Download Quantumdotsbased fluoroimmunoassay for the rapid and sensitive

Research Article
Received: 17 March 2009,
Revised: 25 May 2009,
Accepted: 20 June 2009,
Published online in Wiley Online Library: 21 October 2009
(wileyonlinelibrary.com) DOI 10.1002/bio.1167
Quantum-dots-based fluoroimmunoassay for
the rapid and sensitive detection of avian
influenza virus subtype H5N1
Liping Chena,b, Zonghai Shengc, Anding Zhanga,b, Xuebo Guoa, Jiakui Lib,
Heyou Hanc and Meilin Jina,b*
ABSTRACT: The continuous spread of highly pathogenic avian influenza virus (AIV) subtype H5N1 is threatening the poultry
industry and human health worldwide. Rapid and sensitive diagnostic methods are required for the H5N1 surveillance. In this
study, the fluorescent (FL) probe of CdTe quantum dots (QDs) was designed using covalently linked rabbit anti-AIV H5N1
antibody. Based on these QD–antibody conjugates, a novel sandwich FL-linked immunosorbent assay (sFLISA) was developed
for H5N1 viral antigen detection. The sFLISA allowed for H5N1 viral antigen determination in a linear range of 8.0 ¥ 10-3 to
5.1 ¥ 10-1 mg mL-1 with the limit of detection (LOD) of 1.5 ¥ 10-4 mg mL-1. In comparison with virus isolation for 103 clinic
samples, the sensitivity and specificity of sFLISA were found to be 93.6 and 91.1% respectively. The sFLISA supplied a novel
approach to rapid and sensitive detection of AIV subtype H5N1 and showed great potential for biological applications in
immunoassays. Copyright © 2009 John Wiley & Sons, Ltd.
Keywords: quantum dots; fluoroimmunoassay; avian influenza virus; subtype H5N1
Introduction
Luminescence 2010; 25: 419–423
* Correspondence to: Meilin Jin, Unit of Animal Infectious Diseases, State Key
Laboratory of Agricultural Microbiology, College of Veterinary Medicine,
Huazhong Agricultural University, Wuhan 430070, People’s Republic of
China. E-mail: [email protected]
a
Unit of Animal Infectious Diseases, State Key Laboratory of Agricultural
Microbiology, Huazhong Agricultural University, Wuhan 430070, People’s
Republic of China
b
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan
430070, People’s Republic of China
c
College of Science, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China
Copyright © 2009 John Wiley & Sons, Ltd.
419
Avian influenza viruses (AIV) are agents for fatal poultry diseases.
H5N1, a subtype of AIV, is of particular concern to people because
of its ability to infect humans and the potential for person-toperson transmission. In 1997, 18 people were infected by H5N1
in Hong Kong and six died. (1) The recurrence of H5N1 in Southeast Asia in 2004, which resulted in the culling of millions of
poultry birds and caused gigantic economic losses has attracted
greater attention. (2) It was a shock that 394 people in 15 countries had been infected with H5N1 by 14 January 2009, among
whom 248 had died. (3) H5N1 has not only endangered the
poultry industry, but also poses a potential danger to global
human health. Therefore, a rapid and sensitive detection assay
for the early diagnosis of H5N1 is required to lower the chance
of spread and prevent it from reaching epidemic levels.
With the development of biotechnology, several analytical
methods for the detection of H5N1 have been proposed, such as
virus isolation (VI), standard reverse transcription-PCR (RT-PCR),
real-time RT-PCR and enzyme-linked immunosorbent assay
(ELISA). (4–7) Among then, VI is the gold standard method for the
detection of H5N1. However, VI tends to be costly and slow.
ELISA, a common method used for diagnosing AIV, has a comparatively low sensitivity and specificity.
The emergence of quantum dots (QDs) has provided a novel
tool for effective diagnostic methods in immunoassay. (8,9) Compared with conventional organic dyes and fluorescent protein,
QDs have several excellent luminescent advantages, such as sizetunable light emission, superior signal brightness, resistance to
photobleaching, and simultaneous excitation of multiple fluores-
cent (FL) colors. (10,11) To date, immunoassays based on QDs
have been tested for detection of antigens in multiplex assays.
(12–15) However, until now there have been few reports on
quantitative detection for virus based on QDs. In this strategy,
QDs were conjugated with antibody, and used as an FL probe for
antigen using sandwich immunoassys. Inspired by these, we
attempted to developed an QDs-based-fluoroimmunoassay for
rapid, sensitive and specific for the detection of H5N1.
In the present work, a novel method for the determination of
H5N1 virus was reported basd on QD fluoroimmunoassay. Under
the optimal conditions, the limit of detection (LOD) of the proposed method reached 1.5 × 10−4 μg mL−1 of viral protein in virus
preparations, which was lower than commercial enzyme-linked
immunosorbent assay (ELISA). (4) Moreover, in comparison with
virus isolation, this method has a high efficiency in clinical
samples.
L. Chen et al.
Experimental
Reagents and materials
Thioglycolic acid (90%), Na2TeO3 (99%) and CdCl2.2.5H2O (99%)
were purchased from Sinopharm Chemical Reagent Co. Ltd.
NaBH4 (96%) was obtained from Shanghai Chemical Reagent
Co. Ltd. N-hydroxysulfosuccinimide (NHS) and 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) were purchased from
Sigma-Aldrich. A commercial AIV Ag ELISA test kit was obtained
from Wuhan Keqian Animal Biological Products Co. Ltd, Wuhan,
China. Monoclonal antibody (MAb) against the hemagglutinin
glycoprotein of H5N1 was made by ourselves. (16) AIV subtype
H5N1 was kept in the Unit of Animal Infectious Diseases, State
Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University. The negative control specimen was obtained
from a chicken in an AIV-free herd. Clinical samples were obtained
from naturally infected chickens. All other chemicals were of analytical reagent grade and used without further purification. Ultrapure water obtained from Milli-Q purification system was used in
all experiments.
Instrumentation
Ultraviolet–visible (UV–vis) absorption spectra were obtained on
a Thermo Nicolet Corporation Model Evolution 300 UV–visible
spectrometer. FL and resonance light scattering (RLS) spectra
were acquired on a Perkin-Elmer Model LS-55 luminescence
spectrometer. The TEM image of the QDs was performed on a
transmission electron microscope (JEM-1010FEF, Japan). The
concentration of protein was determined by a DU 800 UV–vis
spectrophotometer (Beckman Coulter, USA). FL imaging (at ×20)
was carried out using an IS70 inverted optical microscope
(Olympus, Japan) equipped with a charge-coupled device
camera (TOTA 500II, Japan) and a 100 W Hg excitation lamp. The
FL intensity was detected by a Biotec Corporation Model Synergy
HT multi-detection microplate reader.
Preparation and purification of positive control antigen
The positive control antigen was produced following a routine
procedure using chicken embryos. The AIV subtype H5N1 strain
A/chicken/HuBei/327/2004 (H5N1) stocks were propagated in
9-day-old embryonated specific-pathogen-free (SPF) chicken
eggs. Allantoic fluid was collected at 24–96 h post-inoculation if
HA titer was higher than 28. Collected allantoic fluid was purified
by centrifugation at 8000 rpm, for 10 min and then ultracentrifuged at 27,000 rpm for 2 h. The viral pellet was resuspended in
10 mM phosphate-buffered saline (PBS; pH 7.4) containing 0.05%
azide. The concentration of the viral protein was determined by
measuring its optical density at 280 nm (OD280) and OD260 and
calculating the content in accordance with the following formula:
milligrams of protein per milliliter = (1.45 × OD280) − (0.74 × OD260).
The purified virus stocks were aliquoted and stored at −70°C until
used.
Production and purified of polyclonal antibody angainst
AIV subtype H5N1
420
Two New Zealand White male rabbits weighing 1 kg were chosen
for preparing polyclonal antibody against AIV subtype H5N1. The
rabbits were first immunized with 0.1 mg purified H5N1 virus in
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an equal volume of Freund complete adjuvant, and immunized
again at the third and fourth week respectively with 5 mg of the
same antigen formulated in an equal volume of Freund incomplete adjuvant. Sera were collected 10 days after the last boost
and the IgG was purified by protein A affinity column. The effect
of purified IgG was evaluated by agarose-gel precipitation (AGP)
and UV–vis spectrum analysis.
Preparation of CdTe QDs and QDs-rabbit anti-AIV H5N1
antibody probe
CdTe QDs were synthesized in a method suggested by Bao (17)
with slight modifications. A 1.6 × 10−4 mol L−1 portion of CdCl2
was dissolved in 50 mL of deionized water in a 100 mL threeneck flask, and thioglycolic acid, trisodium citrate dehydrate,
Na2TeO3 and NaBH4 were added under nitrogen atmosphere. The
mol ratio of Cd2+, thioglycolic acid and Te2− was 3 : 9 : 1. When the
color of the solution was changed to yellow, the solution was
heated to the boil and kept under reflux for 6 h. The prepared
CdTe QDs were characterized by UV–vis absorption and FL
spectra.
QDs-rabbit anti-AIV H5N1 antibody conjugates were prepared
in the following way: the mixture containing 5 × 10−5 mol L−1
CdTe QDs, 0.05 mol L−1 EDC and 5.00 × 10−3 mol L−1 NHS in PBS
buffer (0.05 mol L−1, pH 7.4) was prepared and reacted at room
temperature for 30 min. The residual EDC and NHS were removed
by washing three times in PBS, and centrifuged at 5000 rpm each
for 5 min. After the last wash, the sediment was resuspended in
PBS buffer. With gentle agitation, the rabbit anti-AIV H5N1 antibody in PBS (the molar ratio of antibody to QDs was 1.2) was
added to the above suspension. The mixture was incubated
at room temperature for 2 h and stored at 4°C overnight. Finally,
the solution was centrifuged at 12,000 rpm for 30 min and the
supernatant was stored at 4°C.
Procedure for sFLISA
The principle of sFLISA is shown in Fig. 1. In the study, first, antiAIV H5 monoclonal antibody (MAb) was bound to the polystyrene microplates, then the bound MAb specifically captured
H5N1 viral antigen, and finally, the QDs-rabbit anti-AIV H5N1
antibody was selectively bound to the captured H5N1 viral
antigen. MAb was diluted in sodium carbonate buffer (0.05 mol
L−1, pH 9.6) at the optimal dilution and 100 μL was dispensed in
each well of the 96-well polystyrene microplates which were later
incubated overnight at 4°C. The microplates were washed three
times with washing buffer (0.05% Tween-20 in PBS), and the
excess binding sites were blocked by 5% skimmed milk (300 μL
per well) at 37°C for 1 h. The solution was discarded, and the wells
were washed with washing buffer, and then dried under vacuum
and stored at 4°C. Samples were added to the wells of microplates which were incubated at 37°C for 30 min. After thorough
washing, 100 μL of QDs-rabbit anti-AIV H5N1 antibody at the
optimal dilution were added to each well of the microplates,
which were then incubated at 37°C for another 30 min. After
being washed five times, the microplates were dried. The FL
signals of the microplates were detected by a multi-detection
microplate reader with excitation wavelength at 420 nm and
emission wavelength at 651 nm. The cutoff value of sFLISA was
calculated from the mean FL signal of 60 tissue or swab samples
known to be negative for AIV plus three times the standard
deviation (SD).
Copyright © 2009 John Wiley & Sons, Ltd.
Luminescence 2010; 25: 419–423
Quantum-dots-based fluoroimmunoassay
Figure 1. Schematic representation of the sandwich FL-linked immunosorbent assay (sFLISA) for the detection of AIV
H5N1. (a) Immobilized MAb. (b) MAb-antigen conjugates. (c) MAb–antigen–QD–antibody conjugates.
ELISA procedure
ELISA for detection of H5N1 subtype antigen was performed
according to the manufacturer’s instructions. Briefly, a series of
dilutions of purified H5N1 virus were added to the anti-NP
protein MAb-coated microplates and incubated at 37°C for
30 min. Then rabbit polyclonal IgG was added to each well, and
next the peroxidase-conjugated goat anti-rabbit IgG conjugate.
After thorough washing, tetramethylbenzidine substrate was
added. The reaction was stopped by the addition of 0.25% hydrofluoric acid. The plates were read at 630 nm with an ELISA reader.
VI procedure
Figure 2. UV–vis absorbance (a) and FL (b) spectra of CdTe QDs.
Isolation of influenza virus was performed by inoculating 9-dayembryonated chicken eggs with 0.2 mL tissue suspensions or
swab suspensions via the allantoic cavity. The eggs were incubated for 4 days and candled daily for viability; embryos that died
within 24 h of inoculation were discarded as nonspecific. Allantoic fluid collected 96 h later from dead and surviving embryos
was tested for HA activity. Samples that did not show hemagglutination were reinoculated for a second passage.
Results and discussion
Characterization of CdTe QDs and QDs-rabbit anti-AIV H5N1
antibody probe
Figure 3. TEM image of the CdTe QDs.
Luminescence 2010; 25: 419–423
Figure 4. The resonance light scattering (RLS) spectra of QDs-rabbit anti-AIV H5N1
antibody probe (a) and QDs-antibody probe in the presence of H5N1 viral antigen
(b).
was immobilized as negative sample was almost all dark. (Fig. 5B).
Both the RLS spectra and FL imagings indicated that the asprepared QDs were stably linked to the anti-HA polyclonal IgG,
and formed QDs-rabbit anti-AIV H5N1 antibody conjugates. (20)
Optimization of the sFLISA conditions
For optimization of the dilution times of the antibody (anti-AIV
H5N1 MAb and QDs-rabbit anti-AIV H5N1 antibody) used to
Copyright © 2009 John Wiley & Sons, Ltd.
View this article online at wileyonlinelibrary.com
421
Figure 2 shows UV–vis absorption and FL spectra of CdTe nanocrystals. It can be seen from curve (a) that the absorbance
maximum wavelength was at 556 nm. The average size of the
CdTe QDs was estimated to be 3.3 nm according to the method
reported by Yu et al. (18) Furthermore, the FL spectra of CdTe QDs
(curve b) exhibited a peak at 609 nm when excited at 390 nm.
The sharp peaks of UV–vis and FL spectra indicated that the
prepared CdTe QDs were nearly monodispersed and homogeneous. From the TEM image (see Fig. 3), it can be seen that the
average size of CdTe nanoparticles is about 3 nm and their size
distribution is relatively uniform.
In this research, CdTe QDs were linked to rabbit anti-AIV H5N1
antibody using the coupling reagents EDC and NHS. The QDsrabbit anti-AIV H5N1 antibody probe were characterized by RLS
spectra and FL imaging. As shown in Fig. 4, the RLS intensity of
QDs-rabbit anti-AIV H5N1 antibody was remarkably increased in
the presence of H5N1 viral antigen, which showed that larger
particles were formed based on the immunoreaction between
H5N1 viral antigen and the QDs-rabbit anti-AIV H5N1 antibody
probe. (19) Further evidence for the effect of QDs-rabbit anti-AIV
H5N1 antibody probe was also confirmed by FL imaging. Figure 5
showed two FL imagings from the sandwich conjugates bound
on microplates after sFLISA. The QDs-rabbit anti-AIV H5N1 antibody probe recognized the H5N1 viral antigen specially and
therefore led to extensive aggregation of the QDs and the
microplate well which was immobilized H5N1 viral antigen
showed red fluorescence (Fig. 5A). On the contrary, the well which
L. Chen et al.
Figure 5. FL imaging for the characterization of QDs-rabbit anti-AIV H5N1 antibody probe (examined by FL microscopy
under violet excitation at 20× magification). (A) FL imaging in the presence of H5N1 viral antigen; (B) FL imaging in the
presence of negative sample.
capture and detect antigen, a checkerboard titration was performed. All the steps of sFLISA were performed as above. During
optimization of those reagents, blocking buffer was used with
5% skimmed milk. A dilution of 1 : 3000 was selected for the
monoclonal antibody, thereby ensuring a slight excess of capture
antibody on the plates. A dilution of 1 : 1500 QD–rabbit anti-AIV
H5N1 antibody was selected to maximize the detection of captured antigen. Under the optimal conditions, the cut-off level of
the sFLISA (FL intensity = 20,000) was calculated as 3 standard
deviations above the mean FL value (FL intensity = 16530 + 3790)
obtained with 60 tissue and swab samples known to be negative
for H5N1.
Detection of H5N1 viral antigen
The purified positive antigen (H5N1 virus) (102.4 μg mL−1) was
serially diluted by PBS to different levels, and calibration curve
was constructed using the optimized sFLISA conditions. As
shown in Fig. 6, a good linear correlation between logarithm of
FL intensity and the logarithm of concentration of H5N1 viral
antigen was obtained in the range of 8.0 × 10−3 to 5.1 ×
10−1 μg mL−1, with a regression equation, log(FL) = 4.69869 +
0.14647 logC (where C is the concentration of H5N1 virus), R2 =
0.992. As the the logarithm of concentration of H5N1 viral antigen
increased, the logarithm of FL intensity increased between 8.0 ×
10−3 and 5.1 × 10−1 μg mL−1. The results demonstrate the possibility of detecting H5N1 viral antigen at very low concentrations.
The limit of detection
The limit of detection was obtained by determination of the positive sample (H5N1 virus). According to the above procedure of
sFLISA, H5N1 viral antigen was serially diluted in PBS and the limit
of detection was obtained. Results showed that the limit of
detection for sFLISA was approximately 1.5 × 10−4 μg mL−1 of viral
protein, which was very low compared with other diagnostic
methods.
Comparison of sFLISA with ELISA and VI
422
ELISA and VI have been recognized as standard methods for diagnosing AIV. To evaluate the diagnostic performance of the sFLISA,
it was necessary to compare sFLISA with the reference standards
(VI and ELISA).
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Figure 6. Linear relationship between logarithm of concentration of H5N1 viral
antigen and logarithm of FL intensity.
In present study, a commercially available ELISA was used as a
control method to evaluate the sensitivity of sFLISA. The purified
H5N1 virus was serially diluted in PBS and tested by sFLISA and
ELISA simultaneously. For ELISA, data were analyzed according to
the manufacturer’s instructions. Results showed that the limit of
detection of ELISA was 5 × 10−3 μg mL−1, while that of sFLISA was
1.5 × 10−4 μg mL−1. Compared with ELISA, sFLISA showed higher
sensitivity, which was extremely valuable for the early diagnosis
of H5N1.
In order to verify the application of the proposed method, 103
clinical chicken samples including hearts, livers, spleens, lungs,
kidneys, muscles, tracheas, brains and cloaca swabs obtained
from central China in 2005 were detected using the sFLISA and
VI. The diagnostic performance of sFLISA was compared with that
of VI, the current standard of AIV detection. As shown in Table 1,
44 samples were detected positive among all 103 samples in VI,
but five more samples were detected positive with sFLISA. We
named the 44 samples ture-positive (TP) and five as false-positive
(FP). Similarly, 51 samples were detected negative among 103
samples in VI, but three more samples were detected negative in
sFLISA. We called the 51 samples true-negative(TN), and three
false-negative (FN). According to the following fomular: sensitivity = TP × 100/(TP + FN); specificity = TN × 100/(TN + FP); accuracy
Copyright © 2009 John Wiley & Sons, Ltd.
Luminescence 2010; 25: 419–423
Quantum-dots-based fluoroimmunoassay
Table 1. Comparative results of the sFLISA and VI of 103 virus samples
VI
sFLISA
Positive
Negative
Total
Performance
Positive
Negative
Total
Sensitivity (%)
Specificity (%)
Accuracy (%)
FP rate (%)
FN rate (%)
44
3
47
5
51
56
49
54
103
93.6
91.1
92.2
8.9
6.4
Sensitivity = TP × 100/(TP + FN). Specificity = TN × 100/(TN + FP). Accuracy = (TP + TN) × 100/total. False-positive rate = FP × 100/
(FP + TN). False-negative rate = FN × 100/(TP + FN).
= (TP + TN) × 100/total; false-positive rate = FP × 100/(FP + TN);
false-negative rate = FN × 100/(TP + FN), we obtained the result
that, compared with VI, sFLISA showed high sensitivity (93.6%),
specificity (91.1%) and accuracy (92.2%), moderate false-positive
rate (8.9%) and moderate false-negative rate (6.4%). Compared
with VI, our sFLISA method provides remarkable advantages in
terms of reliability and in practical uses, such as high sensitivity
and specificity.
Conclusions
In conclusion, a novel method was developed for the detection of AIV subtype H5N1. The limit of detection was 1.5 ×
10−4 μg mL−1, which was lower than ELISA. When it was
applied to 103 clinic chicken samples (Table 1), the sFLISA
showed high sensitivity (93.6%) and specificity (91.1%), and the
agreement between sFLISA and VI was found to be 92.2%. The
proposed method can be used to detect other animal disease
biomarkers, and showed great potential for early diagnosis of
major animal diseases.
Acknowledgment
This study was supported by China National Basic Research
Program (China ‘973’ Program 2005CB523003), China National
Scientific and Technical Supporting Programs (no. 2006BAD06A11
and no. 2006BAD06A15).
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