Modular glycosphere assays for high-throughput functional characterization of influenza viruses.
ABSTRACT BACKGROUND: The ongoing global efforts to control influenza epidemics and pandemics require high-throughput technologies to detect, quantify, and functionally characterize viral isolates. The 2009 influenza pandemic as well as the recent in-vitro selection of highly transmissible H5N1 variants have only increased existing concerns about emerging influenza strains with significantly enhanced human-to-human transmissibility. High-affinity binding of the virus hemagglutinin to human receptor glycans is a highly sensitive and stringent indicator of host adaptation and virus transmissibility. The surveillance of receptor-binding characteristics can therefore provide a strong additional indicator for the relative hazard imposed by circulating and newly emerging influenza strains. RESULTS: Streptavidin-coated microspheres were coated with selected biotinylated glycans to mimic either human or avian influenza host-cell receptors. Such glycospheres were used to selectively capture influenza virus of diverse subtypes from a variety of samples. Bound virus was then detected by fluorescently labelled antibodies and analyzed by quantitative flow cytometry. Recombinant hemagglutinin, inactivated virus, and influenza virions were captured and analyzed with regards to receptor specificity over a wide range of analyte concentration. High-throughput analyses of influenza virus produced dose--response curves that allow for functional assessment of relative receptor affinity and thus transmissibility. CONCLUSIONS: Modular glycosphere assays for high-throughput functional characterization of influenza viruses introduce an important tool to augment the surveillance of clinical and veterinarian influenza isolates with regards to receptor specificity, host adaptation, and virus transmissibility.
- Methods in Enzymology 02/1987; 138:162-8. · 2.19 Impact Factor
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ABSTRACT: We describe here the development of a carbohydrate-based microarray to extend the scope of biomedical research on carbohydrate-mediated molecular recognition and anti-infection responses. We have demonstrated that microbial polysaccharides can be immobilized on a surface-modified glass slide without chemical conjugation. With this procedure, a large repertoire of microbial antigens (approximately 20,000 spots) can be patterned on a single micro-glass slide, reaching the capacity to include most common pathogens. Glycoconjugates of different structural characteristics are shown here to be applicable for microarray fabrication, extending the repertoires of diversity and complexity of carbohydrate microarrays. The printed microarrays can be air-dried and stably stored at room temperature for long periods of time. In addition, the system is highly sensitive, allowing simultaneous detection of a broad spectrum of antibody specificities with as little as a few microliters of serum specimen. Finally, the potential of carbohydrate microarrays is demonstrated by the discovery of previously undescribed cellular markers, Dex-Ids.Nature Biotechnology 04/2002; 20(3):275-81. · 39.08 Impact Factor
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ABSTRACT: Human influenza viruses are known to bind to sialic acid linked alpha2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection. Viruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of alpha2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for alpha2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of alpha2-6 and alpha2-3 sialic acids, including human A549 and HeLa cells with high levels of alpha2-6 sialic acid, and CHO cells that have only alpha2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells. Agglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of alpha2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of alpha2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with alpha2-6 receptor specificity.Virology Journal 02/2007; 4:42. · 2.09 Impact Factor
METHODOLOGY ARTICLE Open Access
Modular glycosphere assays for high-throughput
functional characterization of influenza viruses
Sven N Hobbie1, Karthik Viswanathan2, Ido Bachelet2, Udayanath Aich2, Zachary Shriver2, Vidya Subramanian2,
Rahul Raman2and Ram Sasisekharan1,2*
Background: The ongoing global efforts to control influenza epidemics and pandemics require high-throughput
technologies to detect, quantify, and functionally characterize viral isolates. The 2009 influenza pandemic as well as
the recent in-vitro selection of highly transmissible H5N1 variants have only increased existing concerns about
emerging influenza strains with significantly enhanced human-to-human transmissibility. High-affinity binding of the
virus hemagglutinin to human receptor glycans is a highly sensitive and stringent indicator of host adaptation and
virus transmissibility. The surveillance of receptor-binding characteristics can therefore provide a strong additional
indicator for the relative hazard imposed by circulating and newly emerging influenza strains.
Results: Streptavidin-coated microspheres were coated with selected biotinylated glycans to mimic either human
or avian influenza host-cell receptors. Such glycospheres were used to selectively capture influenza virus of diverse
subtypes from a variety of samples. Bound virus was then detected by fluorescently labelled antibodies and
analyzed by quantitative flow cytometry. Recombinant hemagglutinin, inactivated virus, and influenza virions were
captured and analyzed with regards to receptor specificity over a wide range of analyte concentration. High-
throughput analyses of influenza virus produced dose–response curves that allow for functional assessment of
relative receptor affinity and thus transmissibility.
Conclusions: Modular glycosphere assays for high-throughput functional characterization of influenza viruses
introduce an important tool to augment the surveillance of clinical and veterinarian influenza isolates with regards
to receptor specificity, host adaptation, and virus transmissibility.
Influenza viruses are a significant cause of morbidity and
mortality worldwide [1,2]. Besides the seasonal influenza
epidemics caused by H1N1 and H3N2 influenza virus
strains, new strains of influenza virus emerge periodically
with pandemic potential. Despite the extensive network in
place to monitor influenza virus evolution through muta-
tion and recombination, public health laboratories still
fail to detect novel strains of influenza and differentiate
those that are primarily animal-adapted from those with
true pandemic potential. For example, the outbreak of
the swine-origin H1N1 pandemic in spring 2009  hit
the medical community unprepared, even though the
initial transmission from swine to humans occurred months
before, and prior to that it is believed to have circulated
undetected in swine for years . This underscored the
gap in our ability to detect and characterize emerging
strains before the widespread onset of disease in the popu-
lation. Early detection of virus strains with pandemic po-
tential is important, as early detection of an outbreak is
critical to generate and stockpile sufficient quantities of
vaccines and anti-virals to limit the spread of the disease.
One of the challenges in detecting emerging strains is
that the factors leading to the generation of a pandemic
virus are complex and poorly understood. At a functional
level, however, it is clear that for a virus to have pandemic
potential it must be capable of human-to-human aerosol
transmission and there must exist a substantial population
that is immunologically naïve to the strain of virus .
Poor human-to-human transmissibility of avian-adapted
H5N1 strains causing “bird flu”, for example, seems to be
* Correspondence: email@example.com
1Singapore-MIT Alliance for Research and Technology, Singapore 138602,
2Harvard-MIT Division of Health Sciences and Technology, Koch Institute for
Integrative Cancer Research, Department of Biological Engineering,
Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA
© 2013 Hobbie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Hobbie et al. BMC Biotechnology 2013, 13:34
the major impediment to more serious outbreaks [6,7].
Recent news on H5N1 variants capable of efficient aerosol
transmission in ferrets, however, suggest that a few muta-
tions may be sufficient to render bird flu highly transmis-
sible in ferrets and possibly humans [8,9]. Therefore, the
development of assays that identify subtypes and strains
that have the potential to make the jump to humans from
animal reservoirs is vitally important for disease surveil-
lance and public health.
We have previously elucidated the role of the influenza
hemagglutinin (HA) in aerosol transmissibility [7,10-17].
HA binding to cell surface glycans present on cells of
the upper respiratory tract is the key initial step in viral
infection; indeed, HA has been found to be one of the
key viral genes involved in infectivity and transmission
. Together, these studies on seasonal and pandemic
influenza strains have provided a comprehensive under-
standing of how high-affinity binding to the distinct top-
ology of ‘long’ α2,6 linked sialylated glycans is a necessary
step in efficient aerosol transmission (Additional file 1).
Current surveillance methods include genotyping of
viral isolates using PCR to identify their type and subtype,
as well as comparing the antigenicity of newly identified
virus strains to existing strains. Despite comprehensive
genotypic and phenotypic analyses, it is often difficult to
functionally type the virus. Given the observed correlation
between high affinity to long α2-6 sialylated glycans and
efficient transmission, we reasoned that a surveillance
strategy involving the typing of virus strains, and more
specifically, viral HAs based on their affinity to long α2-6
sialylated glycan would provide a robust methodology to
detect and predict the transmissibility and therefore pan-
demic potential of emerging strains.
Traditionally, receptor specificities of avian- and human-
adapted influenza viruses are determined using a red-blood
cell (RBC) agglutination assay. RBCs from species such as
chicken, turkey, horses, guinea pigs and humans have
been used in such assays [18,19]. RBCs have also been
used in conjunction with sialidases and sialyltransferase
to present certain glycan structures, for example exclu-
sively containing either α2-3 or α2-6 linked sialic acid
[20,21]. This type of assay however is inherently limited
in that it fails to account for receptor complexity beyond
the sialic acid linkage, i.e. binding to a distinct topology that
a glycan receptor adopts based on a variety of determinants
such as sequence, linkage, chain length, and branching of
sugar molecules. Moreover, it has been shown recently
that the diversity of sialylated glycans present on RBCs
is significantly different from the glycans present in the
upper respiratory tract of humans . Other methods
such as fetuin capture assays suffer from the same limi-
The continuous progress of chemoenzymatic synthesis
strategies for glycans and development of glycan array
platforms has enabled the study of HA specificity using
chemically defined glycans [24-27]. Intact viruses, recombi-
nantly expressed hemagglutinins, and their mutant forms
from H1, H3, and H5 subtypes have already been analyzed
using glycan arrays [26,28]. While high-quality binding
data can be obtained using such arrays, they do not readily
lend themselves as a routine tool for virus surveillance
due to three major factors: first, the microarrays are syn-
thesized by molecular printing on glass slides using high-
precision equipment, and are still costly to manufacture;
second, the glycans are covalently bound to the glass,
making the array irreversibly rigid and thus not suitable
for rapid construction of a custom-made array; and third,
typical array formats are interpreted in an on/off manner,
rather than through a quantitative readout, thus missing
potentially critical information.
Here we present an alternative to the planar glycan
array using magnetic polystyrene microspheres as a flowing
matrix for a modular glycan array. Suspension bead arrays
offer the advantages of higher flexibility, faster reaction kin-
etics and greater sensitivity owing to the three-dimensional
presentation of glycans. Importantly, flow cytometry en-
ables automated, large-scale sample screening. In the
past, microspheres in conjunction with flow cytometry
have been used for immunoassays, including for the
detection of infectious agents such as influenza .
Using custom-designed glycospheres, we developed an
assay platform for high-throughput functional character-
ization of clinical influenza isolates based on their ability
to bind to certain host-specific glycan motifs.
Results and discussion
Glycan motif selection
We have previously reported the glycan diversity in
human upper respiratory tissue with a predominance
of α2-6 sialylated glycans . Further, analysis of glycan
array data of human-adapted influenza viruses revealed
them to consistently bind to long α2-6 sialylated glycans
(tetrasaccharide or longer), despite showing a remarkable
overall heterogeneity in their glycan binding patterns,
[28,30-36] (summarized in Additional file 2). Together,
based on these findings, a commonly available α2-6-
sialylated tetrasaccharide glycan, LS-tetrasaccharide c
(LSTc), was chosen as a representative influenza recep-
tor of the human upper respiratory tissue. Likewise, a
structurally related α2-3 sialylated glycan, LS-tetrasaccharide
a (LSTa), was chosen as representative receptor for the
binding preference of avian influenza viruses [28,37]
(Additional file 2). LSTc and LSTa were biotinylated with
a long-chain spacer and purified by HPLC to remove
excess biotin. Conjugation of streptavidin-coated micro-
spheres with either of the biotinylated receptors resulted
in receptor-specific glycospheres that were used to capture
recombinant hemagglutinin or virus from a small sample
Hobbie et al. BMC Biotechnology 2013, 13:34
Page 2 of 8
volume. Bound analyte was then labelled with either a
broad-spectrum or subtype-specific antibody and quan-
tified by fluorescent flow cytometry. Figure 1 illustrates
the overall assay schematic.
The glycan density on the microspheres (controlled by
streptavidin density on the microsphere) was found to
be a critical parameter for sensitivity of hemagglutinin
binding. The receptor binding domain of a single HA
monomer binds to sialylated glycans with very weak affin-
ity, and strong avidity to host cell receptors is only achieved
by polyvalent binding of multiple HA trimers [38,39].
Several commercially available preparations of streptavidin-
conjugated microspheres were tested and the microspheres
with a high streptavidin density provided the highest
sensitivity for detection of lectin and virus binding
(Additional file 3).
Functional assessment of glycosphere receptors
Previous studies have reported that glycans adopt a dis-
tinct topology in the presence of HA [11,28,40,41]. The
topology is a function of the linkage of the terminal sialic
acid with the penultimate monosaccharide, the length of
the oligosaccharide, and its binding mode with HA. High-
affinity binding to oligosaccharides with α2-6-linked sialic
acid that are tetrasaccharide or longer (such as LSTc) has
been found to be a key feature for human adaption and
human-to-human aerosol transmission . To provide
proof of concept of our glycosphere assay, we tested the
binding characteristics of two human-adapted and two
avian-adapted hemagglutinins whose glycan-binding char-
acteristics have been studied in detail. LSTc and LSTa
were used to probe the glycan specificity of human-
adapted HAs from the Asian H2N2 pandemic of 1957–58
(A/Albany/6/1958; Alb58) and the H1N1 “swine flu” pan-
demic of 2009 (A/California/04/2009; Ca04). In addition
we tested avian-adapted HAs of the H5N1 bird flu and
an avian H2N2 strain from 2004 (A/Vietnam/1203/04,
VN1203; and A/Chicken/PA/2004, CkPA04, respectively).
Glycospheres with the avian-receptor motif LSTa bound
HAs from bothVN1203 and CkPA04 in a dose-dependent
fashion, with little binding of HAs from Alb58 and Ca04
to the avian receptor (Figure 2A). Conversely, HAs from
human viruses Alb58 and Ca04 bound with high affinity
to LSTc-glycospheres with little or no binding of HAs from
VN1203 and CkPA04 to the human receptor (Figure 2B).
These results are consistent with previous findings on
planar glycan arrays, and thus served to validate the
glycosphere assay as a reliable tool for the functional
characterization of influenza binding to host cell receptors.
While quantitative assessment of the HA-glycan inter-
action for known influenza strains is an important valid-
ation of the assay platform, the glycosphere assays are
designed to probe the glycan specificity of clinical or
veterinarian influenza isolates. A panel of representa-
tive influenza A viruses was applied to the glycospheres
for receptor-specific binding and incubated with clade/
Figure 1 Illustration of an influenza glycosphere assay.
Hobbie et al. BMC Biotechnology 2013, 13:34
Page 3 of 8
subtype-specific antibodies targeting a conserved region
on the stem of HA. Captured virus-antibody complexes
were probed with a phycoerythrin-labeled secondary anti-
body and the glycosphere suspensions were then analyzed
by quantitative flow cytometry (Figure 1). Glycosphere
analysis revealed specific binding of the H1N1 and H3N2
strains to the human receptor motif LSTc (Figure 3),
which is in agreement with the fact that these strains were
originally isolated from human patient samples. Import-
antly, the glycosphere assay worked equally well with live
Figure 2 Binding specificity of the representative receptor motifs used in the glycosphere assay. A, Recombinantly expressed
hemagglutinin of the avian-adapted influenza strains A/Chicken/PA/2004 (CkPA04, H2N2) and A/Vietnam/1203/2004 (Viet04, H5N1) binds
quantitatively and selectively to its cognate avian glycan domain. B, Hemagglutinin of the human pandemic strains A/Albany/6/58 (Alb58, H2N2)
and A/California/04/2009 (Ca04, H1N1) binds with high specificity to only its cognate human glycan domain.
Figure 3 Quantitative receptor binding analysis of a representative panel of influenza viruses. Influenza A subtypes H1N1, H3N2, as well
as influenza B showed a selective and dose-dependent binding to glycospheres coated with the human receptor motif LSTc (red). H5 strains of
both human and avian origin demonstrated highly selective binding to the avian receptor motif LSTa (blue). SM15, A/Singapore/SM15/09; Bb07,
A/Brisbane/59/07; NC99, A/New Caledonia/20/99; SM19, A/Singapore/SM19/09; WY03, A/Wyoming/03/03; HK68, A/Hong Kong/8/68; TK05, A/turkey/
Turkey/1/05; VN04, A/Vietnam/1194/04; SG97, A/duck/Singapore/97; Bb08, B/Brisbane/60/08; Tw62, B/Taiwan/2/62; Lee40, B/Lee/40. Full details of
influenza strains used in this study are provided in Additional file 4.
Hobbie et al. BMC Biotechnology 2013, 13:34
Page 4 of 8
and inactivated virus as it did with purified hemagglutinin
alone (Additional file 4). The functional characterization
of a clinical isolate of the 2009 H1N1 pandemic (SM15)
revealed a binding specificity and relative affinity (Figure 3)
that is consistent with the binding of Ca04 hemagglutinin
(Figure 2). The H5N1 strain A/Vietnam/1194/04(H5N1)
(VN1194) was found to bind to the avian but not the
human receptor motif with a binding pattern (Figure 3)
identical to the related hemagglutinin of VN1203
Assay performance characteristics
To determine the most important assay performance char-
acteristics of the glycosphere assay platform, we studied
the reproducibility, limit of detection, and robustness of
glycosphere assays. Repeating the assay for selected viral
isolates at different times, with independently prepared
reagents and non-identical virus stocks, revealed the
reliability of the assay platform and the reproducibility
of receptor-binding characteristics (Figure 4A).
To determine the limit of detection of the glycosphere
assay, we prepared a serial dilution of influenza A virus
to run low viral titers in the glycosphere array. Since
clinical influenza isolates are routinely quantified by qRT-
PCR, we wanted to specify the limit of detection in units
of threshold cycle (Ct) determined by standard diagnostic
procedures used in the clinic [42,43]. The limit of detec-
tion was consistently in the range of Ctvalues between 25
and 29 (Figure 4B). The relation of Ct values, plaque-
forming units, and TCID50is provided in Additional file 4.
Further, the glycosphere assay was probed for its robust-
ness towards various sample media. Both virus growth
medium and Universal Transport Medium (UTM), which
is used in clinical laboratories to collect nasopharyngeal
and throat swabs, did not significantly interfere with assay
sensitivity and the results were comparable to virus in
PBS (Figure 4C).
We introduce here a high-throughput suspension array
platform using glycan-coated microspheres for the func-
tional characterization of clinical and veterinarian influ-
enza isolates. The use of two distinct influenza receptor
glycans enabled us to rapidly determine the binding pref-
erence of a virus and thus assess the level of adaptation
of the virus hemagglutinin to human host cells. We
anticipate that the assay developed here will complement
traditional genotyping assays and provide a functional
assessment of the viruses for better surveillance of emer-
ging influenza strains.
Biotinylation of glycans
LS-tetrasaccharide c (LSTc) and LS-tetrasaccharide a
(LSTa; Isosep AB) were biotinylated with EZ-Link Biotin-
LC-Hydrazide (Thermo) according to the manufactur-
er’s instructions. Biotinylated glycans were collected on
a GlykoClean G Cartridge (Prozyme), eluted with water,
lyophilized, and further purified by separating the
biotinylated glycans from free biotin by HPLC. HPLC
separation was done with a GLYCOSEP™ N HPLC column
(Prozyme), running an increasing gradient (20-53%) of
50 mM ammonium formate pH 4.4 in acetonitrile. The
collected glycan fraction was lyophilized, reconstituted
twice in water to remove residual salts, and analyzed for
purity and integrity by MALDI-TOF (Additional file 5).
Purified biotinylated glycans in solution were quantified
by both a Sialic Acid Quantification Kit (Prozyme) and a
HABA Biotin Quantitation Kit (AnaSpec), following the
manufacturers’ instructions. The biotin and sialic acid
concentrations differed less than 1% from each other.
Preparation and analysis of glycospheres
Glycospheres were prepared by incubating streptavidin-
functionalized polystyrene beads with biotinylated glycans
Figure 4 Assay performance characteristics. A, Assay reproducibility as determined by three independent receptor binding assays performed
in different weeks with independently prepared reagents and non-identical virus stocks of A/Fort Monmouth/1/1947(H1N1). B, Limit of detection
as determined by comparing the fluorescent signals of increasing viral titers of A/Hong Kong/8/1968(H3N2) with the no-virus control (NV).
Statistical significance was determined by an unpaired t-test and categorized as not significant (ns), p<0.05 (*), p<0.01 (**), or p<0.001 (***). C, Assay
robustness as determined by comparing the signal intensity of identical viral titers in different assay media. A/Fort Monmouth/1/1947(H1N1) was well
detected over a range of viral titers when serially diluted in phosphate buffered saline (PBS), virus growth medium (MEM), or Universal Transport
Hobbie et al. BMC Biotechnology 2013, 13:34
Page 5 of 8
for one hour at room temperature. The glycosylated
beads were washed twice with assay buffer (PBS-1% BSA)
to remove excess glycans. The glycospheres were then
incubated with recombinant hemagglutinin, live virus,
or inactivated virus as described in detail below. After
analyte binding, the glycospheres were washed twice
each with wash buffer (PBS 0.1% Tween) and assay buf-
fer. Glycospheres were then analyzed on a BD LSRII flow
cytometer with blue (488 nm, 100 mW, LP505, BP525/50)
and yellow-green (561 nm, 50 mW, LP570, BP585/15)
laser. A comparison of different streptavidin microspheres
revealed a remarkable range of biotin-binding capacities
(Additional file 3). Singlets of high-capacity paramagnetic
glycospheres were gated from duplets and multiplets by
forward and side scatter (Additional file 6), and the mean
signal intensity of microsphere singlets was further ana-
lyzed. Non-glycosylated microspheres were used as a
negative control to assess non-specific binding of ana-
lyte. Every binding assay was performed over a range of
analyte concentration and each concentration was tested
in at least three independent assay reactions. The absolute
and relative binding signal intensity (mean ± standard
deviation, n ≥ 3) was plotted against analyte concentra-
tion. Concentration-dependent binding was verified by
a linear response when plotting on double-logarithmic
scale. GraphPad Prism 5.0 was used for data plotting
and statistical analyses.
Hemagglutinin and influenza virus
Soluble hemagglutinin was recombinantly expressed in a
baculovirus system as described previously . Details
of viruses used in this study are listed in Additional file 4.
Clinical isolates A/Singapore/SM15/2009(H1N1) and A/
Singapore/SM19/2009(H3N2) were a kind gift of Julian
Tang, Evelyn Koay, and Paul Tambyah from the National
University Health System (NUHS), Singapore. Influenza
viruses A/New Caledonia/20/99(H1N1), A/Vietnam/1194/
2004(H5N1), and A/duck/Singapore/97(H5N3) were pur-
chased as inactivated virus (Fitzgerald Inc.; NIBSC). All
other viruses were obtained from the American Type
Culture Collection (ATCC). Virus propagation in MDCK
cells, harvesting, and titer determination by both plaque
and TCID50 assays were performed using established
standard procedures , with the exception of using
Avicel RC-591F (FMC BioPolymer) instead of agarose
overlay in the plaque assays . Viral RNA was quantified
by qRT-PCR according to the protocol by Spackman et al.
, with slight adaptations in primer sequence for
quantification of 2009 H1N1 strains .
Antibodies for the quantitative detection and labelling of
captured virus were selected based on their ability to
bind to a conserved region on a wide variety of strains
within a subtype or clade. Further, the antibodies were
chosen such that the antibody binding does not interfere
with receptor binding function of the hemagglutinin on
the virus. The modular design of the glycosphere assay
allows for a choice of subtype specific, clade specific, or
universal HA antibodies that target a conserved region
on the stem of HA. In the present study, mouse mono-
clonal antibody clones C179 and F49 (Takara) were used
as clade 1 (H1N1, H2N2, H5N1) and clade 2 (H3N2)
specific antibodies, respectively. A rabbit polyclonal anti-
body against human influenza A and B (Takara) was used
to detect influenza B virus. Goat anti-mouse secondary
antibody conjugated to R-phycoerythrin (Invitrogen) was
used as fluorescent marker for flow cytometry analysis.
To account for the avidity in hemagglutinin binding to
sialylated glycans, recombinant hemagglutinin was pre-
complexed with primary and secondary antibody in a 4:2:1
ratio as described previously . In brief, hemagglutinin,
mouse anti-6X His IgG (Abcam), and RPE-conjugated goat
anti-mouse Ab were mixed in a ratio of 4:2:1 and kept
on ice for 20 min. The resulting precomplex was then
topped up with assay buffer to a final HA concentration
of 4 μg of HA per assay. A half-log serial dilution of the
precomplex in assay buffer was prepared and applied to
the glycospheres. After gently rotating the beads for two
hours at room temperature, the beads were washed and
analyzed as described above.
Undiluted culture supernatant was used in the glycosphere
assays to determine glycan binding at the highest viral titer
available for each strain. Twofold or halflog serial dilutions
of virus in assay buffer were incubated with 1 pmol pri-
mary antibody per assay for 1 hour at room temperature.
Virus-antibody aggregates were mixed thoroughly and ap-
plied to the glycospheres on ice. After overnight incuba-
tion at 4°C, the glycospheres were washed twice each with
cold wash and assay buffer, and RPE-conjugated goat
anti-mouse IgG (Invitrogen) was added at 0.2 μg per assay.
Following incubation at 4°C for another 2 hours, the
glycospheres were washed and analyzed as described above.
Additional file 1: Table S1. Correlation of receptor binding avidities
with virus transmissibility.
Additional file 2: Table S2. Glycan array affinities of influenza virus to
human and avian receptor glycans.
Additional file 3: Figure S1. Comparison of different streptravidin-
conjugated microspheres with regards to their biotin-binding capacities
and performance in glycosphere assays.
Additional file 4: Table S3. Influenza viruses used in this study.
Hobbie et al. BMC Biotechnology 2013, 13:34
Page 6 of 8
Additional file 5: Figure S2. MALDI-MS spectra of purified LST-LC-biotin.
Additional file 6: Figure S3. Gating of paramagnetic microspheres
during flow cytometry analysis.
The authors declare that they have no competing interests.
SH, KV, IB, ZS and RS wrote the manuscript. SH, KV, IB, UA, VS and RS
contributed reagents/materials/analysis tools. SH, KV, IB, ZS, RR and RS
analyzed the data. SH, IB and UA performed the experiments. SH, KV, IB, UA,
ZS, VS, RR and RS conceived and designed the experiments. All authors read
and approved the final manuscript.
Influenza A strains SM15 and SM19 were kindly provided by Julian Tang,
Evelyn Koay, and Paul Tambyah of the National University Health System
(NUHS), Singapore. Avicel was a kind gift of FMC BioPolymer. The authors
thank Ong Waichung and Loh Siew Chin for outstanding technical
assistance. This research was supported by the National Research Foundation
Singapore through the Singapore-MIT Alliance for Research and
Technology's Infectious Diseases research programme.
Received: 8 November 2012 Accepted: 18 March 2013
Published: 15 April 2013
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Cite this article as: Hobbie et al.: Modular glycosphere assays for high-
throughput functional characterization of influenza viruses. BMC
Biotechnology 2013 13:34.
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