Research Article (Open access) |
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SSR Inst. Int. J. Life Sci., 6(2):
2520-2527,
March 2020
Application of
Antibody-Conjugated CdSe/MSA Quantum Dots on Immunohistochemistry
Hong-Diep
Thi Tran1,2, Khanh-Thien Le11,2, Thuy-Dung
Mai-Hoang1,2, Thuoc Linh Tran1,2, Hieu
Tran-Van1,2,*
1Department of Molecular and Environmental
Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City,
Vietnam
2Vietnam National University, Ho Chi Minh City, Vietnam
*Address for
Correspondence: Dr. Hieu Tran-Van, Associate Professor, Department
of Molecular and Environmental Biotechnology, University of Science, Ho Chi
Minh City, Vietnam
E-mail: tvhieu@hcmus.edu.vn;
Fax Number: +842838350096
ABSTRACT-
Background- Quantum dots (QDs) are
recently conjugated to antibody for using in biological labeling applications.
In previous studies, we developed CdSe/MSA QDs, which were coated with protein
A/G (pA/G) for antibody conjugation, and evaluated their cell staining
application. Here, we expanded their applications into immunohistochemistry
(IHC) by investigating their storage time by accelerated heat aging method, and
comparing them with FITC in sample labeling.
Methods-
Cytokeratin 6A (KRT6A) in animal skin tissue section
were stained by pA/G-coated QDs conjugated to anti-KRT6A antibody and TF-1
human erythroleukemia cells were stained by pA/G-coated QDs conjugated to
anti-CD34 antibody.
Results-
The
results indicated that our pA/G-coated QDs effectively stained KRT6A in
epidermis of skin tissue section when they were probed with specific antibody.
Besides, pA/G-coated QDs still maintained their labeling ability in cell
staining and IHC formats after 12-month storage at 4oC. In
comparison with FITC (relatively almost the same emission fluorophore), our QDs showed a significantly stronger fluorescent signal.
Conclusion- In conclusion, these
results indicated that our pA/G-coated QDs were potentially fluorescent nanomaterials
and could be applied in a wide range of biological labeling applications.
Key-words- Biological labeling, Cell staining, Fluorescent nanomaterials,
Immunohistochemistry, Quantum dots, QDs
INTRODUCTION- Quantum dots
have been recently developed as new fluorescent nanomaterials for biological
labeling applications [1,2]. These nanomaterials have unique optical
properties such as photo stability, high fluorescence intensity, and low level
of fluorescence spectral overlap [3-5]. In biological labeling, QDs
are commonly conjugated to antibody in order to direct them to antigen [6].
Among the many strategies for antibody conjugation to nanoparticles, we exploited protein
A/G (pA/G) and coated it onto QDs’ surface due to its high affinity with the
antibody Fc region [7-10]. The advantage of this method is that QDs
are only conjugated to antibody Fc region so antibody Fab region always turns
outwards from QDs’ surface and antigen binding ability is maintained [11,12].
In previous studies, we reported one-step synthesis of pA/G-coated
CdSe/MSA QDs and evaluated their cell staining ability when they were
conjugated to antibody. The results indicated that our QDs efficiently stained
cells when they were probed with specific antibody [13]. For more
biological labeling applications, in this study, we applied our QDs in IHC.
Besides, we performed a comparison between the same emission fluorophore FITC
and our QDs and investigated their storage time by accelerated heat aging
method [14]. QDs as well as other nanoparticles are commonly stored
at 4oC. Therefore, we identified if the labeling ability of our
pA/G-coated QDs would be preserved after 6, 9, and 12 months at 4oC.
However, instead of keeping pA/G-coated QDs undergoing a long time at 4oC,
we accelerated their aging process by incubating them at 37oC to
save time. pA/G-coated QDs after aging were evaluated their labeling ability in
cell staining and IHC.
MATERIALS AND METHODS- All experiments of this study were performed from
October, 2019 to January, 2020 at the Department of Molecular and Environmental
Biotechnology, University of Science, Ho Chi Minh City, Vietnam. CdSe/MSA QDs were provided by the Department of
Applied Physics, Faculty of Physics and Engineering Physics, University of
Science, Ho Chi Minh City, Vietnam.
Protein A/G was
purchased from Bio Basic. Anti-human CD34 mouse monoclonal antibody was
purchased from BioLegend. Anti-cytokeratin 6A (KRT6A) rabbit polyclonal
antibody was purchased from Proteintech. Goat anti-mouse and goat anti-rabbit
antibodies conjugated FITC were purchased from Abcam.
Murine skin was collected from sacrificed mice in
educational courses in University of Science, Vietnam National University Ho
Chi Minh City, Vietnam. IHC using QDs was performed with paraffin-embedded
murine skin tissue sections. The tissue sections were incubated with BSA 10%
dissolved in TBS, 0.2% Triton X-100 overnight at 4oC. 50 μl of
pA/G-coated QDs was conjugated to 400 ng of anti-KRT6A rabbit polyclonal
antibody, spotted on the tissue section and incubated for 30 minutes at room
temperature. Then, the tissue section was washed with TBS 0.5% Triton X-100,
0.2% Tween 20 for 3 times, 20 minutes per each. Finally, mounting medium was
spotted on the tissue section and it was observed under fluorescence
microscope. For cell staining, 50 μl of pA/G-coated QDs was conjugated to
1 μg of anti-human CD34 mouse monoclonal antibody. TF-1 human
erythroleukemia cells were harvested from culture medium. Every 2x105 cells were re-suspended in 400
μl of RPMI 1640 medium added 10% FBS and 100 μl of
antibody-conjugated QDs. Finally, the tube was continuously rotated on
end-over-end rotator for 1 hour at room temperature and then centrifuged. The
medium was discarded and the cells were re-suspended in 20 μl RPMI 1640
medium added 10% FBS, spotted on lame and observed under fluorescence
microscope. Likewise, cell staining and IHC using FITC-conjugated secondary
antibody were similar to using QDs, with the same amount of primary antibody
conjugated to QDs and FITC-conjugated secondary antibody concentration of 2
μg/ml. For storage time investigation, pA/G-coated QDs was incubated for 18.6, 27.8, and 37.1 days at 37oC,
corresponding to 6, 9, and 12 months at 4oC, respectively.
The accelerated
aging time (AAT) was calculated by the formula below [15]-
AAT= RT/Q10(T2-T1)/10
Whereas,
RT- Desired real time; T2- Accelerated aging temperature; T1-
Ambient temperature; Q10- Aging factor, common value is 2.
After each time point,
QDs were conjugated to the relevant antibody for cell staining and IHC testing.
RESULTS
The
results of IHC testing showed that pA/G-coated QDs probed with anti-KRT6A antibody
effectively stained KRT6A in the epidermis (Fig. 1C), while QDs (Fig.
1A) and QDs (without pA/G) mixed with antibody (Fig. 1B) did not show signal in this region.
Fig. 1: KRT6A in murine skin
tissue sections stained by using QDs.
A: QDs; B: QDs + IgG; C: IgG-pA/G-QDs. (E: Epidermis; D: Dermis)
During the storage time investigation, pA/G-coated QDs showed their
stability after aging process in both of cell staining and IHC. In cell
staining with conjugation with anti-CD34 antibody, pA/G-coated QDs after 0
month of storage showed strong fluorescent signal that all TF-1 cells were
stained (Fig. 2B). After 6, 9, and 12 months, pA/G-coated QDs
showed reduction of their staining efficiency, but they still remained the
yield of about 10 stained cells in field containing about 15 cells (Fig.
2D, F, H). In IHC testing, staining
ability of pA/G-coated QDs was almost maintained after 0, 6, or 9 months (Fig.
3B, D, F) although it was noticeably
reduced after 12 months (Fig. 3H).
Fig. 2: TF-1 cells stained with anti-CD34 antibody
conjugated to pA/G-coated QDs after month 0 (A, B);
6 (C, D); 9 (E, F); and 12 (G, H). A, C, E, G:
pA/G-QDs; B, D, F, H: IgG-pA/G-QDs
Fig. 3: KRT6A in murine skin tissue sections stained
with anti-KRT6A antibody conjugated to pA/G-coated QDs after month 0 (A, B); 6
(C, D); 9 (E, F); and 12 (G, H). A, C, E, G: pA/G-QDs; B, D, F, H:
IgG-pA/G-QDs. (E: Epidermis; D: Dermis)
In comparison
with FITC, our pA/G-coated QDs showed significantly stronger fluorescent signals
in both of cell staining (Fig. 4)
and IHC (Fig. 5) than FITC.
Furthermore, goat anti-rabbit antibody conjugated FITC generated non-specific
background signal in the epidermis (Fig.
5A), while this phenomenon was not recorded when we used QDs (Fig. 5C).
Fig. 4: TF-1 cells stained with anti-CD34 antibody and goat
anti-mouse antibody conjugated FITC (A, B) and with pA/G-coated QDs conjugated
to anti-CD34 antibody (C, D). A: negative control without primary antibody; B:
positive control with primary antibody; C: pA/G-QDs; D: IgG-pA/G-QDs
Fig. 5: KRT6A
in murine skin tissue stained by anti-KRT6A antibody and goat anti-rabbit
antibdoy conjugated FITC (A, B) and with pA/G-coated QDs conjugated to
anti-KRT6A antibody (C, D). A:
negative control without primary antibody; B:
positive control with primary antibody; C:
pA/G-QDs; D: IgG-pA/G-QDs. (E:
Epidermis; D: Dermis)
DISCUSSION- In all IHC experiments using QDs of this study, it
was recorded that background signal was visually seen, especially in the dermis. This
phenomenon could be caused by interactions between metal surfaces of QDs and
protein in the tissue sections [16]. This problem could be solved by
increasing BSA concentration for blocking tissue sections, increasing
surfactant concentration and time in the washing step. However, the results of
IgG-pA/G-QDs were significantly different from the ones of negative control
groups that only IgG-pA/G-QDs generated fluorescent signal in the epidermis
which expressed cytokeratin in the skin.
In IHC, polyclonal
secondary antibody is more preferred than monoclonal secondary antibody because
polyclonal antibody can effectively amplify the signal. Nevertheless,
polyclonal antibody can generate non-specific signal [17]. Using
QDs, we not only limited the non-specific signal but also obtained strong
fluorescent signal which was not even seen by using
polyclonal secondary antibody.
Various studies developed
antibody-conjugated QDs for immunofluorescence. For example, Tsuboi et al. [4] coated their QDs
with immunoglobulin binding domain of protein G for antibody conjugation as the
way we exploited pA/G. They effectively stained KPL-4 cells by using their QDs
conjugated to anti-Her2 antibody. Interestingly, their QDs even labeled breast
tumor in mice implanted KPL-4 cells [4]. Wu et al. [18] were
successful in staining membrane and intracellular antigen by their
streptavidin-conjugated QDs incorporated with biotinylated antibodies.
Similarly, Kaul et al. [19] used their antibody-conjugated
QDs to stain mortalin in cancer cells. For IHC applications, Zhou et al.
[20] exploited QDs to stain cells in lymph node tissue specimens.
Prost et al. [21] used commercial QDs for kidney and liver
tissue sections staining. However, there was no research in the world which
determined the storage time of QDs before this study. Besides, our results of
comparison between our QDs and FITC were in line with other studies that QDs
generated stronger fluorescent signal than FITC did due to their high
photostability [22,23].
CONCLUSIONS-
In this study, we demonstrated that our pA/G-coated CdSe/MSA QDs
could be applied in IHC. In comparison with FITC, our ready-to-use QDs showed
stronger fluorescent signal in both of cell staining and IHC and more specific
signal than its
counterpart FITC did. Furthermore, our pA/G-coated QDs
were
stable after 12 months of storage at 4oC
by using the accelerated heat aging method. This study laid groundwork for
the application
of QDs in other biological labeling
applications.
Further studies are warranted to evaluate other applications of our QDs such as in Western Blot, ELISA, in-vivo
labeling or synthesize
QDs conjugated to DNA, ligands, or small peptides for antibody-independent
labeling applications.
ACKNOWLEDGEMENTS-
This research has supported by Ho Chi Minh City Department of Science and
Technology, Vietnam under the research code 105/2017/HD-SKHCN.
CONTRIBUTION OF AUTHORS
Research concept- Hieu Tran-Van
Research design-
Hong-Diep Thi Tran, Khanh-Thien Le, Hieu Tran-Van
Supervision- Hieu Tran-Van
Materials- Hieu Tran-Van, Thuoc Linh Tran
Data collection- Hong-Diep Thi Tran, Khanh-Thien Le, Thuy-Dung Mai-Hoang
Data analysis and
interpretation- Hong-Diep Thi
Tran, Khanh-Thien Le
Literature search- Hong-Diep Thi Tran, Khanh-Thien Le
Writing article- Hong-Diep Thi Tran, Khanh-Thien Le
Critical review- Hieu Tran-Van, Hong-Diep Thi Tran, Khanh-Thien Le
Article editing- Hieu Tran-Van, Hong-Diep Thi Tran, Khanh-Thien Le
Final approval-
Hieu
Tran-Van
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