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DOI: 10.1039/C7CC03043F
Chem. Comm.
COMMUNICATION
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on a
transilluminator and after Coomassie brilliant blue (CBB)-
staining, we confirmed that the fluorescent dye was efficiently
conjugated to the scFv through UV exposure, while no
labelling was observed without IBA nor UV exposure (Fig. 1D).
Based on the CBB-stained and fluorescent band densities of
these gels, the photocrosslinking yield (number of TAMRA dye
incorporated per scFv) was calculated as 0.379.
We mixed the anti-HSA antibody and IBA-C8-TAMRA and
incubated the mixture at room temperature for 1 h. After UV
light exposure, we denatured the sample by heating with the
addition of dithiothreitol and subjected it to SDS-PAGE. We
confirmed the fluorescence labelling using a transilluminator
and then stained the gel using CBB (Fig. 2B). The disulphide
bonds between the heavy and light chains as well as those
between the hinge regions of IgG should be reduced under
this condition to yield two bands of about 25 kDa and 50 kDa
at a molar ratio of approximately 1:1, which was confirmed
after CBB staining. The brightness of the upper band (heavy
chain) was higher than that of the lower band (light chain) on
the transilluminator, suggesting that more IBA-C8-TAMRA was
When we measured the fluorescence intensity of the Q-
body with the addition of various concentrations of BGP
peptide, the maximum fluorescent response was improved up
to 9-fold (Figs. 1E and F). This fluorescent response in the
presence of antigen was more than 2-fold higher than that of
our previous TAMRA-labelled scFv-type anti-BGP Q-body,
covalently conjugated to the VH side than the V side of the
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which was made using the Cys-labelling technique
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NBS. We measured the fluorescent intensities of the anti-HSA
Q-body in the presence of varied concentrations of HSA or the
same volumes of PBST. As a result, the fluorescent intensity
increased only in the presence of antigen, while the intensity
was unchanged or slightly decreased as expected owing to the
increased volume of the reaction mixture (Figs. S2A and B).
The fluorescence titration curve plotted from the emission
maxima of each spectrum clearly indicated an HSA-
concentration dependent increase in the fluorescence
intensity (Fig. 2C). The fluorescent response was similar to
that of a conventional anti-HSA Q-body that was site-
specifically TAMRA-labelled using an amber codon-based cell-
Although the reason for this difference in the fluorescent
response cannot be precisely determined, we assume two
possibilities as below. First, the location of NBS is closer to the
antigen binding site than the N-terminus of scFv, where Cys-
tag is appended. Second, the fluorescent response is
influenced by the distance between the antibody-binding
moiety and the fluorescence dye of the probe. We previously
constructed scFv-type anti-BGP Q-bodies with spacers of
three different lengths between maleimide, which binds to
the Cys-tag, and TAMRA, and compared the fluorescent
responses in the presence of both the target antigen and a
denaturing reagent. The highest response was observed for
the Q-bodies with the longer spacers (maleimide-C0-TAMRA <
maleimide-C2-TAMRA < maleimide-C5-TAMRA, where each
number after C indicates the number of the carbon atom
between maleimide and TAMRA). Although the method and
the point of labelling is different, since the spacer in this study
was C8 (IBA-C8-TAMRA), the dye might be able to go close
enough to interact with the internal Trp residues in the scFv
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free transcription-translation system . These results indicate
that the IBA-conjugated fluorescent dye was successfully
incorporated into both the scFv fragment and the full-length
antibody, and the quenching was removed in an antigen-
dependent manner.
Taken together, the results presented in this study
demonstrated the feasibility of a method involving the
specific crosslinking of an IBA-conjugated fluorescent dye to
the NBS of an antibody or antibody fragment for the one-step
(Fig. 1A), which would contribute to the efficient quenching
and antigen-dependent release of the dye. The EC50 and limit
of detection values of the Q-body were 116.8 ± 32.0 and 0.56
construction of a Q-body. The simple ‘stapling’ of a
fluorescent dye to the highly-conserved NBS is expected to
provide the potential for performing an ideal immunoassay
without numerous reaction steps.
±
0.20 nM, respectively, indicating a high sensitivity and
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practical utility of this Q-body as a sensor for BGP
.
Next, we applied the photochemical crosslinking strategy
to convert a full-sized antibody to a Q-body (Fig. 2A). Instead
of an anti-BGP antibody, which is not commercially available,
we used rabbit IgG against human serum albumin (HSA),
which exists at high concentration in plasma and is considered
This work was partly supported by Grants-in-Aid for
Scientific Research (No. 26889028 to HJJ, No. 15H04191 to
HU, and No. 26420793 to JD) from the Japan Society for the
Promotion of Science, and the Cooperative Research Program
of the Network Joint Research Center for Materials and
Devices, MEXT, Japan. We also thank Dr. Hiroyuki Ohashi in
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to be a biomarker for nutritive status and oxidative stress
.
Fig. 2 (A) Schematic representation of the process for constructing an IgG-type Q-body using photochemical crosslinking. (B) Coomassie brilliant blue-stained (left) and
fluorescence (right) images of SDS-PAGE for the Q-body. (C) HSA concentration-dependent fluorescent response of the Q-bodies. Error bars represent ± 1 SD (n = 3).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Jeong, Hee-Jin
