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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Dual Recognition Element Lateral Flow Assay Toward
Multiplex Strain Specific
Influenza Virus Detection</title><author><name sortKey="Le, Thao T" sort="Le, Thao T" uniqKey="Le T" first="Thao T." last="Le">Thao T. Le</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>Imperial College London</institution>, London SW7 2AZ,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Chang, Pengxiang" sort="Chang, Pengxiang" uniqKey="Chang P" first="Pengxiang" last="Chang">Pengxiang Chang</name><affiliation><nlm:aff id="aff2">Avian Viral Diseases Program,<institution>Pirbright Institute</institution>, Woking GU24 0NF,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Benton, Donald J" sort="Benton, Donald J" uniqKey="Benton D" first="Donald J." last="Benton">Donald J. Benton</name><affiliation><nlm:aff id="aff3">Worldwide Influenza Centre,<institution>Francis Crick Institute</institution>, London NW1 1AT,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Mccauley, John W" sort="Mccauley, John W" uniqKey="Mccauley J" first="John W." last="Mccauley">John W. Mccauley</name><affiliation><nlm:aff id="aff3">Worldwide Influenza Centre,<institution>Francis Crick Institute</institution>, London NW1 1AT,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Iqbal, Munir" sort="Iqbal, Munir" uniqKey="Iqbal M" first="Munir" last="Iqbal">Munir Iqbal</name><affiliation><nlm:aff id="aff2">Avian Viral Diseases Program,<institution>Pirbright Institute</institution>, Woking GU24 0NF,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Cass, Anthony E G" sort="Cass, Anthony E G" uniqKey="Cass A" first="Anthony E. G." last="Cass">Anthony E. G. Cass</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>Imperial College London</institution>, London SW7 2AZ,<country>U.K.</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">28558471</idno><idno type="pmc">5514394</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514394</idno><idno type="RBID">PMC:5514394</idno><idno type="doi">10.1021/acs.analchem.7b01149</idno><date when="2017">2017</date><idno type="wicri:Area/Pmc/Corpus">000086</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000086</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Dual Recognition Element Lateral Flow Assay Toward
Multiplex Strain Specific
Influenza Virus Detection</title><author><name sortKey="Le, Thao T" sort="Le, Thao T" uniqKey="Le T" first="Thao T." last="Le">Thao T. Le</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>Imperial College London</institution>, London SW7 2AZ,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Chang, Pengxiang" sort="Chang, Pengxiang" uniqKey="Chang P" first="Pengxiang" last="Chang">Pengxiang Chang</name><affiliation><nlm:aff id="aff2">Avian Viral Diseases Program,<institution>Pirbright Institute</institution>, Woking GU24 0NF,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Benton, Donald J" sort="Benton, Donald J" uniqKey="Benton D" first="Donald J." last="Benton">Donald J. Benton</name><affiliation><nlm:aff id="aff3">Worldwide Influenza Centre,<institution>Francis Crick Institute</institution>, London NW1 1AT,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Mccauley, John W" sort="Mccauley, John W" uniqKey="Mccauley J" first="John W." last="Mccauley">John W. Mccauley</name><affiliation><nlm:aff id="aff3">Worldwide Influenza Centre,<institution>Francis Crick Institute</institution>, London NW1 1AT,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Iqbal, Munir" sort="Iqbal, Munir" uniqKey="Iqbal M" first="Munir" last="Iqbal">Munir Iqbal</name><affiliation><nlm:aff id="aff2">Avian Viral Diseases Program,<institution>Pirbright Institute</institution>, Woking GU24 0NF,<country>U.K.</country></nlm:aff></affiliation></author><author><name sortKey="Cass, Anthony E G" sort="Cass, Anthony E G" uniqKey="Cass A" first="Anthony E. G." last="Cass">Anthony E. G. Cass</name><affiliation><nlm:aff id="aff1">Department of Chemistry,<institution>Imperial College London</institution>, London SW7 2AZ,<country>U.K.</country></nlm:aff></affiliation></author></analytic><series><title level="j">Analytical Chemistry</title><idno type="ISSN">0003-2700</idno><idno type="eISSN">1520-6882</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="ac-2017-01149n_0005" id="ab-tgr1"></graphic></p><p>Different influenza
virus strains have caused a number of recent
outbreaks killing scores of people and causing significant losses
in animal farming. Simple, rapid, sensitive, and specific detection
of particular strains, such as a pandemic strain versus a previous
seasonal influenza, plays a crucial role in the monitoring, controlling,
and management of outbreaks. In this paper we describe a dual recognition
element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer
with an antibody for use as a point-of-care platform which can detect
particular strains of interest. The combination is used to overcome
the individual limitations of antibodies’ cross-reactivity
and aptamers’ slow binding kinetics. In the detection of influenza
viruses, we show that DRELFA can discriminate a particular virus strain
against others of the same subtype or common respiratory diseases
while still exhibiting fast binding kinetic of the antibody-based
lateral flow assay (LFA). The improvement in specificity that DRELFA
exhibits is an advantage over the currently available antibody-based
LFA systems for influenza viruses, which offer discrimination between
influenza virus types and subtypes. Using quantitative real-time PCR
(qRT-PCR), it showed that the DRELFA is very effective in localizing
the analyte to the test line (consistently over 90%) and this is crucial
for the sensitivity of the device. In addition, color intensities
of the test lines showed a good correlation between the DRELFA and
the qRT-PCR over a 50-fold concentration range. Finally, lateral flow
strips with a streptavidin capture test line and an anti-antibody
control line are universally applicable to specific detection of a
wide range of different analytes.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Taubenberger, J" uniqKey="Taubenberger J">J. Taubenberger</name></author><author><name sortKey="Morens, M D" uniqKey="Morens M">M. D. Morens</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kilbourne, D E" uniqKey="Kilbourne D">D. E. Kilbourne</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Boivin, G" uniqKey="Boivin G">G. Boivin</name></author><author><name sortKey="Cote, S" uniqKey="Cote S">S. Côté</name></author><author><name sortKey="Dery, P" uniqKey="Dery P">P. Déry</name></author><author><name sortKey="De Serres, G" uniqKey="De Serres G">G. De Serres</name></author><author><name sortKey="Bergeron, M G" uniqKey="Bergeron M">M. G. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Anal Chem</journal-id><journal-id journal-id-type="iso-abbrev">Anal. Chem</journal-id><journal-id journal-id-type="publisher-id">ac</journal-id><journal-id journal-id-type="coden">ancham</journal-id><journal-title-group><journal-title>Analytical Chemistry</journal-title></journal-title-group><issn pub-type="ppub">0003-2700</issn><issn pub-type="epub">1520-6882</issn><publisher><publisher-name>American
Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28558471</article-id><article-id pub-id-type="pmc">5514394</article-id><article-id pub-id-type="doi">10.1021/acs.analchem.7b01149</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dual Recognition Element Lateral Flow Assay Toward
Multiplex Strain Specific
Influenza Virus Detection</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Le</surname><given-names>Thao T.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Chang</surname><given-names>Pengxiang</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Benton</surname><given-names>Donald J.</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath4"><name><surname>McCauley</surname><given-names>John W.</given-names></name><xref rid="aff3" ref-type="aff">§</xref></contrib><contrib contrib-type="author" id="ath5"><name><surname>Iqbal</surname><given-names>Munir</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath6"><name><surname>Cass</surname><given-names>Anthony E. G.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref></contrib><aff id="aff1"><label>†</label>Department of Chemistry,<institution>Imperial College London</institution>, London SW7 2AZ,<country>U.K.</country></aff><aff id="aff2"><label>‡</label>Avian Viral Diseases Program,<institution>Pirbright Institute</institution>, Woking GU24 0NF,<country>U.K.</country></aff><aff id="aff3"><label>§</label>Worldwide Influenza Centre,<institution>Francis Crick Institute</institution>, London NW1 1AT,<country>U.K.</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>E-mail: <email>t.cass@imperial.ac.uk</email>. Phone: <phone>+44-20-7594-5195</phone>.</corresp></author-notes><pub-date pub-type="epub"><day>30</day><month>05</month><year>2017</year></pub-date><pub-date pub-type="ppub"><day>20</day><month>06</month><year>2017</year></pub-date><volume>89</volume><issue>12</issue><fpage>6781</fpage><lpage>6786</lpage><history><date date-type="received"><day>28</day><month>03</month><year>2017</year></date><date date-type="accepted"><day>30</day><month>05</month><year>2017</year></date></history><permissions><copyright-statement>Copyright © 2017 American Chemical Society</copyright-statement><copyright-year>2017</copyright-year><copyright-holder>American Chemical Society</copyright-holder><license><license-p>This is an open access article published under a Creative Commons Attribution (CC-BY) <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html">License</ext-link>, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="ac-2017-01149n_0005" id="ab-tgr1"></graphic></p><p>Different influenza
virus strains have caused a number of recent
outbreaks killing scores of people and causing significant losses
in animal farming. Simple, rapid, sensitive, and specific detection
of particular strains, such as a pandemic strain versus a previous
seasonal influenza, plays a crucial role in the monitoring, controlling,
and management of outbreaks. In this paper we describe a dual recognition
element lateral flow assay (DRELFA) which pairs a nucleic acid aptamer
with an antibody for use as a point-of-care platform which can detect
particular strains of interest. The combination is used to overcome
the individual limitations of antibodies’ cross-reactivity
and aptamers’ slow binding kinetics. In the detection of influenza
viruses, we show that DRELFA can discriminate a particular virus strain
against others of the same subtype or common respiratory diseases
while still exhibiting fast binding kinetic of the antibody-based
lateral flow assay (LFA). The improvement in specificity that DRELFA
exhibits is an advantage over the currently available antibody-based
LFA systems for influenza viruses, which offer discrimination between
influenza virus types and subtypes. Using quantitative real-time PCR
(qRT-PCR), it showed that the DRELFA is very effective in localizing
the analyte to the test line (consistently over 90%) and this is crucial
for the sensitivity of the device. In addition, color intensities
of the test lines showed a good correlation between the DRELFA and
the qRT-PCR over a 50-fold concentration range. Finally, lateral flow
strips with a streptavidin capture test line and an anti-antibody
control line are universally applicable to specific detection of a
wide range of different analytes.</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>ac7b01149</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>ac-2017-01149n</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id="sec1">Over the
years, influenza virus
pandemics and outbreaks have resulted in high morbidity and mortality
in both people and animals. The most deadly pandemic of modern times
was the “Spanish flu” 1918-19 a H1N1 strain which caused
tens of millions of deaths worldwide.<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> Other
pandemics of the 20th century that caused hundreds of thousands of
deaths were the “Asian flu” 1957-58 a H2N2 strain and
the “Hong Kong flu” 1968-69 a H3N2 strain.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> This century the 2009 “swine flu”
pandemic infected tens of millions of people, with tens of thousands
of lab-confirmed deaths.<sup><xref ref-type="bibr" rid="ref3">3</xref></sup> Outbreaks of
H5N1 strains has resulted in mass culling of millions of farmed birds,<sup><xref ref-type="bibr" rid="ref4">4</xref>,<xref ref-type="bibr" rid="ref5">5</xref></sup> and according to WHO’s January 2017 report,<sup><xref ref-type="bibr" rid="ref6">6</xref></sup> 856 cases of zoonotic human infection were reported, of
which 452 were fatal. Diagnosis of influenza using PCR and virus culture
assays are highly sensitive and specific methods that have been used
as standards, with the former allowing identification of specific
strains and with a detection limit of tens of viral RNA copies.<sup><xref ref-type="bibr" rid="ref7">7</xref>,<xref ref-type="bibr" rid="ref8">8</xref></sup> These methods are, however, laboratory-based and therefore are unable
to meet the need for field diagnostic tests during a pandemic outbreak.
Therefore, methods for detection of viruses that meet the WHO ASSURED
criteria<sup><xref ref-type="bibr" rid="ref9">9</xref></sup> play a crucial role not only
in diagnosis during a pandemic but also in control and management
of less serious outbreaks.</p><p>Point of care immunoassays are built
primarily around the lateral
flow assay (LFA) format, typically employing antibodies with visual
detection of the endpoint immune complex formation through the use
of a nanoparticle label.<sup><xref ref-type="bibr" rid="ref10">10</xref>,<xref ref-type="bibr" rid="ref11">11</xref></sup> Antibodies have been
well established for several decades as a class of recognition molecules
and used for many applications due to their high binding affinity
and fast association kinetics. These characteristics mean that antibodies
have been used in many diagnostic tests both laboratory-based, e.g.,
ELISAs<sup><xref ref-type="bibr" rid="ref12">12</xref>,<xref ref-type="bibr" rid="ref13">13</xref></sup> and in decentralized environments, e.g.,
LFA.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup></p><p>Another notable class of recognition
molecules is that of aptamers.
Since the first aptamers were reported in 1990,<sup><xref ref-type="bibr" rid="ref15">15</xref>,<xref ref-type="bibr" rid="ref16">16</xref></sup> hundreds have been selected for both diagnostic and therapeutic
applications.<sup><xref ref-type="bibr" rid="ref17">17</xref>,<xref ref-type="bibr" rid="ref18">18</xref></sup> Aptamers have frequently been
compared to antibodies in regards of their affinity and specificity
toward their targets as well as in their potential uses. An additional
advantage of aptamers is that they can function under conditions that
often cause antibodies to become irreversibly inactivated.<sup><xref ref-type="bibr" rid="ref19">19</xref></sup></p><p>Here we focus on another advantage of
aptamers that is important
in diagnostic applications, the ability to select high specificity
variants by employing counter-selection to eliminate cross reactivity
with closely related molecules. Examples of this specificity include
discrimination between theophylline and caffeine despite the two molecules
differing only by a methyl group,<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> an
aptamer that enantioselectivly binds <sc>l</sc>-arginine over <sc>d</sc>-arginine,<sup><xref ref-type="bibr" rid="ref21">21</xref></sup> and an aptamer that
can distinguish between the human glioblastoma cell lines U87MG and
T98G,<sup><xref ref-type="bibr" rid="ref22">22</xref></sup> where they only differ by a mutation
in p53. This distinctive feature led us to employ aptamers to achieve
strain-specific discrimination in LFAs for influenza virus detection.
Currently available LFAs based on antibodies against influenza virus
may have type or subtype specificity depending on which antibodies
have been chosen and what the assay is designed to achieve, for example,
distinguishing influenza A from B.<sup><xref ref-type="bibr" rid="ref23">23</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup></p><p>Aptamers,
because of the nature of the SELEX (systematic evolution
of ligand by exponential enrichment) process, can take advantage of
counter-selection steps during their production and therefore can
provide the required discrimination between strains. Aptamers showing
discrimination between strains of a given subtype have been demonstrated
where, for example, a RNA aptamer has been selected to bind to one
H3N2 strain, A/Panama/2007/99, but not to other strains of the H3N2
subtype.<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> This RNA aptamer binds to virus
envelope’s most abundant protein, the hemagglutinin, H3, and
is able to distinguish between the particular strain it selected for
and other strains of the H3N2 subtype. This discrimination was achieved
by counter-selection against whole virus particles of a closely related
H3N2 strain during the SELEX process. The strain specificity of this
aptamer was demonstrated using surface plasmon resonance with other
H3N2 strains (A/Wyoming/3/2003, A/Sydney/05/97, and A/Wuhan/359/95),<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> but it has not previously been employed in LFAs
to achieve strain-specific detection.</p><p>The rapid nature of LFAs
for influenza virus detection is of great
benefit for transmission control and prevention, as the viruses can
spread in a variety of locales and in many cases are highly contagious
through air and physical contact routes. LFAs as point-of-care devices
have long been considered to be useful for the detection of viral
infections.<sup><xref ref-type="bibr" rid="ref26">26</xref></sup> Antibodies for influenza
viruses can show different, and often broad, ranges of specificity.<sup><xref ref-type="bibr" rid="ref27">27</xref>,<xref ref-type="bibr" rid="ref28">28</xref></sup> This is likely due to the monoclonal antibodies arising from an
immune response to the antigen of interest but without a counter-selection
mechanism to allow discrimination against other strains.<sup><xref ref-type="bibr" rid="ref29">29</xref></sup> Influenza viruses are known to acquire amino
acid substitutions with great ease. These substitutions can lead to
a significant difference in virulence, transmission, and/or mortality
rate so that distinguishing between more and less virulent strains
is important. An illustration of this is the 2009 influenza A (H1N1pdm09)
“swine flu” pandemic strain that emerged to replace
the classic seasonal flu, also a H1N1 subtype, and at one stage the
two viruses were cocirculating.<sup><xref ref-type="bibr" rid="ref30">30</xref></sup></p><p>A LFA for detection of small molecules (adenosine and cocaine)
that completely replaced antibodies by aptamers was first introduced
by Lu et al.,<sup><xref ref-type="bibr" rid="ref31">31</xref>,<xref ref-type="bibr" rid="ref32">32</xref></sup> but since then only a handful
of aptamer LFAs have been described.<sup><xref ref-type="bibr" rid="ref33">33</xref>,<xref ref-type="bibr" rid="ref34">34</xref></sup> One major
drawback of using aptamers in LFAs is that the control line employed
for assay assurance often yields a broad, indistinct shape, owing
to the slow kinetics of target binding by immobilized aptamers.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> It would be highly advantageous to combine the
specificity of aptamers with the rapid surface binding kinetics of
the streptavidin–biotin interaction,<sup><xref ref-type="bibr" rid="ref36">36</xref>,<xref ref-type="bibr" rid="ref37">37</xref></sup> and we have employed this combination in DRELFA (<xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>).</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Schematic diagram of
a DRELFA for detection of a virus. An anti-virus
antibody (Y-shape) is conjugated with GNPs (golden oval). An aptamer
(black line) has a biotin (blue star) at its 5′-or 3′-end.
(a) In the presence of the specific virus, both the aptamer and the
gold nanoparticle (GNP)-conjugated antibody bind to the virus (brown-textured
sphere) and the biotin on the aptamer enables the complex to be bound
onto the streptavidin test line (purple line) and the detection can
be made by the color of GNPs. (b) Absence of the specific virus would
result in the capture of GNPs on the secondary antibody control line
(red line) only.</p></caption><graphic xlink:href="ac-2017-01149n_0001" id="gr1" position="float"></graphic></fig><p>To illustrate a DRELFA
that can detect a specific virus strain
of interest, we paired a highly strain-specific aptamer with a biotin
at either the 5′ or 3′ end with a less specific antibody
labeled with gold nanoparticles (GNPs). The biotin-tagged aptamer
is captured by streptavidin printed at the test line and this yields
rapid capture kinetics. The excess of GNP-labeled monoclonal antibody
is captured by a secondary antibody printed at the control line which
also has fast kinetics. In the presence of the virus, both the aptamer
and the monoclonal antibody bind to the virus to form a complex which
is then captured by streptavidin through the biotinylated aptamer
and shown by the color of the complexed antibody-GNPs conjugates on
the test line. In the absence of virus, the complex is not formed
and therefore no signal of GNPs is shown on the test line. This approach
enables fast capture kinetics on both test and control lines and has
been used in double antibody-based LFAs.<sup><xref ref-type="bibr" rid="ref10">10</xref></sup></p><sec id="sec2"><title>Materials and Methods</title><sec id="sec2.1"><title>Materials and Reagents</title><p>All the oligonucleotides
were
synthesized and HPLC purified by Integrated DNA Technologies. and
the sequences are listed in the Supporting Information,<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">SI Table</ext-link>. Anti-hemagglutinin H3 antibody (ab82454),
anti-hemagglutinin H5 antibody (ab82455), and goat anti-mouse antibody
(ab6708) were from Abcam. Anti-hemagglutinin H7 antibody (ABIN573415)
from Antibodies-Online and anti-hemagglutinin H9 antibody (ID, 955HA9)
was produced at the Pirbright Institute (U.K.).<sup><xref ref-type="bibr" rid="ref38">38</xref></sup> HAuCl4 was purchased from Sigma. HiFlow Pus membrane, glass
fiber conjugate sheet, and cellulose fiber roll were from Merck Millipore.
Other common reagents were bought from Sigma unless specified.</p></sec><sec id="sec2.2"><title>Methods</title><sec id="sec2.2.1"><title>Preparation
of the RNA Aptamers</title><p>The ssDNA template
sequence for the A/Panama/2007/99 (H3N2) was <underline>TCTAA TACGA
CTCAC TATA</underline>G GGAGA ATTCC GACCA GAAGG GTTAG CAGTC GGCAT
GCGGT ACAGA CAGAC CTTTC CTCTC TCCTT CCTCT TCT (underlined for T7 promoter).
This ssDNA template was then amplified using a forward primer (H3P07)
TCTAA TACGA CTCAC TATAG GGAGA ATTCC GACCA GAAG and a reverse primer
(RevH3P07), OneTaq DNA polymerase (New England Biolabs), and the dNTP
mix (Promega) to generate and amplify dsDNA. The obtained dsDNA was
purified by ethanol precipitation and transcribed into the RNA using
an AmpliScribe T7-Flash transcription kit from Epicenter. The RNA
obtained from the transcription was then purified using 10% polyacrylamide
TBE-urea gel electrophoresis. The RNA sequence obtained (GGGAG AAUUC
CGACC AGAAG GGUUA GCAGU CGGCA UGCGG UACAG ACAGA CCUUU CCUCU CUCCU
UCCUC UUCU), the RNA aptamer for the A/Panama/2007/99 (H3N2) (RNAH3P07Apt),
was hybridized with excess DNA biotin linker (ApH3P07Linker, 5′-biotin-TEG-
AGAAGAGGAAGGAGAGAGG) with a molar ratio of RNA aptamer–DNA
biotin linker = 1:1.2 to add a biotin on the RNA aptamer 3′-end.
The excess amount of DNA biotin linker was removed using 10 kDa Amicon
centrifugal filters (Millipore) prior to being used in the DRELFA.</p><p>For other aptamers, their sequences and the corresponding ssDNA
templates and the primer pairs used for PCR were specified in <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">SI Table</ext-link> in the Supporting Information.</p></sec><sec id="sec2.2.2"><title>Synthesis
of Gold Nanoparticles (GNPs)</title><p>GNPs were synthesized
by the sodium citrate method.<sup><xref ref-type="bibr" rid="ref39">39</xref></sup> GNPs were
synthesized in a 50 mL batch using a 100 mL glass flash bottle with
a glass-coated stir bar. First, 49 mL of deionized ultrafiltered water
was poured into the flash bottle with the stir bar, and then 1 mL
of freshly made 12.7 mM chloroauric acid solution was added. The bottle
was placed on a magnetic stirrer hot plate (200 °C, 150 rpm)
and brought to boiling. A variable volume of 38.8 mM of trisodium citrate solution was added, and the solution
was kept at boiling for a further 10 min. It was then cooled down
to room temperature with stirring (100 rpm). Volumes of 0.8 mL for
GNPs with nominal diameters of 25 nm, 0.5 mL for GNPs of 40 nm, and
0.4 mL for GNPs of 60 nm were used. The diameters were then determined
experimentally using Nanoparticle Tracking Analysis (NTA) and gave
values of 37, 55, and 72 nm, respectively.</p></sec><sec id="sec2.2.3"><title>Conjugation of Antibodies
to GNPs</title><p>Antibodies were transferred
to 2 mM borax buffer (pH 9.0) using the Zebra spin desalting column
(7 kDa MWCO) from ThermoFisher. Suitable volumes of a borax stock
solution (500 mM, pH 9.0) to a final concentration of 2 mM were added
to the freshly made GNPs. Anti-hemagglutinin H3 antibody and anti-hemagglutinin
H5 antibody were conjugated with the 37 nm GNPs, anti-hemagglutinin
H7 antibody was conjugated with the 55 nm GNPs, and anti-hemagglutinin
H9 antibody was conjugated with the 72 nm GNPs. Antibody stock solutions
were added to a final concentration of 5 μg/mL in a solution
of GNPs having an o.d. of 1 at the wavelength
of maximum absorbance. The solutions were placed on a tube rotator
at room temperature for 4 h and then in a cold room (4 °C) overnight.
Finally, 5% BSA in 2 mM borax (pH 9.0) was added to give a final concentration
of 0.5% BSA and rotated at room temperature for 30 min. The antibody-GNP
conjugates were then washed 3 times using centrifugation with buffer
(0.5% BSA, 4% sucrose, 0.02% Tween20 in 20 mM HEPES, pH 7.3).</p></sec><sec id="sec2.2.4"><title>Preparation
of the Lateral Flow Test Strips</title><p>A lateral
flow strip was assembled from 3 parts (bottom up): a glass fiber conjugate
pad (26 mm), HiFlow Plus membrane (25 mm), and wicking pad (15 mm).
Before assembling, the HiFlow Plus membrane was printed with a test
line and a control line and the conjugate pad was treated as described
below.</p></sec><sec id="sec2.2.5"><title>Conjugate Pad Treatment</title><p>Conjugate pads were dipped
into a solution containing 1% BSA, 0.2% Tween20, 4% sucrose in 20
mM HEPES, pH 7.3 for 30 min. The pads were then taken out and left
to dry overnight.</p></sec><sec id="sec2.2.6"><title>Printing of Test and Control Lines</title><p>The membrane was
printed with a streptavidin test line and an anti-mouse antibody control
line using a Scienion sciFLEXARRAYER S3 with a type 4 piezo. The test
line consisted of 10 lines of 50 μm pitch and were printed at
a speed of 0.1 μL/cm of 2 mg/mL streptavidin in 10 mM acetate
buffer pH 5.0. The control line consisted of 5 lines of 100 μm
pitch printed with 0.1 μL/cm of 1 mg/mL of goat anti-mouse antibody
in 10 mM borax buffer pH 8.5.</p></sec><sec id="sec2.2.7"><title>Assembling the Strip</title><p>The HiFlow Plus membrane of 25
mm width was already placed on a 60 mm adhesive plastic backing card.
The HiFlow Plus membrane is cellulose ester with a nominal capillary
flow of 180 s/4 cm. Conjugate pads were cut from a Millipore glass
fiber sheet with a width of 26 mm and the wicking pads were cut from
Millipore cellulose fiber sheet to bands with a width of 15 mm. The
conjugate pads and the wicking pads were attached to the HiFlow Plus
membrane card with a 2 mm overlap between pads. The assembled card
was then placed between 2 clean plastic sheets and pressed lightly
with a roller. The assembled card was cut to 5 mm wide strips using
a Fellowes Astro Cutter. The strips were then housed in plastic cassettes
and stored in an amber tinted desiccator cabinet with humidity around
20%.</p></sec><sec id="sec2.2.8"><title>Lateral Flow Assay</title><p>The assay was run with 100 μL
samples (in 20 mM Hepes, 0.1% Triton X-100, pH 7.4). Typically, the
time between adding the samples and recording the images was 15 min.
The images were taken with a digital camera.</p></sec></sec></sec><sec id="sec3"><title>Results and Discussion</title><sec id="sec3.1"><title>Influenza
Virus Strain Specificity of the Aptamer</title><p>Enzyme
linked oligonucleotide assay (ELONA) data shown in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>a confirms that aptamers with
a high level of discrimination can be specifically selected for the
A/Panama/2007/99 strain of H3N2 virus over other H3N2 strains by suitable
choice of counter-selection regimes.<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> A
commercially available anti-H3 HA monoclonal antibody, on the other
hand, binds all of the three tested strains of the H3N2 subtype as
shown in <xref rid="fig2" ref-type="fig">Figure <xref rid="fig2" ref-type="fig">2</xref></xref>b.
We aim to transfer this high specificity of aptamers into a LFA diagnostic
platform using a format we call dual recognition element lateral flow
assay (DRELFA).</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>(a) Binding of an RNA aptamer to whole virus particles
of 3 different
strains of the influenza A H3N2 using ELONA: A/Panama/2007/99 (Panama/99),
A/Aichi/2/68 (Aichi/68), and A/Udorn/307/72 (Udorn/72). The aptamer
can discriminate one strain from others of the H3N2 subtype. (b) A
monoclonal antibody shows binding to all three strains of the influenza
A H3N2: Panama/99, Aichi/68, and Udorn/72. In all cases 7 × 10<sup>8</sup> virus particles were added to each well for immobilization
of the virus on the microtiter plate.</p></caption><graphic xlink:href="ac-2017-01149n_0002" id="gr2" position="float"></graphic></fig></sec><sec id="sec3.2"><title>Strain-Specific Detection of Influenza Virus Using DRELFA</title><p>To demonstrate strain-specific detection in the proposed format,
a DRELFA was built from paring a Panama/99-specific RNA aptamer<sup><xref ref-type="bibr" rid="ref25">25</xref></sup> having a biotin at its 5′-end<sup><xref ref-type="bibr" rid="ref40">40</xref></sup> with an anti-H3 antibody conjugated with 37
nm GNPs. As shown in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>a this DRELFA detected the Panama/99 strain without any observable
cross-reactivity with two other strains of the H3N2 subtype, Aichi/68
and Udorn/72. On the other hand, as shown in <xref rid="fig3" ref-type="fig">Figure <xref rid="fig3" ref-type="fig">3</xref></xref>b, a conventional antibody-based LFA with
these virus strains show cross-reactivity between the different strains
of this subtype. Conventional LFA systems built from different antibodies
would show different results depending on how broad a cross-reactivity
they exhibit.</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Specificity of LFA’s (a) DRELFA: The sample with
Panama/99
has 2 lines showing visual detection of this virus strain while measurements
for Udorn/72 and Aichi/68 samples have only 1 line (control quality)
indicating that this DRELFA for Panama/99 virus does not have any
cross reactivity with other strains of this H3N2 subtype. (b) Antibody-LFA:
all 3 samples of the influenza A H3N2 displayed 2 lines showing visual
detection of the viruses, indicating that the conventional LFA does
not differentiate between the three virus strains of the H3N2 subtype.
The measurements were performed with samples of 10<sup>8</sup> virus
particles. The sample volume was 100 μL.</p></caption><graphic xlink:href="ac-2017-01149n_0003" id="gr3" position="float"></graphic></fig><p>Furthermore, the DRELFA system were also used to assess nonspecific
binding with three different viruses (Newcastle disease virus (NDV)
and infectious bronchitis virus (IVB) that cause common viral respiratory
diseases and an influenza B virus (Florida/04/2006). As shown in the
Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">SI1 Figure</ext-link>, the
DRELFA demonstrated that even for samples of high concentrations of
virus particles, 100% specificity with no visually detectable signal
against any of these three viruses. This is probably due to the aptamer
produced with counter-selection for high specificity can also reduce
nonspecific binding. Finally, the production of DRELFA strips can
be readily adapted to a wide range of targets as the test line is
always streptavidin and the control line is always a secondary antibody.</p><p>To assess the distribution of virus particles on the DRELFA strips
they were analyzed using quantitative real time-PCR (qRT-PCR). Four
different regions of the LF membrane were taken: before the test line,
the test line, between the test and control lines, and the control
line. RNA was extracted from these regions, and a probe for the M
gene was used to reverse transcribe it into cDNA for quantitation
by qRT-PCR as described by Londt et al.<sup><xref ref-type="bibr" rid="ref41">41</xref></sup> The corresponding virus copies of the extracted regions of the samples
are shown in <xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>a. The blue bars represent the number of copies of viral RNA at each
location indicating that the great majority (consistently over 90%)
of the viral RNA is at the test line. This confirmed that DRELFA is
very effective in localizing the analyte to the test line and this
is crucial for the sensitivity of the device.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>(a) Distribution of the
virus particles on the lateral flow membranes
were analyzed using qRT-PCR. It shows that consistently >90% of
the
virus particles are captured on the test line. (b) Comparison of the
intensity of the test lines’ signals to the virus copies obtained
from the RNA using qRT-PCR at different virus dilutions. The image
intensity and the RNA copy number have a correlation coefficient of
0.995.</p></caption><graphic xlink:href="ac-2017-01149n_0004" id="gr4" position="float"></graphic></fig><p>The color of the test line is
visible down to 50-fold dilution,
equivalent to 2 × 10<sup>6</sup> virus particles. The typical
influenza virus viral load range 10<sup>9</sup> to 10<sup>3</sup> copies/mL
in respiratory samples depending on the time when the samples were
taken and the virus strains.<sup><xref ref-type="bibr" rid="ref42">42</xref>−<xref ref-type="bibr" rid="ref44">44</xref></sup> The DRELFA can therefore detect
the samples taken in the early days of the symptoms (where the viral
loads are higher). We also used simple image analysis to compare the
color intensities of the visible test lines with the number of virus
RNA copies obtained from the qRT-PCR. <xref rid="fig4" ref-type="fig">Figure <xref rid="fig4" ref-type="fig">4</xref></xref>b shows a very good correlation between the
two, indicating consistency between the qRT-PCR results and visual
signals.</p></sec><sec id="sec3.3"><title>Choice of Aptamers and the Specificity of DRELFAs</title><p>The
specificity of DRELFAs depends on the specificity of the aptamer employed.
As shown in <xref rid="fig1" ref-type="fig">Figure <xref rid="fig1" ref-type="fig">1</xref></xref>, strain-specific aptamers can be used; however, aptamers can also
be made for a broader range of influenza viruses (e.g., the H5N1 and
H7N1 subtypes)<sup><xref ref-type="bibr" rid="ref45">45</xref>,<xref ref-type="bibr" rid="ref46">46</xref></sup> depending on how the selections
and counter selections are designed. We performed ELONA binding assays
on an aptamer with cross-subtype binding, and the data are shown in
the Supporting Information <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">SI2 Figure</ext-link>.
The DRELFAs are based on aptamers with different levels of specificity
such as strain-specific binding to influenza A/Panama/2007/99 and
more general for subtypes H5N, H7N1, and H9N2. Using the same type
of DRELFA strips that have test lines printed with streptavidin and
the control lines printed with a secondary antibody, we also have
prepared DRELFA devices to detect virus strains of other subtypes
as shown in the <xref rid="tbl1" ref-type="other">Table <xref rid="tbl1" ref-type="other">1</xref></xref> and in the Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">SI3 Figure 1,2</ext-link>.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><title>Detection of Viruses Using DRELFAs</title></caption><table frame="hsides" rules="groups" border="0"><colgroup><col align="left"></col><col align="left"></col><col align="left"></col><col align="left"></col></colgroup><thead><tr><th style="border:none;" align="center">virus strain</th><th style="border:none;" align="center">H3N2 DRELFA<xref rid="t1fn1" ref-type="table-fn">a</xref></th><th style="border:none;" align="center">H5N1 DRELFA<xref rid="t1fn2" ref-type="table-fn">b</xref></th><th style="border:none;" align="center">H9N2 DRELFA<xref rid="t1fn3" ref-type="table-fn">c</xref></th></tr></thead><tbody><tr><td style="border:none;" align="left">A/Panama/2007/99 (H3N2)</td><td style="border:none;" align="left">+</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">–</td></tr><tr><td style="border:none;" align="left">A/Aichi/2/68 (H3N2)</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">–</td></tr><tr><td style="border:none;" align="left">A/Udorn/307/72 (H3N2)</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">–</td></tr><tr><td style="border:none;" align="left">A/Vietnam/1203/2004 (H5N1)</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">+</td><td style="border:none;" align="left">–</td></tr><tr><td style="border:none;" align="left">A/turkey/Turkey/1/2005 (H5N1)</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">+</td><td style="border:none;" align="left">–</td></tr><tr><td style="border:none;" align="left">A/turkey/Italy/1279/99 (H7N1)<xref rid="t1fn4" ref-type="table-fn">d</xref></td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">+</td><td style="border:none;" align="left">–</td></tr><tr><td style="border:none;" align="left">A/chicken/Pakistan/UDL/2008 (H9N2)</td><td style="border:none;" align="left">–</td><td style="border:none;" align="left">+</td><td style="border:none;" align="left">+</td></tr></tbody></table><table-wrap-foot><fn id="t1fn1"><label>a</label><p>Pairing an RNA aptamer for Panama/99
(H3N2) and an anti H3 antibody.</p></fn><fn id="t1fn2"><label>b</label><p>Paring an RNA aptamer for H5N1 and
an anti H5 (*H7) antibody.</p></fn><fn id="t1fn3"><label>c</label><p>Pairing a DNA aptamer for H9N2 and
an anti H9 antibody.</p></fn><fn id="t1fn4"><label>d</label><p>See
the Supporting Information, <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">SI3 Figure 1,2</ext-link>.</p></fn></table-wrap-foot></table-wrap></sec></sec><sec id="sec4"><title>Conclusion</title><p>We
have demonstrated that pairing an aptamer and an antibody in
a DRELFA format offers direct detection of whole virus particles with
high specificity, thereby allowing discrimination of a specific influenza
strain (A/H3N2/Panama/2007/99) as a model target. The assay had a
detection limit of 2 × 10<sup>6</sup> virus particles. DRELFA
also showed no detectable signal for with neither NDV and IBV (common
respiratory viruses) nor with an influenza B virus. Using quantitative
real-time PCR (qRT-PCR), we showed that the DRELFA is very effective
in capturing the analyte at the test line (capture efficiencies consistently
over 90%), which is crucial for the sensitivity of the device. In
addition, color intensities of the test lines in DRELFA showed a good
correlation with qRT-PCR over a concentration range of 50-fold. Finally
a lateral flow format that combinines a biotinylated aptamer with
a streptavidin test line and a secondary antibody control line, as
with double antibody LFAs, avoids the need to optimize the deposition
of different capture antibodies.</p></sec></body><back><notes id="notes-1" notes-type="si"><title>Supporting Information Available</title><p>The Supporting Information
is available free of charge on the ACS Publications Web site. SI Table
for . The Supporting Information is available free of charge on the <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org">ACS Publications website</ext-link> at
DOI: <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/abs/10.1021/acs.analchem.7b01149">10.1021/acs.analchem.7b01149</ext-link>.<list id="silist" list-type="simple"><list-item><p>Oligonucleotide
sequences, cross-reactivity test of
DRELFA, specificity of aptamers, and other influenza virus subtypes’
DRELFA (<ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/doi/suppl/10.1021/acs.analchem.7b01149/suppl_file/ac7b01149_si_001.pdf">PDF</ext-link>)</p></list-item></list></p></notes><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sifile1"><media xlink:href="ac7b01149_si_001.pdf"><caption><p>ac7b01149_si_001.pdf</p></caption></media></supplementary-material></sec><notes notes-type="" id="notes-2"><title>Author Contributions</title><p>All
authors
have given approval to the final version of the manuscript.</p></notes><notes notes-type="COI-statement" id="notes-3"><p>The authors
declare no competing financial interest.</p></notes><ack><title>Acknowledgments</title><p>The work
was supported by BBSRC ZELS Grant BB/L018853/1. J.W.M.
and D.J.B. were supported by the Francis Crick Institute and receive
their core funding from Cancer Research UK (Grant FC001030), the UK
Medical Research Council (Grant FC001030), and the Wellcome Trust
(Grant FC001030). The authors would like to thank Dr. Christopher
Johnson for taking photos of the LFA devices and Ms. Rachel Sim for
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| This area was generated with Dilib version V0.6.33. |  |