BACKGROUND
foot-and-mouth disease  is one of most contagious diseases of cloven-hoofed animals including cattle, pigs, sheep, buffalo, and approximately  <dig> 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  <dig>  <cit> , and in the united kingdom in  <dig>  <cit> .
every outbreak of disease causes enormous economic loss and significant increases in public awareness.
the causative agent is fmd virus , 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  <dig>   <dig> and  <dig> , and asia  <dig>  asia  <dig> virus initially occurred in  <dig> in okara, punjab, paskistan in asia  <cit> .
fmd asia  <dig> virus is its own unique serotype, and outbreaks due to asia  <dig> have been reported sporadically in the past few decades.
analyses of asia  <dig> indicate that some strains have been spread across large distances between countries in asia within a short time .
conventional technologies, such as the virus neutralization test   <cit> , antigen enzyme linked immunosorbent assays   and reverse transcription polymerase chain reaction   <cit>  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  against fmd asia  <dig> virus, and constructed the specific probe based on conjugation of sdab with cdse/zns quantum dots  which were used to trace and image the subcellular location of asia  <dig> virus in bhk- <dig> cells  <cit> .
herein, we extend the application of sdab in fmdv diagnostic research.
specific asia  <dig> virus sdabs were conjugated onto the surface of carboxyl superparamagnetic beads  to form immunomagnetic beads  to enable them to effectively capture asia  <dig> 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  <dig> virus with high sensitivity.
RESULTS
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 .
in this study, we developed a convenient mnb assay based on a sdab used to specifically capture, recognize, and enrich detection of fmd asia  <dig> virus .
to confirm successful conjugation of sdab onto the surface of the functionalized mnbs capable of binding fmd asia  <dig> virus, the polyclonal serum against asia  <dig> antigen can be used form a sandwich elisa with sdab coated on the surface of mnbs in the presence of asia  <dig> antigen, with the appearance of a hrp signal to indicate the successful conjugation of sdab with mnb forming the functionalized imnbs.
the reliability and specificity of the method were further carried out by rt-pcr.
this demonstrated that the complexes of virus homogeneously mixed with mnbs as all had corresponding vp <dig> gene fragments, which suggests that the influence of mnbs was insignificant on the extraction of total viral rna, and was amplified template in rt-pcr reaction.
the size of the 460 bp vp <dig> fragment of asia  <dig> virus was observed in the positive samples, while the amount of pcr products of complexes of beads-virus was higher than only the culture samples.
the tested samples included fmdv serotype asia  <dig>  a, o and were synthetic samples detected by rt-pcr after imnb capture.
results were shown in table  <dig>  the imnb has the ability to specifically bind fmdv asia  <dig>  moreover the type a and o samples were all negative in this study.
the results suggests that imnb may bind specifically to fmdv asia  <dig>  and well enhance sensitivity when diagnosing fmdv asia  <dig> using rt-pcr.fig.
<dig> principle for fmd asia  <dig> virus detection using sdab based on immunomagnetic separation.
carboxyl-adembead are functionalized with anti- fmdv asia  <dig> sdab forming the immunomagnetic beads, and used to capture fmdv asia  <dig> in solution.
the complexes of beads-sdab-virus are easily separated from solution using an ordinary magnet.
the isolated complexes may be detected by the antigen in both an elisa and rt-pcr, and can also be utilized in virus isolation and for purifying virus
p* and n* represent asia  <dig> positive and negative, respectively
the conventional rt-pcr and real-time rt-pcr have been widely used to specifically detect fmdv  <cit> .
the purpose of this assay was to estimate the binding target virus efficiency of mnbs by real-time rt-pcr and conventional rt-pcr.
in the first real-time rt-pcr assay, amplification of the 3d fragment, the taqman® amplification plot started in the  <dig> th cycle  which was as template with complexes of beads-virus, and comparison of the original virus as template with the amplification plot appeared in the 22nd cycle  in the second real-time rt-pcr assay.
the parallel conventional rt-pcr tests were performed to amplify the vp <dig> gene with the same samples.
the results demonstrated that the reaction of viruses mixed with the imnb all received vp <dig> fragments  , which suggested the brightness of dna strips of complexes were more than the original virus .
however, it is envisioned that the method of capture of fmd asia  <dig> virus with imnb will be used in conjunction with real-time or conventional rt-pcr methods to increase the concentration of target antigen in samples per unit volume in diagnostic process and enhance the sensitivity and reliability for fmdv diagnosis.fig.
<dig> serial dilution of primary fmd asia  <dig> virus rna in vitro was amplified to 3d fragment using real-time pcr and general rt-pcr amplifying vp <dig> fragment based on immunomagnetic to conjugate with sdab.
a fmd asia  <dig> virus mixed with imnbs at the concentration of 10− <dig> to 10− <dig> tcid50/ml; b fmd asia  <dig> virus was diluted at the concentration of 10− <dig> to 10− <dig> tcid50/ml  lane n, negative control using sterile pbs as template; lane p, positive control of primary fmd asia  <dig> virus  reacted with mnb; lanes 1– <dig>  asia  <dig> virus mixed with mnb at the concentration of 10− <dig> to 10− <dig> tcid50/ml, respectively; lane m, dl <dig> dna marker  <dig>   <dig>   <dig>   <dig>   <dig>  100 bp;  lane n, negative control using sterile pbs as template; lane p, positive control of primary fmd asia  <dig> virus ; lanes 1– <dig>  fmd asia  <dig> virus at the concentration of 10− <dig> to 10− <dig> tcid50/ml, respectively; lane m, dl2000
to determine the sensitivity of imnb, it was directly conjugated with the labeled horseradish peroxidase  sdab-c <dig>  reached a stable plateau after 35 min incubation with different concentrations of viruses from 10− <dig> to 10− <dig> tcid50/ml, and the absorbance values for the negative control remained unchanged .
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  ratio was obtained in pbs .
we found the ability to capture antigen of imnb could be further reconfirmed by calculating the change of concentration of fmd asia  <dig> virus vlp antigens after incubation.
the p1-2a precursor is processed by viral protease 3c to produce the structural proteins vp <dig>  vp <dig>  and vp <dig> which then self-assemble to form icosahedral, empty capsid particles.
<cit> .
the recombinant fmdv vlp contains three structural proteins, vp <dig>  vp <dig> and vp <dig> in vivo when observed by sds-page  <cit> .
we calculated that  <dig>  μg of mnb  are able to bind  <dig>  μg target antigen.
additionally, our capture efficiency assay demonstrated that the antigen complex could be dissociated by boiling, and fmd vlps were lysed into vp <dig> and  and vp1/vp <dig>  .
the capture of the viruses with the imnbs was observed by tem.
the blackish spots are the beads, while the protuberant spots  are characteristic of virion morphology around the beads, because the fmdv particle is too small, at approximately 25 ~ 30 nm in diameter  <cit>  compared with the beads at about 200 nm in diameter, which leads to the virus particles not being obvious in the image.
the evidence of binding virus was also suggested by the increase in the hydrated particle size of mnbs from 198 nm to 640 nm with dynamic light scatting  .
binding of the target virus to the functionalized imnbs could be easily realized by mixing with qds-c <dig> and co-localization analysis of beads, fmd asia  <dig> virus and fluorescent qds resulting in qds-labeled virions.
the functionalization of mnbs with sdab incubated with fmd asia  <dig> virus, were then reacted with qds-c <dig>  which produced the qds-labeled virions.
the labeled virions were evaluated by confocal fluorescence microscopy, and an overlap of functionalized mnbs between the bright field and fluorescent image  which indicated that functionalized mnbs had successfully captured fmd asia  <dig> virus was generated.
the number of molecules sdab conjugated to each mnb was estimated by the fluorescence intensity of the fluorescein-labeled vhh molecular conjugated to magnetic beads  <cit>  and was calculated about 900– <dig> protein molecular on a bead.
the amount of sdab molecule per bead showed sufficient binding antigen sites to the coupling of target virus to ensure the capture capacity of imnbs.
the combination of qds and imnbs offers researchers a new pathway to both separate and identify targets of interest via the use of a simple magnet, and is particularly advantageous in the detection and quantification of soluble biomarkers during which excess qd-antibody constructs must be removed for further analysis  <cit> .fig.
<dig> characterzaitons of imnbs by dynamic light scatting.
the hydrated particle sizes of beads , bead-virus complexes , and the bead-virus-polyclonal ab complexes
fig.
<dig> confocal microscopic images of fluorescent, mnbs, and complexes of bead-virus.
b the fluorescence of qds-c <dig> from the labeled target virus , a the beads in bright field image , c the merged image of complexes of beads-virus- indicating successful capture of target virus onto the mnb , d the aggregation of complexes of beads-virus-
these results suggest that mnbs have a specific capture capacity of fmd asia  <dig> 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.
CONCLUSIONS
in summary, we have successfully used a specific sdab conjugated with superparamagnetic beads forming imnb for the rapid detection of fmd asia  <dig> virus.
in this method, the imnb has the ability to efficiently capture the fmd asia  <dig> 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  <dig>

 foot-and-mouth disease is one of most contagious diseases of cloven-hoofed animals including cattle, pigs, sheep, buffalo, and approximately 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, and in the united kingdom in. every outbreak of disease causes enormous economic loss and significant increases in public awareness. the causative agent is fmd virus, 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 and, and asia asia virus initially occurred in in okara, punjab, paskistan in asia. fmd asia virus is its own unique serotype, and outbreaks due to asia have been reported sporadically in the past few decades. analyses of asia indicate that some strains have been spread across large distances between countries in asia within a short time. conventional technologies, such as the virus neutralization test, antigen enzyme linked immunosorbent assays and reverse transcription polymerase chain reaction 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 against fmd asia virus, and constructed the specific probe based on conjugation of sdab with cdse/zns quantum dots which were used to trace and image the subcellular location of asia virus in bhk- cells. herein, we extend the application of sdab in fmdv diagnostic research. specific asia virus sdabs were conjugated onto the surface of carboxyl superparamagnetic beads to form immunomagnetic beads to enable them to effectively capture asia 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 virus with high sensitivity.  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. in this study, we developed a convenient mnb assay based on a sdab used to specifically capture, recognize, and enrich detection of fmd asia virus. to confirm successful conjugation of sdab onto the surface of the functionalized mnbs capable of binding fmd asia virus, the polyclonal serum against asia antigen can be used form a sandwich elisa with sdab coated on the surface of mnbs in the presence of asia antigen, with the appearance of a hrp signal to indicate the successful conjugation of sdab with mnb forming the functionalized imnbs. the reliability and specificity of the method were further carried out by rt-pcr. this demonstrated that the complexes of virus homogeneously mixed with mnbs as all had corresponding vp gene fragments, which suggests that the influence of mnbs was insignificant on the extraction of total viral rna, and was amplified template in rt-pcr reaction. the size of the 460 bp vp fragment of asia virus was observed in the positive samples, while the amount of pcr products of complexes of beads-virus was higher than only the culture samples. the tested samples included fmdv serotype asia a, o and were synthetic samples detected by rt-pcr after imnb capture. results were shown in table  the imnb has the ability to specifically bind fmdv asia moreover the type a and o samples were all negative in this study. the results suggests that imnb may bind specifically to fmdv asia and well enhance sensitivity when diagnosing fmdv asia using rt-pcr.fig. principle for fmd asia virus detection using sdab based on immunomagnetic separation. carboxyl-adembead are functionalized with anti- fmdv asia sdab forming the immunomagnetic beads, and used to capture fmdv asia in solution. the complexes of beads-sdab-virus are easily separated from solution using an ordinary magnet. the isolated complexes may be detected by the antigen in both an elisa and rt-pcr, and can also be utilized in virus isolation and for purifying virus p* and n* represent asia positive and negative, respectively the conventional rt-pcr and real-time rt-pcr have been widely used to specifically detect fmdv. the purpose of this assay was to estimate the binding target virus efficiency of mnbs by real-time rt-pcr and conventional rt-pcr. in the first real-time rt-pcr assay, amplification of the 3d fragment, the taqman® amplification plot started in the th cycle which was as template with complexes of beads-virus, and comparison of the original virus as template with the amplification plot appeared in the 22nd cycle in the second real-time rt-pcr assay. the parallel conventional rt-pcr tests were performed to amplify the vp gene with the same samples. the results demonstrated that the reaction of viruses mixed with the imnb all received vp fragments, which suggested the brightness of dna strips of complexes were more than the original virus. however, it is envisioned that the method of capture of fmd asia virus with imnb will be used in conjunction with real-time or conventional rt-pcr methods to increase the concentration of target antigen in samples per unit volume in diagnostic process and enhance the sensitivity and reliability for fmdv diagnosis.fig. serial dilution of primary fmd asia virus rna in vitro was amplified to 3d fragment using real-time pcr and general rt-pcr amplifying vp fragment based on immunomagnetic to conjugate with sdab. a fmd asia virus mixed with imnbs at the concentration of 10− to 10− tcid50/ml; b fmd asia virus was diluted at the concentration of 10− to 10− tcid50/ml lane n, negative control using sterile pbs as template; lane p, positive control of primary fmd asia virus reacted with mnb; lanes 1– asia virus mixed with mnb at the concentration of 10− to 10− tcid50/ml, respectively; lane m, dl dna marker 100 bp; lane n, negative control using sterile pbs as template; lane p, positive control of primary fmd asia virus; lanes 1– fmd asia virus at the concentration of 10− to 10− tcid50/ml, respectively; lane m, dl2000 to determine the sensitivity of imnb, it was directly conjugated with the labeled horseradish peroxidase sdab-c reached a stable plateau after 35 min incubation with different concentrations of viruses from 10− to 10− tcid50/ml, and the absorbance values for the negative control remained unchanged. 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 ratio was obtained in pbs. we found the ability to capture antigen of imnb could be further reconfirmed by calculating the change of concentration of fmd asia virus vlp antigens after incubation. the p1-2a precursor is processed by viral protease 3c to produce the structural proteins vp vp and vp which then self-assemble to form icosahedral, empty capsid particles.. the recombinant fmdv vlp contains three structural proteins, vp vp and vp in vivo when observed by sds-page. we calculated that  μg of mnb are able to bind  μg target antigen. additionally, our capture efficiency assay demonstrated that the antigen complex could be dissociated by boiling, and fmd vlps were lysed into vp and and vp1/vp. the capture of the viruses with the imnbs was observed by tem. the blackish spots are the beads, while the protuberant spots are characteristic of virion morphology around the beads, because the fmdv particle is too small, at approximately 25 ~ 30 nm in diameter compared with the beads at about 200 nm in diameter, which leads to the virus particles not being obvious in the image. the evidence of binding virus was also suggested by the increase in the hydrated particle size of mnbs from 198 nm to 640 nm with dynamic light scatting. binding of the target virus to the functionalized imnbs could be easily realized by mixing with qds-c and co-localization analysis of beads, fmd asia virus and fluorescent qds resulting in qds-labeled virions. the functionalization of mnbs with sdab incubated with fmd asia virus, were then reacted with qds-c which produced the qds-labeled virions. the labeled virions were evaluated by confocal fluorescence microscopy, and an overlap of functionalized mnbs between the bright field and fluorescent image which indicated that functionalized mnbs had successfully captured fmd asia virus was generated. the number of molecules sdab conjugated to each mnb was estimated by the fluorescence intensity of the fluorescein-labeled vhh molecular conjugated to magnetic beads and was calculated about 900– protein molecular on a bead. the amount of sdab molecule per bead showed sufficient binding antigen sites to the coupling of target virus to ensure the capture capacity of imnbs. the combination of qds and imnbs offers researchers a new pathway to both separate and identify targets of interest via the use of a simple magnet, and is particularly advantageous in the detection and quantification of soluble biomarkers during which excess qd-antibody constructs must be removed for further analysis.fig. characterzaitons of imnbs by dynamic light scatting. the hydrated particle sizes of beads, bead-virus complexes, and the bead-virus-polyclonal ab complexes fig. confocal microscopic images of fluorescent, mnbs, and complexes of bead-virus. b the fluorescence of qds-c from the labeled target virus, a the beads in bright field image, c the merged image of complexes of beads-virus- indicating successful capture of target virus onto the mnb, d the aggregation of complexes of beads-virus- these results suggest that mnbs have a specific capture capacity of fmd asia 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 virus. in this method, the imnb has the ability to efficiently capture the fmd asia 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 
after magnetic separation, complexes of mnb-sdab-virus were detected with either a sandwich elisa or qds-c probe under a fluorescence microscope, and the complexes were used as templates for extraction of total rna for amplification of the vp or 3d gene fragments using rt-pcr and real-time rt-pcr. moreover, this method has been successfully used to capture asia antigen in synthetic samples.
 immunomagnetic nanobead 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 labeled with a single-domain antibody for capturing foot-and-mouth disease asia virus. after magnetic separation, complexes of mnb-sdab-virus were detected with either a sandwich elisa or qds-c probe under a fluorescence microscope, and the complexes were used as templates for extraction of total rna for amplification of the vp or 3d gene fragments using rt-pcr and real-time rt-pcr. the asia vlps were efficiently captured through imnb with a high binding rate of  μg of antigen/μl of bead suspension. moreover, this method has been successfully used to capture asia antigen in synthetic samples.  ultimately, a specific and highly sensitive capture fmdv asia tool has been established that has the potential to enhance the sensitivity and reliability when diagnosing fmdv asia electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 