Characterization of Asia 1 sdAb from Camels Bactrianus (C. bactrianus) and Conjugation with Quantum Dots for Imaging FMDV in BHK-21 Cells
et al. (2013) Characterization of Asia 1 sdAb from Camels Bactrianus (C. bactrianus) and Conjugation with
Quantum Dots for Imaging FMDV in BHK-21 Cells. PLoS ONE 8(5): e63500. doi:10.1371/journal.pone.0063500
Characterization of Asia 1 sdAb from Camels Bactrianus (C. bactrianus ) and Conjugation with Quantum Dots for Imaging FMDV in BHK-21 Cells
Shuanghui Yin. 0
Shunli Yang. 0
Youjun Shang 0
Shiqi Sun 0
Guangqing Zhou 0
Ye Jin 0
Hong Tian 0
Jinyan Wu 0
Xiangtao Liu 0
Adam Hall, University of North Carolina Greensboro, United States of America
0 State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Lanzhou, Gansu , China
Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious viral disease affecting cloven-hoofed animals. Camelids have a unique immunoglobulin profile, with the smallest functional heavy-chain antibodies (sdAb or VHH) naturally devoid of light chains with antigen-binding capacity. We screened and characterized five sdAbs against FMDV by immunized library from C. bactrianus with Asia 1 virus-like particles (VLPs). Three of five recombinant sdAbs were stably expressed in E.coli, remained highly soluble, and were serotype-specific for VP1 protein of FMDV Asia 1 by ELISA. These failed to completely neutralize the Asia 1 virus. According to the KD value of binding affinity to three sdAbs, which ranged from 0.44 to 0.71 nm by SPR, sdAb-C6 was selected and conjugated with Zn/CdSe quantum dots (QDs) to form a QDs-C6 probe, which was used to trace and image the subcellular location of FMDV in BHK-21 cells. The results show that FMD virions were observed from 3 h.p.i., and most of virions were distributed on one side of the nucleus in the cytoplasm. We demonstrate the utility of sdAbs as functionalized QDs are powerful tools for FMDV research.
. These authors contributed equally to this work.
Foot-and-mouth disease (FMD) is a highly contagious viral
disease of cloven-hoofed livestock, caused by FMD virus (FMDV),
a member of the genus Aphthovirus, in the family Picronaviridae. It
consists of a single-stranded, positive-sense RNA genome of
approximately 8.5 kb surrounded by four structural proteins (VP1
to 4), which form viral capsid proteins that are considered
antigenic, in addition to 15 different, mature non-structural
proteins (Nsp) . There are currently seven known serotypes: A,
O, C, Asia 1, and three South African Territories (STA1, SAT2
and SAT3), and multiple subtypes exist within each serotype [2,3].
The recent outbreaks of FMD, especially in a number of
FMDfree regions, such as Taiwan (1997)  and the United Kingdom
(2001) , have caused huge economic losses and significantly
increased public awareness. However, in FMD-free countries,
livestock, including cloven-hooved animals, are extremely
susceptible to FMDV due to the non-prophylactic-vaccination policy,
which results in no humoral response against FMDV. Globally,
FMDV serotypes O and the A are the most prevalent [6,7].
However, Asia has its own unique serotype, and outbreaks due to
Asia 1 have been reported only sporadically in the past few
decades. Analysis of Asia 1 indicates that some strains have been
spread across large distances between countries in Asia within a
short time .
Camelids produce conventional antibodies, but they also
contain a high quantity of naturally occurring, functional
antibodies devoid of light chains . The evolutionary advantage
of such heavy chain antibodies (HcAb) is still unclear. In HcAb,
the fragment antigen binding (Fab) region is reduced to a single
variable domain (sdAb, VHH, or Nanobodies, Nbs) which is easily
cloned and produced by standard genetic techniques . The
sdAb displays very significant characteristics in many selection
systems such as phage, yeast, or ribosomal display . Of
particular note is the small size and strict monomeric behavior,
and when combined with other biochemical properties such as
high solubility while readily expressed in Escherichia coli (E. coli), a
high specificity and affinity makes an advantageous tool for many
medical and biotechnological applications, including proteomics
Quantum dots (QDs) are semiconductor nanocrystals (2
to10 nm), such as CdSe/ZnS cores, which emit fluorescence
depending on their particle size, provide brighter and more stable
fluorophores that are comparable with fluorescent proteins
[12,13]. QDs are luminescent inorganic fluorophores which allow
for multicolor imaging, but orthogonal targeting approaches must
be developed to label different proteins with different color QDs.
Antibody-conjugated-QDs represent one approach for protein
labeling. However, compared to conjugating full-length
monoclonal antibodies (mAb) and single chain antibody fragments (ScFv)
Note: 0a day serum before the primary vaccination; 21b days, 35c days, 49d days
which were after the primary immunization, respectively; *Peripheral blood was
collected 7 days after the 3rd inoculation.
with sdAbs, which is a relatively easy process because sdAbs are
the smallest (<15 kDa) known natural domain with full available
antigen-binding capacity . Due to their small size, sdAb
molecules have been shown to act as strong enzyme inhibitors by
interacting with enzyme pockets not accessible to common
antibodies , and have the ability to efficiently target and aid
in imaging of specific immune cell types in vivo . Recombinant
sdAb fragments are emerging as new tools for sensitive diagnostics
methods, molecular probes, and infectious disease therapy.
Here, we describe the preparation, isolation, selection, and
characterization of FMDV serotype Asia 1 by binding
recombinant sdAbs derived from peripheral mononuclear lymphocytes
(PMLC) of Camelus bactrianus (C. bactrianus) and generating five
sdAbs with high affinity with FMDV antigen by phage display.
These were expressed in E. coli and purified, and three of the five
sdAbs (sdAb-C4,C5 and C6) expressed soluble protein. The
identical results demonstrated that these are type-specific against
FMDV Asia 1 by ELISA. Furthermore, we used CdSe/ZnS
coreshell QDs with emission wavelengths of 605 nm (QD605, red)
conjugated with the sdAb-C6, a highly sensitive, newly developed
nanoprobe, to achieve target detection and fluorescent spectral
analysis of FMDV infection in BHK-21 cells.
Detection for camels FMDV Asia 1 antibody
Two Bactrian camels were appropriated from the Gobi desert
before starting the experiment, and they nor their parents were
vaccinated against FMDV, and were as such considered natural
specific pathogen free (nSPF) animals. The camels were
immunized with recombinant VLPs expressing antigenic epitopes
of FMDV Asia 1. Blood samples were collected and serum was
isolated from both camels at several time points throughout the
study. The serum samples were tested by LPB-ELISA for
timesensitive evaluation of the animals immune reaction against the
antigen. The results indicate that a continuous and high immune
reactivity was observed, and that seroconversion occurred after
secondary inoculations. Interestingly, antibody titration of the
female camel demonstrated a higher reactivity than the male
Construction of VHH library
To construct a VHH library for FMDV Asia 1, the 66108
PBMC of the camels, underwent extraction of total RNA from
which cDNA synthesized. The VH and VHH regions were
amplified by three rounds PCR. The cDNA was a template in the
first PCR with the first PCR primers, based on available protein
sequence information and on the camel VHH clone VH-CH2, and
was predicted to anneal to codons 1 through 10 of the camel VHH
of subgroup III, the only family reported to be present in camels.
The 600 bp fragment (VHH-CH2 without the CH1 exon) was
purified from 1.8% agarose gel after separation from the 900 bp
fragment (VH-CH1-CH2 exons). The VHH fragments in the
600 bp gene were a template for amplification by using the second
PCR primers, which anneal at the framework 1 and framework 4
regions. The third PCR primers, which contain the SfiI/NotI
restriction sites and the 450 bp gene fragments were cloned into
the phagemid vector pHEN2, and were transformed into E. coli
TG1. A strong enriching effect was observed, and dilution plating
of the cultured library indicated a total of 1.916105 colonies. By
coating the intact virus directly, a polyclonal phage ELISA was
conducted. Twenty-four clones were then picked up from the third
elution and subjected to monoclonal phage ELISA to bind the
intact virus positively and specifically. By local DNA sequencing, 5
Figure 1. Multiple amino acid sequence alignment of FMDV Asia 1 specific sdAbs antibody clones. Amino acid sequences were aligned
according to the Kabat numbering. The specific VHH amino acids were thick-lined boxed, the FRs were thin-lined boxed.
unique sdAb genes were identified (GenBank: sdAb-C3/C4/C5/
C6/C20, KC816013/ KC816014/ KC816015/ KC816016/
In this work, we first screened five specific sdAb antibodies
against FMDV Asia 1 from camels by phage display. Analysis of
sdAb amino acid fragments showed that the four conservative
hallmark residues of sdAb in FR2 were Phe37, Glu44Gly/Ala,
Arg45 and Ala47Gly. With the exception of three samples,
residues in FR2 of one sequence were mutated into a Ala in
sdAbC5 and sdAb-20, and these substitutions of otherwise conserved
amino acids are hallmarks of sdAb originating from heavy-chain
homodimer immunoglobulins . Furthermore, the CDR3
consisted of lengths of 19 and 20 amino acids, and two of the
five clones contain a Cys107 (sdAb-C3 and 20) which could
possibly form a disulfide bond with a second cysteine located in the
CDR1(Fig. 1). In contrast to human VHs, the camelid VHHs are
generally well expressed in E. coli, highly soluble, and stable at high
concentrations in aqueous solutions. Comparison of the crystal
structures of cAbAn33 and its humanized derivative reveals steric
hindrance exerted by VHH-specific residues Tyr37 and Arg45,
which prevent the VL domain pairing, whereas Glu44 and Arg45
are key elements to avoid insolubility of the domain . In our
study, although sdAb-C3 and 20 have distinguished features of
sdAb-derived from camels and also were expressed in E. coli, the
former is a soluble protein that could not bind the resin and the
latter is insoluble.
Expression and purification soluble sdAb antibody
The three unique sdAb fragments were cloned into vector
pSMK, and transformed into BL21-codon-Plus (DE3)-RIL strains,
aVNT: All sdAb fragments failed to neutralize the FMDV, and were only able to
delay cytopathic effect to 34 h p.i. when compared to the control. b ELISA: the
OD450 values were obtained from sdAb screening of all clones binding to
inactivated Asia 1 antigen by
which were induced with IPTG for 20 h at 20uC. Solubility of the
recombinant sdAb protein were indentified by SDS-PAGE, and
confirmed its apparent molecular weight and that the recombinant
proteins of clone 4/5/6 express solubility behavior (Fig. 2A).
Unfortunately, clone 3/20 both were inclusion bodies (data not
shown). The purified recombinant proteins of clone 4/5/6 were
stained using NTA affinity resins which have the ability to
specifically bind to His6 tag polypeptides (Fig. 2A). The fusion tag
of all three clones (Clone 4/5/6) may be recognized with an
antiHis antibody and observed by Western blotting (Fig. 2B).
Identification of Asia 1 sdAb
The dasELISA assay and guinea pig-antisera as a parallel
positive control was able to demonstrate the ability of sdAb to bind
FMDV Asia 1 antigen, as indicated by sdAb having similar
characteristics with polyclonal anti-sera, and both were able to
capture target antigen. There was no significant difference in
reading, suggesting that the sdAb is effective when used as a
The same dasELISA method was used to study the
serotypespecific capability of sdAb with FMDV serotypes A, O, and Asia 1
inactive antigens. Our results indicate that sdAb has high
specificity against Asia 1 and exhibits no cross-reactivity with
serotypes O and A antigen (Fig. 3).
VNT results demonstrated that all sdAb failed to neutralize the
FMDV Asia 1, and sdAb was only able to delay cytopathic effect
within the cell cultures for 34 h when compared to the positive
control. The reactivity of the sdAbs to recombinant FMDV-Asia
1-VP1 and VP0-3 was examined using an indirect ELISA. Our
results indicate that three sdAbs bound only to recombinant Asia
1-VP1 only (Table 2).
The affinities of sdAb ranged from 0.44 to 0.71 nM (Table 2),
which is within the range of KD values most sdAbs have for their
targeted antigens. The results demonstrate the KD value of binding
affinity of sdAbs to native antigen, which is consistent with
previous screening sdAbs by bELISA (Table 2). Therefore, these
data on identify clone 6 as an optimal candidate for further
Characteristics of CdSe/ZnS ODs conjugated anti-Asia1
Absorption spectra were acquired on a Perkin Elmer LS-55
UV/vis spectrometer run on the Olympus BX51, DP72. (Fig. 4),
and demonstrates the optical properties including absorption and
emission of the CdSe/ZnS QDs conjugated with anti-Asia 1 sdAb
dispersed in 50mM borate at pH 8.4. We also tested whether these
sdAbs are labeled successfully with QDs by 1% agarose gel and
these coatings are run relatively slowly because of bigger molecular
weight than QDs alone (data no show), indicating sdAbs
conjugation with QDs.
ODs-sdAb probe imaging FMDV Asia1 in BHK-21 cells
To establish the kinetics of FMDV infection in BHK-21 cells
and study the interactive characteristics of QDs-sdAb probe,
FMDV-infected BHK-21 cells were monitored for the presence of
virions over time by immunofluorescence (IF) microscopy.
The first and second images indicate that the FMD virions
cannot be observed with QD-sdAb staining (Fig. 5) at 1 and 2 h
p.i., although we inspected for virions in all fields on all coverslips.
With lastingness of infection, the minority of cells in cultures had
developed virion at 3 h p.i. the image in Fig. 5 shows that FMDV
virions appeared sporadically in perinuclear sites (red).
At 4 h p.i., the majority of BHK-21 cells demonstrated
detectable FMD viral capsid protein labeling with QD-sdAb
probe indicating that the viral protein isolated to a specific region
on one side of the nucleus. As the time reached 5 h p.i. and 6 h
p.i., the majority of cells, both infected and non-infected, had
detached from the coverslips, and consequently the detectable
number of cells became less visible.
To identify the QD-sdAb probe in vitro, a standard fluorescence
detection probe, AF488-C6, was conjugated with sdAb-C6, and
dubbed AF488-C6 probe (Fig. 6). This was used to demonstrate
that the FMD viral proteins were visible, through the green
AF488-C6 probe in BHK-21 cells at 4 h p.i. The green AF488-C6
probe was able to stain the FMD virions in the same location with
QD-sdAb probe in cells. This demonstrated that the QD-sdAb
nanoprobe specifically bound FMD Asia 1 virions. In addition, IF
studies identified strong staining with QD-sdAb probe for newly
synthesized capsid proteins of FMDV, and provided clear
discrimination between the virions and nucleus.
Primer sequence (5939)
CTTCGTCTCAAGGTGATGTGCAGCTGGTGGAG AGTGGATCCTCATGAGGAGACAGTGACCTGG CTTCGTCTCAAGGTCATGTGCAGCTGGTGGAG AGTGGATCCTCATGAGGAGACAGTGACCTGG
The Office International des E pizooties (OIE) Code chapter on
FMD includes camelids as being susceptible species to FMD,
giving the impression that they are similar to cloven-hoofed
animals in their potential involvement in the epidemiology of
FMD . In fact, dromedary camels are not susceptible to
FMDV type A infection and to neither be a reservoir of FMDV
nor transmit this virus to susceptible species. When compared with
the non-susceptible dromedaries, Bactrian camels showed
moderate to severe clinical signs of FMD and developed high titers of
antibodies to FMDV 710 days post-inoculation . Days
postinoculation . C bactrianus are more likely to induce strong
passive immune reactions against FMDV antigen than other
camels in our experience. C bactrianus produce high titers of
antibodies post-immunization with Asia 1 antigen rendering them
the ideal candidate animal for the production of sdAb against
In this report, we demonstrate that mature virions can be
reliably recognized and labeled with QD-sdAb probe in BHK-21
cells infected with FMDV Asia 1 up to 3 h.p.i. There is a dramatic
distribution of the cytoplasmic contents, leading to a very defined
location pattern that has been associated with the viral replication
complex. For many positive-strand viruses, a common feature of
infection is the extensive rearrangement of host cell membranes,
and generation of cytoplasmic vesicles which are apparently
required for replication. Most research performed on
picornaviruses is based on analysis of cells infected with poliovirus .
FMDV, as member of the Picornaviridae, has a similar genome
organization and is believed to follow a similar replication strategy
to poliovirus . In contrast to poliovirus, replication and
translation of FMDV RNA occurs in the cytoplasm , and a
replication complex of FMDV and its possible association with
cellular membranes has not yet been described in detail. However,
our research with a QD-sdAb probe demonstrates that mature
FMD virions are located on one side of the cell nucleus as the time
after inoculation increases. This has been similarly reported for
serotype O FMDV, and since the site is often occupied by the
Golgi apparatus where it is described as the virus replication site, it
has become the primary focal point of the subsequent events that
take place within the FMDV-infected cell . Differences in the
kinetics of expression and cell distribution among FMDV Nsp
have been observed in BHK-21-infected cells. 3Dpol was the first
protein detected at 1.5 h p.i., and it appeared in a perinuclear
distribution. Between 2 to 2.5 h p.i., 2B, 2C, 3B, and 3C were
detected, mostly exhibiting a punctated and scattered pattern,
while 3A and 3Dpol showed concentration to one side of the
FMDV mRNA is translated by a cap-independent mechanism
that utilizes an internal ribosomal entry site, and taking over the
host cell protein synthesis machinery  which antagonizes the
cellular innate immune and inflammatory responses to the viral
infection and produces virus progeny . The leader proteinase
(Lpro) of FMDV is involved in overcoming the innate immune
response by blocking the expression of interferon (IFN) and
disturbing IFN-b transcription during FMDV infection.
Interestingly, FMDV Lpro is localized to similar nuclear regions globally,
and the transcription factors are situated in close proximity during
viral infection . BHK-21 or IBRS-2 cell lines are typically used
for the propagation of FMDV. Analyses of both cell lines indicates
that they harbor either an impairment in type IIFN production or
IFN response, and do not allow a clear phenotypic differentiation
between wild-type virus and leaderless viruses . Therefore,
FMDV, as with other RNA viruses, dependents on various host
cell factors for their replication, and as modify a variety of cellular
signal transduction pathways. To counter some of the host
immune responses, viruses have developed very sophisticated
mechanisms to subvert the host defense and to support their own
The sdAb fragment is a VH variable domain that is
approximately one-tenth the size of the conventionally assumed
humoral respondent IgG (15 kDa vs 150 kDa) , and are called
nanobodies due to their lack of the Fc fragment which renders
them unable to exhibit nonspecific binding to target antigen. This
feature is likely causing the weak virus neutralizing activity of the
anti-FMDV sdAbs in vitro. However, blocking of viral cell entry,
which is the predominant mechanism of in vitro FMDV
neutralization, requires far higher antibody concentration than
are normally reached in vivo . While sdAb are capable of
binding their antigens with affinities comparable to conventional
IgG, its size allows it to bind epitopes inaccessible to conventional
IgG. sdAbs are characterized by a low tendency to aggregate ,
exist as monomers, disseminate much better in tissues than full-size
IgG , increase the ability withstand harsh physiochemical
treatments, maintains solubility under certain non-physiological
conditions, and resume normal function following exposure to
chemical heat or heat denaturing [35,36]. All these advantages
make sdAb the best capture molecules due to the great deal of
flexibility in their chemistry for bioconjugation to QD-based
fluorescent nanoprobes for biodetection and diagnostics. Zamman
et al (2009) developed sdAb-conjugated QDs as nanoprobes for
cellular imaging of cancers cells, which bind strongly to epidermal
growth factor (EGF) receptor, a protein which is widely accepted
as a tumor marker . QD-sdAb probes have a highly oriented
manner, which make them an attractive alternative for the
generation of ultra-small targeted nanoprobes in high-throughput
diagnostic platforms .
sdAb is as a new class of molecular tracers, which are routinely
indentified with nanomolar affinity for their target, and are easily
tailored for molecular imaging and drug delivery application.
Molecular imaging is aimed at the noninvasive investigation of
cellular and molecular events in living subjects. The key advantage
of this imaging methodology is that a biological process can be
investigated in its native environment in an intact living individual
(animal or human) . Rothbauer et al (2006) prepared a GFP
sdAb from alpacas (Lama pacos) as a probe of chromobodies for
targeting and tracing antigens in different subcellular compartments
in living cells, which can be targeted and traced for any potentially
antigenic cellular structure . Additionally, sdAbs can
successfully target brain epitopes by transmigrating through blood-brain
barrier. , can be used for tuning and detecting the activity of cell
proteins in vivo [42,43,44], provide better diffusion in fixed cells in
comparison to conventional antibodies , and can simplify the
generation of anti-idiotypic antibodies suitable for vaccination .
Finally, the latest antibody engineering designs for sdAb, such as
diabodies, minibodies, and labeling tags, have achieved impressive
stability and specifity, and are now perfectly positioned to take full
advantage of the next-generation position-labeling chemistries and
QD conjugations to open new opportunities in a variety of
research and amplification fields.
We have defined a panel of specific sdAb (Nanobody) for FMDV
serotype Asia 1 from C. bactrianus by Western blotting, ELISA,
VNT and SPR. The sdAb-C6 was selected as a candidate antibody
to conjugate with QDs forming QDs-sdAb probe, which was used to
locate and image the subcellular distribution of FMDV Aisa1 in
BHK-21 cells. The results demonstrate clearly visible FMD virions
from 3 h.p.i., onward and most virions were distributed to one side
of the nucleus in the cytoplasm. Herein, we establish the utility of
sdAbs as functionalized QDs are powerful tools for FMDV research
and others pathogens. The confluence of nanotechnology and
biomaterials, which helps researchers understand the many basic
biological processes and phenomena in level of visible by nano
luminescent materials to label nanobody, then conjugate products
to bind potential targets. Therefore, the new biomaterial probe is
helpful example for strengthening the sdAbs applications for tracing
the virus or cancer cells or others, and our research also expand the
range of nanobody as in vivo tools for virus infection and functional
advantages of sdAb.
All animals were handled in strict accordance with good animal
practice according to the Animal Ethics Procedures and
Guidelines of the Peoples Republic of China, and the study was
approved by the Animal Ethics Committee of Lanzhou Veterinary
Research Institute, Chinese Academy of Agricultural Sciences
(No. LVRIAEC2012-006). In this study, camles were permitted as
experimental animal by the owner.
Materials and Methods
All of the FMDV strains, sera, and ELISA assay used in this
study are reference strains obtained from OIE/CHINA National
Foot and Mouth Disease Reference Laboratory.
The virus-like particles (VLPs) from Asia 1 (Strain:1/Jiangsu/
China/2005; Genbank: EF149009) were used as antigen in
immune animals, and were developed from a recombinant
expression of specific structural proteins (VP1 and VP0-VP3) in
an E. coli expression vector.
C. bactrianus immunization and antibody detection
Bactrian camel feeding, management, immunization, and
sample collections were conducted by trained personnel under
the supervision of a veterinarian.
Two Bactrian camels (one year old male and ten month old
female) were immunized with Asia 1 VLP antigen preparations,
emulsified in a 206 adjuvant. Camels were inoculated
intramuscularly in the back, both sides of the neck, and on both hind legs at
different sites on days 0, 21, 28, 35 and 42. Serum samples from
peripheral blood via jugular venipuncture were collected on day 0
and subsequently after each inoculation. The sera antibody
response was monitored by FMDV type Asia 1 liquid-phase
blocking (LPB) ELISA.
A total of 600 ml of peripheral blood was collected from the
external jugular vein in EDTA coated tubes, 7 days after the final
inoculation (300 ml from each camel and pooled). Total PBLCs
were isolated by Ficoll-Paque gradient centrifugation; cell pellets
were suspended in MEM medium, then transferred into 6-cell
plates and incubated for 30 min at 37uC to eliminate cell debris.
Monoclonal cells were divided into aliquots of 66108 PBLC, and
were subsequently stored in liquid nitrogen.
RNA extraction, PCR amplification and sdAb antibody
The total RNA was extracted by using a commercially availabe
RNA extraction kit (Nucleospin RNA II; Macherey Nagel).
Firststrand cDNA was synthesized by using Superscript III reverse
transcriptase with oligo(dT)1218 primers (Invitrogen). The cDNA
encoding sdAb and VH was specifically amplified with the first
PCR primers H1 and G1 (Table 3), annealing at both the leader
and CH2 sequences. The 600 bp gene encoding for sdAb
fragments was the template for amplification by using the second
PCR primers, H2 and H3, which anneal at the framework 1 and 4
regions, and were followed by the third PCR primers, P1 and TN,
which contain the restriction sites for further cloning steps. The
final PCR fragments contained the SfiI/NotI restriction sites, and
were ligated into the phage vector pHEN2. Ligated material was
transformed in triplicate into E. coli TG1 cells. The colonies from
the plated cells were collected, washed, and stored at 80uC in LB
medium, supplemented with glycerol equivalent to 50% of the
Specific sdAbs against FMDV type Asia 1 were selected from
the immune sdAb library using phage display technology. The
library was infected with M13K07 helper phages, and phage
particles expressing the sdAb repertoire were rescued and
precipitated with polyethylene glycol. Enrichment of specific
binding points was performed by three rounds of in vitro selection.
The VLPs antigen of serotype Asia 1 was coated directly and a
pre-made phage display camel sdAb library was screened to find
specific binding points. After three rounds of screening, a strong
enriching effect was observed by quality control polyclonal phage
ELISA. While screening for Asia 1 specific sdAb fragments, phages
displaying the selected sdAb were selected and separated from
monoclonal TG1 E. coli clones by phage ELISA. Unique sdAb
were identified by local DNA sequencing.
Expression and purification of recombinant sdAb
sdAb cDNA of the unique clones was amplified by PCR, and
were sub-cloned into p-SMK vector with a fusion tag of small
ubiquitin-like modifiers (Sumo) and expressed in E. coli .
Briefly, five pairs of expression primers of sdAb were designed for
amplification of sdAb sequences (Table 4), which was purified and
digested with restriction enzymes, cloned into p-SMK. The
recombinant plasmids were transformed into BL21-codon-Plus
(DE3)-RIL strain (Stratagene). The culture was induced by
isopropyl-b-thiogalactopyranoside (IPTG) and incubated for
20 h at 20uC with constant agitation of 180 rpm. Cells were
separated by centrifugation and the pellet was resuspended in
50 mM Tris-Cl buffer (pH 8.0). The cells were then cracked with
an ultrasonic cell crusher, and the collected supernatant was
purified using NTA affinity resins. The results were analyzed
through 12% SDS-PAGE and Western blotting.
Three pairs of soluble recombinant sdAb antibody protein were
transferred to an Immobilon-P Transfer membrane (Millipore
Corporation, USA) in transfer buffer (20mM Tris-HCl, 190 mM
glycine, 0.1% SDS, 20% Methanol, pH 8.3). The membrane was
blocked for 1 h with PBST (containing 0.05% Tween 20, 5% skim
milk) and incubated with 1:1000 anti-His monoclonal antibody by
a peroxidase-conjuated goat-anti-mouse antibody (antiHis-HRP,
Sigma, USA) diluted by PBST. The assay was used to develop the
signal by DAB reagent.
Characterization of FMDV Asia1 sdAb
Identification of serotype-specific using double antibody
sandwich ELISA. The double antibody sandwich ELISA
(dasELISA) assay was carried out for identification of sdAb against
specific serotypes, including FMDV Asia 1 as well as A and O.
96well microtiter plates were coated with capture antibodies,
including sdAb against FMDV Asia 1, rabbit-antisera of Asia 1,
A, and O diluted at1:1000 for serotype Asia 1, A, and O; sdAbs of
C4, C5, and C6 were diluted to 2.12 mg/ml, 3.75 mg/ml, and
0.84 mg/ml, respectively, in 0.06 M carbomate/bicarbonate buffer
(pH 9.6). They were then incubated overnight at 4uC. Viral
antigens of FMDV Asia 1 (inactivated FMDV), A, and O were
added into wells. Three serotype-specific guinea pig antiseras of
FMDV were added to the corresponding wells as positive controls,
and two wells were selected to be coated with the capture
antibodies and the Sumo recombinant protein (1:2000,
antiHisHRP against Sumo His-tag) as negative controls. The
corresponding the serotype-specific rabbit antisera against FMDV were
added to all wells. The HRP conjugated goat anti-rabbit HRP IgG
was added to all wells. After colorimetric reaction with TMB as
substrate, the reaction was stopped with 2.0 M sulfuric acid, and
the absorbance was measured at 450 nm.
Neutralization capacity of sdAb. A virus neutralization test
(VNT) was performed as described previously .The LPB
ELISA was carried out according to standard procedure for sdAbs
as previously described . The dilutions of antisera required to
affect 50% inhibition were calculated for all the viruses .
Indirect ELISA for locating the sdAb origin. An indirect
ELISA is the common method of detection, and was performed.
FMDV Asia 1 recombinant antigens (VP1 at 6.47 mg/ml,
VP0VP3 7.85 mg/ml) were coated onto 96-well microtiter plates and
were incubated overnight at 4uC. The plates were blocked with
PBST buffer (5% skim milk, 10% E. coli lysate with 0.05% Tween
20) at room temperature for 1 h. The sdAbs were then added to
the plates. After this incubation period, an antiHis-HRP mAb, at
dilution of 1:2000, was added followed by TMB substrate, and the
reaction was stopped with 2.0M sulfuric acid. The optical
absorbance was measured at 450 nm.
Affinity of the sdAb for FMDV. The affinity of the sdAb s
selected to bind Asia 1 antigen was determined by surface plasmon
resonance (SPR) on a Biacore 3000 (Biacore, Uppsala, Sweden).
Asia 1 VLPs_antigen was directly immobilized to 1000 resonance
units on the reactive surface of the CM5 sensor chip. The antibody
fragments were diluted in HEPES-buffered saline at
concentrations between 25 and 500 nM, and were subsequently injected at a
flow rate of 30 ml/min over the sensor chip to record their binding
kinetics toward the antigen . The obtained data were analyzed
with BIA evaluation software version 4.1. The kinetic rate
constants (Ka and Kd), were recorded to calculate the equilibrium
dissociation constant (KD).
CdSe/ZnS ODs conjugated anti-Asia1 sdAb
ZnS-capped-CdSe ODs (QDs, 605 nm) were synthesized by
Wuhan Jiayuan QuantumDots Co. LTD, Wuhan, China .
These activated QDs modified with thioglycolic acid were
dissolved in 50 mM borate (8 mM, pH 8.4) containing 50mmol
hydrochloride, .98%). The resulting QDs were used to observe the
optimized wide ranging criteria. The QDs were reacted with 20 ml
sdAb-C6, which were conjugated with solution of high-quality
oilsoluble core-shell QD605 (QDs-C6), at room temperature (RT) in
a shaking incubator for 2 to 4 h. The final QD bioconjugates were
purified by centrifugation at 6000 g for 10 min, and the
supernatant was transferred into an ultra-filtration tube (MW
30 kDa) to retain 50 kDa molecular and remove unreacted sdAb
and impurities. The samples were then dialyzed at 4uC and
underwent centrifugation at 2000 g until the appropriate volume
was reached. The results were determined and visualized by using
the optimized wide-ranging criteria and agarose gel. The QDs-C6
probes were stored at 4uC.
QDs-C6 based fluorescence probe detection was also compared
to the standard fluorescence probe, Alexa
Fluor-488-conjugatedsdAb-C6 (AF488-C6), to further determine the sdAb-C6
conjugated antibody specificity against FMDV in cells. The standard
procedure was used to label the sdAb antibodies with AF488-C6 as
QDs-C6 probes imaging FMDV Asia1 in BHK-21 cells
Monolayers of baby hamster kidney-21 (BHK-21) cells were
seeded on coverslips in 6-well plates, and were infected with 106
TCID50 Asia 1virus diluted in DMEM as previously described
. After 1 h of adsorption, the culture supernatant was removed
and the monolayer was washed twice with fresh medium
containing 0.5% fetal calf serum, and cells were incubated at
37uC with 5% CO2. Cell samples were harvested at 1 h, 2 h, 3 h,
4 h, 5 h, and 6 h post-infection (p.i.). All samples on coverslips
were fixed with 4% paraformaldehyde, rinsed with 25 mM glycine
(pH 7.4), and permeabilized with 0.1% Triton X-100, 1% bovine
serum albumin, and 1M glycine in PBS for 15 min at RT. These
were incubated in blocking buffer (5% normal goat serum, 2%
bovine serum albumin, 10 mM glycine) to reduce unspecific
binding for 2 h at RT.
The processed cells were then incubated with QDs-sdAb diluted
in 1% BSA in PBS for 1 h in a humidified chamber at RT. After
washing three times in PBS, nuclei were stained with 1 mg/ml of
DAPI (4,6-diamidino-2-phenylindole, Invitrogen). Following
immunochemical staining, the coverslips were mounted using the
Prolong Antifade Kit (Molecular Probes), and observed under an
Olympus BX51 fluorescence microscope coupled to a digital
Conceived and designed the experiments: SHY SLY YJS SQS XTL.
Performed the experiments: SHY SLY YJS GQZ YJ HT JYW XTL.
Analyzed the data: SHY SLY GQZ YJ XTL. Contributed reagents/
materials/analysis tools: SQS GQZ YJ HT JYW. Wrote the paper: SHY
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