- Research article
- Open Access
Specific detection of foot-and-mouth disease serotype Asia 1 virus by carboxyl-magnetic beads conjugated with single-domain antibody
© Yang et al. 2015
Received: 1 April 2015
Accepted: 31 August 2015
Published: 15 September 2015
Immunomagnetic nanobead (IMNB) labeled with specific antibody, has been demonstrated to be useful for the capturing and detection of viruses.
In this study, we developed an imunomagnetic bead based on carboxyl-magnetic beads (MNB) labeled with a single-domain antibody (sdAb) for capturing foot-and-mouth disease (FMD) Asia 1 virus. After magnetic separation, complexes of MNB-sdAb-virus were detected with either a sandwich ELISA or QDs-C5 probe under a fluorescence microscope, and the complexes were used as templates for extraction of total RNA for amplification of the VP1 or 3D gene fragments using RT-PCR and real-time RT-PCR. The Asia 1 VLPs were efficiently captured through IMNB with a high binding rate of 5.09 μg of antigen/μl of bead suspension. Moreover, this method has been successfully used to capture Asia 1 antigen in synthetic samples.
Ultimately, a specific and highly sensitive capture FMDV Asia 1 tool has been established that has the potential to enhance the sensitivity and reliability when diagnosing FMDV Asia 1.
Foot-and-mouth disease (FMD) is one of most contagious diseases of cloven-hoofed animals including cattle, pigs, sheep, buffalo, and approximately 70 wildlife species. The disease has been identified worldwide where livestock are raised. In the last 20 years, there have been massive outbreaks of FMD in countries formerly free of the disease, such as in Taiwan in 1997 , and in the United Kingdom in 2001 . Every outbreak of disease causes enormous economic loss and significant increases in public awareness. The causative agent is FMD virus (FMDV), which consists of a single stranded positive-sense RNA genome, encoding a viral polyprotein, and is a member of the genus Aphthovirus within the family Picornavirvidae. FMDV exists in seven different serologically distinct, serotypes: O, A, C, southern African Territories 1, 2 and 3 (SAT1-3), and Asia 1. Asia 1 virus initially occurred in 1954 in Okara, Punjab, Paskistan in Asia [3, 4]. FMD Asia 1 virus is its own unique serotype, and outbreaks due to Asia 1 have been reported sporadically in the past few decades. Analyses of Asia 1 indicate that some strains have been spread across large distances between countries in Asia within a short time [5–7].
Conventional technologies, such as the virus neutralization test (VNT) , antigen enzyme linked immunosorbent assays (ELISAs) [9–11] and reverse transcription polymerase chain reaction (RT-PCR) [12, 13] have markedly improved the detection of FMDV antigen. However, there still exists a general need for sensitive, reliable, and fast specific viral pathogen diagnosis, isolation and identification of the different serotypes of the viruses involved in FMD outbreaks. This is crucial to the prevention and control the spread of FMD, and for minimizing the serious economic consequences that arise from an outbreak at the beginning of the pandemic. In our previous work, we screened and defined a panel of single-domain antibodies (sdAbs) against FMD Asia 1 virus, and constructed the specific probe based on conjugation of sdAb with CdSe/ZnS Quantum Dots (QDs) which were used to trace and image the subcellular location of Asia 1 virus in BHK-21 cells . Herein, we extend the application of sdAb in FMDV diagnostic research. Specific Asia 1 virus sdAbs were conjugated onto the surface of carboxyl superparamagnetic beads (MNB) to form immunomagnetic beads (IMNBs) to enable them to effectively capture Asia 1 virus, forming the complexes of beads-sdAb-virus which were easy to specifically and rapidly detect using ELISA and RT-PCR. The bead- sdAb-virus complex was also labeled with QDs to measure the stable fluorescence when binding the target virus. These advantages make this a novel assay that can be used for the timely detection of FMD Asia 1 virus with high sensitivity.
Results and discussion
A critical step is rapid and accurate detection, identification, and isolation of a target pathogen from clinical samples when there is an outbreak of disease. Magnetic beads are a powerful tool with unique advantages, such as controlled surfaces, flexible functionalization, convenience to be manipulated due to their magnetic property, and large surface-to-volume ratios where they can be widely applied to the development of analysis methods. Superparamagnetic beads conjugated with antigens and antibodies are commonly utilized in immunoassays for the enhancement of pathogen detection and separation due to their efficient enrichment and separation capability [15–18].
Detection of the FMDV samples using IMNB
Background of Samples
Asia 1 Results
synthetic sample 1
synthetic sample 2
synthetic sample 3
To determine the sensitivity of IMNB, it was directly conjugated with the labeled horseradish peroxidase (HRP) sdAb-C6 (HRP-C6. The HRP-C6 was used as a second antibody to monitor the absorbance values in the sandwich ELISA. The absorbance values (OD450) reached a stable plateau after 35 min incubation with different concentrations of viruses from 10−1 to 10−6 TCID50/mL, and the absorbance values for the negative control remained unchanged (Additional file 1: Figure S1).
Some situations that may lead to the nonspecific adsorption of IMNBs when it alone was used to detect the target virus in mixtures, include natural phenomena such as hydrophobic and electrostatic interactions in this polydispersity synthetic system. In response to this, the pH and salt concentration of the reaction buffer were optimized to reduce the nonspecific background of MNBs, and to enhance the sensitivity. We also found that the largest sample to positive (S/P) ratio was obtained in PBS (0.1 mol/L, pH 7.4).
We found the ability to capture antigen of IMNB could be further reconfirmed by calculating the change of concentration of FMD Asia 1 virus VLP antigens after incubation. The P1-2A precursor is processed by viral protease 3C to produce the structural proteins VP0, VP1, and VP3 which then self-assemble to form icosahedral, empty capsid particles. . The recombinant FMDV VLP contains three structural proteins, VP0, VP1 and VP3 in vivo when observed by SDS-PAGE . We calculated that 1.0 μg of MNB (approximately 1.2 × 108 beads) are able to bind 5.09 μg target antigen. Additionally, our capture efficiency assay demonstrated that the antigen complex could be dissociated by boiling, and FMD VLPs were lysed into VP0 and (~32 kDa) and VP1/VP3 (~24 kDa) (Additional file 2: Figure S2).
These results suggest that MNBs have a specific capture capacity of FMD Asia 1 virus, while it should be noted that future work towards the translation of this method into the field application must take into account the evaluation of a large volume of actual clinical samples.
In summary, we have successfully used a specific sdAb conjugated with superparamagnetic beads forming IMNB for the rapid detection of FMD Asia 1 virus. In this method, the IMNB has the ability to efficiently capture the FMD Asia 1 virus to use in a MNB-virus-polyclonal antibody sandwich ELISA, in general RT-PCR and real-time RT-PCR. It was applied to synthetic samples of different serotypes to validate its specificity. Moreover, this method may well enhance the sensitivity and reliability when detecting FMDV Asia 1.
FMD Asia 1 virus strain (Genbank: EF149009), sera, BHK-21 cell, real-time RT-PCR, general RT-PCR and ELISA kit used in this research were obtained from OIE/CHINA National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, CAAS. The sdAbs of clone 5 (C5) and 6 (C6) anti-FMD Asia 1 virus VP1 protein were expressed in E.coli and purified using Ni-NTA agarose . Carboxyl-adembeads super-paramagnetic beads (200 nm) were purchased from Ademtech SA (Pessac, France), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Sigma-Aldrich (St.louis, MO, USA), QDs (505 nm, green) were obtained from Wuhan Jiayuan Quantum Dots Co.Ltd. (Wuhan, China) which were conjugated with sdAb-C5 to form QDs-sdAb clone 5 (QDs-C5).
Fabrication of immunomagnetic Beads
According to the product instructions, the carboxyl-adembeads (33.33 μL/mg) were washed 4 times with 0.01 M phosphate buffer solution (PBS, pH 6.8), then monodispersed in 600 μL of 0.01 M PBS. The washed beads were activated in 100 mM EDC and 100 mM NHS in 400 μL of 0.01 M PBS for 30 min at 37 °C with gentle shaking. Then, they were washed with 0.01 M PBS (pH 7.4) 3 times to remove the excess reagents, and dispersed in 800 μL 0.01 M PBS. The activated beads were mixed with 150 μg of sdAb-C6 antibody in 1 mL 0.01 M PBS at room temperature for 7 h. Afterward, the immunomagnetic bead-C6 (IN-C6) was washed by 0.01 M PBS 5 times, and they were blocked with 1 mL 3 % BSA for 30 min at 37 °C. Finally, the functional beads were stored in 0.01 M PBS with 1 % BSA (m/v) and 0.05 % NaN3 at 4 °C for use.
Detection of FMD Asia 1 virus using ELISA and general RT-PCR
The titer of Asia 1 virus was quantified in BHK-21 cell by 50 % tissue culture infective dose (TCID50). Three mg/mL(theoretically calculated value was approximately 3 × 108 beads) and 1 mL inactivated virus samples of 106 TCID50/mL Asia 1 were simultaneously mixed in 1 mL of PBS (0.1 mol/mL, pH 7.4) for a 1 h incubation at 37 °C with gentle shaking. The bead-virus complexes were separated by a magnetic scaffold to remove the suspension and washed 3 times using 0.05 % tween-20 in PBS, then resuspended in 300 μL PBS. One hundred microliter complexes were detected with a sandwich ELISA. The complexes were added to 100 μL anti-Asia 1 guinea pig serum (1:1000) for 45 min incubation at 37 °C. After washing 3 times, 100 μL of anti-guinea pig IgG-HRP at a dilution of 1:100 was added and incubated for 45 min at 37 °C. After an additional washing step, TMB substrate was mixed with complexes for 10 min at 37 °C, and the reaction was stopped with 2.0 M sulfuric acid. The optical absorbance was measured at 450 nm. Another 200 μL of complexes of bead-virus were used as the template to extract the total RNA for the general RT-PCR to amplify the VP1 fragment using the a pair of primers, Vp1F:5′-actaccaccactggcgag-3′ and Vp1R:5′- gccggttgttcactctgcg-3′. After optimizing the reacting real-time RT-PCR efficiency using the 10-fold serially diluted 106 TCID50/mL culture virus at 100, and from10−1 to 10−6 in PBS (0.1 mol/mL, p H 7.4), 200 μL and 600 μL diluent were transferred to new tubes, respectively, the former as a template to extract RNA and detect the 3D gene which was for the development of a standard curve by real-time RT-PCR, and the latter mixed with MNBs to concentrate target virus according to the process described above. The total RNA was extracted from bead-virus complexes of 600 μL sample and virus sample of 200 μL which was used as the template in the reaction of the real-time RT-PCR and general RT-PCR, respectively. The negative control was PBS buffer without target RNA in each RT-PCR assay.
Characterizations of immunomagnetic beads
The co-localization analysis of bead-virus complexes was performed with interaction with QDs-C5 (505 nm, green). 1 mL of Asia 1 inactivated virus sample (106 TCID50/mL) was mixed with 20 μL of 3 mg/mL MNBs in PBS (pH 7.4) for 45 min at 37 °C. After washing by magnetic separation, the complexes were reacted with QDs-C5 for 45 min at 37 °C with gentle shaking. After washing, the immune complexes of beads-virus-QDs-C5 were observed by a confocal fluorescence microscope (Leica, TCS SP8). The transmission electron microscopy (TEM) image of bead-virus complexes was observed by a JEM-1230 electron microscope.
All the concentrations of the beads, the hydrated particle size of the NBs, the MNB-virus complexes, and the MNB-virus-polyclonal Ab complexes, were diluted 1:100 (approximately 108 beads/mL) in 0.5 mL of PBS (0.1 mol/mL, pH 7.4) according to the manufacturer’s directions (ZetasizerNano ZS90, Malvern Instruments), and were measured with dynamic light scattering.
Capture efficiency assay
The recombinant Asia 1 VLPs at a concentration of 2.775 mg/mL in 200 μL of PBS (pH 7.4) were incubated with 50 μL of 3 mg/mL MNBs at 4 °C for overnight. After removing the supernatant under magnet separation, the beads-virus complexes were washed 3 times with PBS, added to 19 μL of PBS and 1 μL of 4× loading buffer, and processed through 12 % SDS-PAGE gel. The capture efficiency was quantified and calculated by measurement of absorbance with a NanoDrop 2000 spectrophotometer ( Thermo, Rockford, IL, USA ).
Detection of the FMDV samples using IMNB
Tested samples or cell-culture-adapted strains of FMDV Asia 1/YNBS/58 (GenBank: AY390432), Asia 1/HNK/CHA/05 (GenBank: EF149010), Asia 1/HeB/China/1/2005 (GenBank: EF187274), Asia 1/HN/2006 (GenBank: KC412634), O/China/5/99(GenBank:HQ009509), A/GS/LX/62 (GenBank:AJ131666) and three mixtures (synthetic sample 1: Asia 1/YNBS/58 and O/China/5/99; synthetic sample 2: Asia 1/YNBS/58 and A/GS/LX/62; synthetic sample 3: O/China/5/99 and A/GS/LX/62) were used to mimic real samples, which were provided by OIE/CHINA National Foot and Mouth Disease Reference Laboratory, which were inactivated using binaryethyleneimine to evaluate the diagnostic sensitivity and specificity of IMNB.
This work was supported by the China Agriculture Research System (CARS-39) and Basic Research Project (no.2013FY113300). We would like to thank assistant researcher Zhenli Gong of the instrument center of our institute for her professional taking photos by confocal fluorescence microscope.
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