kubota, K.; Suzuki, E.; Nagamune, T.; Nishimura, H.; Schueler,
P. A.; Winter, G.; Kumagai, I.; Mahoney, W. C. Nat. Biotechnol. 1996, 14, 1714-1718.
(26) Aburatani, T.; Ueda, H.; Nagamune, T. J. Biochem. 2002, 132, 775-782.
(27) Silva, A. P. d.; Fox, D. B.; Moody, T. S.; M.Weir, S. Trends Biotechnol 2001, 19, 29-34.
(28) Koide, Y.; Urano, Y.; Kenmoku, S.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007,
129, 10324-10325.
(29) Torimura, M.; Kurata, S.; Yamada, K.; Yokomaku, T.; Kamagata, Y.; Kanagawa, T.;
Kurane, R. Anal. Sci. 2001, 17, 155-160.
(30) Sakamoto, K.; Murayama, K.; Oki, K.; Iraha, F.; Kato-Murayama, M.; Takahashi, M.;
Ohtake, K.; Kobayashi, T.; Kuramitsu, S.; Shirouzu, M.; Yokoyama, S. Structure 2009, 17, 335-
344.
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(31) Neri, D.; Momo, M.; Prospero, T.; Winter, G. J Mol Biol 1995, 246, 367-373.
(32) Iijima, I.; Hohsaka, T. ChemBioChem 2009, 10, 999-1006.
(33) Laemmli, U. K. Nature 1970, 227, 680-685.
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Table 1. Effect of antigen and denaturant (7M GdnHCl, 100 mM DTT) on TAMRA fluorescence.
* Recovery ratio = A / (B / 1.1) × 100
λmax
Normalized intensity at λmax
PBST
+ BGP-C7
PBST
+Denaturant
(A)
+ BGP-C7
(B)
+ Denaturant
Recovery
ratio (%)*
5-TAMRA
573
580
1.0
1.1
-
w.t.
580
585
5.0
5.5
100
W33HF
580
585
3.8
4.2
100
W36HF
580
585
4.0
4.6
96
W47HF
580
585
2.2
2.6
93
W103HF
580
585
3.0
3.3
100
W35LF
580
585
2.4
3.0
88
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Table 2. Fluorescence lifetimes τi, and corresponding amplitudes, ai, of TAMRA labeled anti-
BGP scFv in the absence and presence of 1 µM BGP-C7 and denaturant 7M GdnHCl and 100
mM dithiothreitol.
τ1(ns) / a1
τ2(ns) / a2
no BGP-C7
1.20 / 0.48
3.92 / 0.52
+ BGP-C7
1.46 / 0.21
3.75 / 0.79
Denaturant
3.14 / 1.00
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Table 3. Locations of Trp residues in the scFvs used. Numbered according to Kabat15.
Residue number
CDRH1
CDRH3
CDRL3
H33
H34
H35
H36
…
H47
…
H95
…
H103
…
L35
…
L47
…
L91
L92
…
L94
αBGP
W
I
H
W
W
S
W
W
L
T
T
V
αBisphenol A
Y
Y
W
W
W
V
W
W
I
S
W
I
αHEL
Y
W
S
W
Y
W
W
W
L
S
N
W
αBSA/HSA
A
M
A
W
W
S
W
W
L
A
D
S
αEstradiol
T
I
H
W
W
Y
W
W
W
W
S
Y
αMorphine
W
I
E
W
W
W
W
W
L
W
Y
N
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Figure Legends!
Figure 1. Synthesis of scFv containing TAMRA at the N-terminal region. (A) Illustration of the
incorporation of TAMRA-C6-AF into scFv in response to a UAG codon in a cell-free translation
system. (B) Fluorescence image of SDS-PAGE for the expression of anti-BGP scFv containing
TAMRA-C6-AF. Fluorescence of TAMRA shown in red was detected with excitation at 532nm
and emission at 580 nm. Fluorescent marker shown in green was detected with excitation at 488
nm and emission at 520 nm. (C) Western blot analysis using an anti-His-tag antibody.
Figure 2. Antigen-dependent fluorescence enhancement of TAMRA-labeled anti-BGP scFv. (A)
Fluorescence spectra of TAMRA-scFv with excitation at 550 nm in the absence and presence of
BGP-C7 peptide. (B) Titration curve of the fluorescence intensity at 580 nm. The intensities are
relative values with respect to that in the absence of BGP-C7 peptide. (C) Fluorescence imaging
of TAMRA-scFv on a microplate in the presence of BGP-C7 peptide with excitation at 532 nm
and emission at 580 nm. (D) Titration curves of TAMRA-scFv for BGP-C10 and BGP-C10dV
peptides.
Figure 3. (A) The model of TAMRA-scFv. Structure for scFv was built using WAM antibody
modeling server (http://antibody.bath.ac.uk) with CONGEN sidechain building and Accessibility
profile screen methods. Trp residues, Trp33H, Trp36H, Trp47H and Trp103H and Trp35L are
colored green. (B) Titration curves of the Trp-to-Phe mutants. Fluorescence intensities are shown
as relative values with respect to those in the absence of the antigen. (C) Normalized titration
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curves.
Figure 4. (A) Schematic structure of scFv containing flexible peptide linkers. (B) Titration
curves of TAMRA-labeled anti-BGP scFvs with and without peptide linkers with excitation at
550 nm. Fluorescence intensities are relative values with respect to those in the absence of the
antigen. (C) Titration curves of TAMRA-labeled anti-bisphenol A scFvs with and without
peptide linkers with excitation at 550 nm. Fluorescence intensities are relative values with respect
to those in the absence of the antigen.
Figure 5. Titration curves of TAMRA-labeled scFvs. Fluorescence intensities are relative values
with respect to those in the absence of the antigen. (A) HyHEL-10 for HEL, (B) 29IJ6 for BSA
and HSA, (C) ES1-11 for estradiol. (D) Microplate-based imaging assay for BGP-C7 peptide
either in 50% human plasma in PBST or in PBST buffer performed with TAMRA-labeled anti-
BGP scFv. The TAMRA-scFv for A-C contain (Gly3Ser)2 linker, while that for D contains no
linker.
Figure 6. (A) Titration curves of TAMRA-labeled anti-morphine scFv for morphine and related
drugs. Fluorescence intensities are relative values with respect to those in the absence of the
antigen. (B) Molecular structure of the drugs with its molecular weight.
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ProX tag VH VL Linker
+
His tag
UAG
HN
NH
O
O
H2N
OO
O
N
N+
O
O-
p-(TAMRA-aminocaproyl)
-aminophenylalanine
(TAMRA-C6-AF)
AUC
(Gly4Ser)3
Linker
Cell-free
translation
TAMRA-scFv
A
B C
M
32.5
47.5
25
16.5
(kDa)
w.t.-scFv
no tRNA
TAMRA-scFv
40
32
21
(kDa)
M
w.t.-scFv
no tRNA
TAMRA-scFv
Figure 1
O
N
N+
O
-O
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0
1 × 10-9
3 × 10-9
1 × 10-8
3 × 10-8
1 × 10-7
1 × 10-6
A
C
B
Wavelength (nm)
0
1
2
3
4
5
6
565 610 655 700
[BGP-C7]
0 M
1 × 10-9 M
3 × 10-9 M
1 × 10-8 M
3 × 10-8 M
1 × 10-7 M
1 × 10-6 M
Normalized FI
BGP-C7 (M)
0
1
2
3
4
5
6 BGP-C10
BGP-C10dV
Peptide (M)
0 10-9 10-8 10-7 10-6
Normalized FI
D
Figure 2
0
1
2
3
4
5
6
Antigen (M)
0 10-9 10-8 10-7 10-6
Normalized FI
10-
5
BGP-C7
Intact BGP
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B C
A
0
1
2
3
4
5
6 w.t.
W33HF
W36HF
W47HF
W103HF
W35LF
BGP-C7 (M)
0 10-9 10-8 10-6
10-7 10-5 10-4
Normalized FI
0
20
40
60
80
100
BGP-C7 (M)
w.t.
W33HF
W36HF
W47HF
W103HF
W35LF
0 10-9 10-8 10-6
10-7 10-5 10-4
Response (%)
Antigen
Antibody VH VL
Trp103H
Fluoresce
Trp33H
Trp47H
Trp36H Trp35L
Figure 3
Quenched
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GGGSGGGSGGGS
scFv
ProX tag His tag
No linker
G3S(1)
G3S(2)
G3S(3)
G3S(5)
GGGSGGGS
GGGS
A B
0
1
2
3
4
5
6
BGP-C7 (M)
No linker
G3S(1)
G3S(2)
G3S(3)
0 10-9 10-8 10-7 10-6
Normalized FI
C
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Normalized FI
10-9
Bisphenol A (M)
0 10-8 10-7 10-6
No linker
G3S(2)
G3S(3)
G3S(5)
GGGSGGGSGGGSGGGSGGGS
Figure 4
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0.9
1.0
1.1
1.2
1.3
1.4
1.5
HEL (M)
10-9 10-8 10-7 10-6
0
Normalized FI
HEL
0
1
2
3
4
5
6
7
Estradiol (M)
Normalized FI
10-3
10-4
10-5
10-6
10-7
10-8
10-9
0
Estradiol
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
HSA
BSA
Antigen (M)
10-8 10-7 10-6 10-5 10-4
0
Normalized FI
A
C
B
D
PBST
Normalized FI
1
2
3
4
5
6
7
0
BGP-C7 (M)
0 10-9 10-8 10-7 10-6
50% plasma
Figure 5
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Figure 6
B
A
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
Drug (M)
0 10-9 10-8 10-6
10-7 10-5
Morphine
Heroin
Codeine
Naloxone
Cocaine
Ketamine
dMAMP
Normalized FI
Morphine (ng/mL)
0 0.29 2.9 290
29 2,900
HO
O
N
H
H
HO
CH3
Morphine
(MW: 285.4)
O
O
N
H
H
O
CH3
H3C
O
H3C
O
Heroin
(MW: 369.4)
Codeine
(MW: 313.4)
HO
O
N
O
OH
Naloxone
(MW: 327.4)
O
O
N
O
O
Cocaine
(MW: 303.4)
O
H
N
Cl
Ketamine
(MW: 237.7)
HN
Methamphetamine
(dMAMP)
(MW: 149.2)
H3CO
O
N
H
H
HO
CH3
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TOC graphic
Antigen
Antibody VH VL
Trp103H
Fluoresce
Trp33H
Trp47H
Trp36H Trp35L
Quenched
Quenchbody
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