Biosensors and Bioelectronics 21 (2006) 1668–1673

Short communication

A cowpea mosaic virus nanoscaffold for multiplexed antibodyconjugation: Application as an immunoassay tracer

Kim E. Sapsforda,∗, Carissa M. Sotob, Amy Szuchmacher Blumb, Anju Chatterjic,Tianwei Linc, John E. Johnsonc, Frances S. Liglerb, Banahalli R. Ratnab,∗∗

a George Mason University, 10910 University Blvd, MS 4E3, Manassas, VA 20110, USAb Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue, SW, Washington, DC 20375, USA

c Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA

Received 17 June 2005; received in revised form 30 August 2005; accepted 5 September 2005Available online 10 October 2005

Abstract

Cowpea mosaic virus (CPMV), an icosahedral 30 nm virus, offers a uniquely programmable biological nanoscaffold. This study reports initialoptimization of the simultaneous modification of two CPMV mutants with AlexaFluor® 647 fluorescent dyes and either IgG proteins or antibodies ats specifically,w scent dyesr ye-labeleda©

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pecific sites on the virus scaffold. The capacity of CPMV as a simultaneous carrier for different types of molecules was demonstrated,hen applied as a tracer in direct and sandwich immunoassays. The ability to label the virus capsid with antibody and up to 60 fluore

esulted in an improved limit of detection in SEB sandwich immunoassays, when used as a tracer, relative to a mole equivalent of dntibody.2005 Elsevier B.V. All rights reserved.

eywords: Cowpea mosaic virus; Antibody; Immunoassay; Array biosensor

. Introduction

Bionanotechnology, encompassing concepts from chem-stry, physics and biology to develop programmable nanoscaleevices, is a newly emerging and exciting area of research, andas been the subject of a number of excellent reviews (Storhofft al., 1997; Choi et al., 2004; Oakley and Hanna, 2004; Clarkt al., 2004). The basis of the technology is the controlled molec-lar self-assembly of simple building blocks into complex struc-

ures. These simple building blocks can take a variety of formsncluding metal colloids (Storhoff et al., 1997; Park et al., 2004),emiconductor nanoparticles (Storhoff et al., 1997), polystyreneeads (Soto et al., 2002) and biological species (Clark et al.,004). Further modification of these building blocks with bio-

ogical species, such as biotin–avidin complexes or DNA–DNA

∗ Corresponding author at: Naval Research Laboratory, Center for BioMolec-lar Science and Engineering, 4555 Overlook Avenue, SW, Washington, DC0375, USA. Tel.: +1 202 404 6129; fax: +1 202 7679594.

∗∗ Corresponding author. Tel.: +1 202 404 6061.E-mail addresses: ksap@cbmse.nrl.navy.mil (K.E. Sapsford),

dimers, drives the process of self-assembly into complex stures (Storhoff et al., 1997). Cowpea mosaic virus (CPMV) reresents one such example of a uniquely programmable biolobuilding block and has been well characterized in the litera(Lomonossoff and Johnson, 1996; Lin et al., 1999; Wang e2002). CPMV displays icosahedral symmetry, formed bycopies of two different protein subunits, creating 30 nm vparticles. Site-directed and insertional mutagenesis can rebe used to generate a variety of mutant viruses with predicand programmable surface chemistry (Wang et al., 2002). Thesurface of CPMV has previously been functionalized with a vety of species such as dyes (Chatterji et al., 2004b), gold colloids(Blum et al., 2004), quantum dots (Portney et al., 2005), pro-teins (Chatterji et al., 2004a) and DNA (Strable et al., 2004).Besides the potential use as a building block for the devment of nanoassemblies, CPMV labeled with signal transduspecies, such as fluorescent dyes, and multiple analyte-spbinding molecules such as antibodies, peptides, DNA or chydrates could be used in multiplexed biosensor applicatThe aim of this study is to demonstrate the bi-functionalizaof CPMV with functional antibodies and a signal transduc

rr@cbmse.nrl.navy.mil (B.R. Ratna). species, in this case a dye, to demonstrate its potential appli-

956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2005.09.003

K.E. Sapsford et al. / Biosensors and Bioelectronics 21 (2006) 1668–1673 1669

cation as a tracer for immunoassays. The virus nanoscaffoldallows labeling of the capsid surface with a large number ofdyes, which, when used as a tracer, may result in improved sen-sitivity relative to plain dye-labeled antibodies. Two mutantsof CPMV were used in this study (provided by The ScrippsResearch Institute), EF-CMPV which has a single cysteine groupinserted (at residue 98) per subunit, generating a total of 60thiols on the surface of the capsid, and DM-CPMV in whichtwo cysteines are inserted (at residues 228 and 2102) per sub-unit, giving a total of 120 surface thiols (Wang et al., 2002;Blum et al., 2004). In both mutants described, there are fourlysines per subunit, each with varying reactivity (Chatterji et al.,2004b), generating a total of 240 per capsid. Initial studies tooptimize the antibody-CPMV coupling chemistry were carriedout with IgG proteins which are cheap, well characterized andstructurally equivalent to antibodies. Following immobilizationof functional antibodies on the CPMV surface, optimization ofthe dye-to-virus ratio, to improve immunoassay sensitivity, wasinvestigated.

2. Experimental

2.1. Functionalization of EF-CPMV with dye and IgGprotein

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1 mg/ml. The bifunctional linkerN-(�-maleimidobutyryloxy)succinimide ester (GMBS) (Fluka; Buchs SG, Switzerland),1 mg/ml in dimethyl sulfoxide (DMSO) (Sigma–Aldrich, St.Louis, MO), was added (90�l) to each of the IgG solutions(1 ml). After 1 h at room temperature (the succinimide esterof the GMBS reacts with the lysine groups on the IgG),the solutions were passed down a BioGel P-10 column (Bio-Rad; Hercules, CA) to remove unreacted GMBS. The GMBS-functionalized IgGs were mixed with the purified Alexa-EF-CPMV sample, at a ratio of∼100 IgG to 1 virus and left toreact at 4◦C for 2–3 days, allowing the maleimide groups onthe GMBS activated IgGs to react with the thiols present on thesurface of CPMV. Unreacted IgG was removed from the IgGfunctionalized Alexa-EF-CPMV using a Q-10 Sepharose ion-exchange column (Amersham Biosciences Corp.; Piscataway,NJ) and a NaCl gradient (100 mM Phosphate buffer + 0.05%Tween + 0–1.0 M NaCl). The virus–IgG complex was found toelute at 0.5 M NaCl, while the free IgG eluted at 0.2 M NaCl. Thecollected fractions were concentrated using a 100 kDa MWCOAmicon Centriplus® centrifugal filter (Millipore Corp.; Bed-ford, MA) and characterized using UV–vis and fluorescencespectroscopy.

2.2. Direct immunoasssays with Alexa-EF-CPMV-IgG

previ-o bledw neTe s-s abbita sonITT assayP thece -

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The EF-CPMV was first labeled via the lysines with the sinimide ester functional dye, AlexaFluor® 647 (Alexa) (Moleclar Probes Inc.; Eugene, OR), as outlined inFig. 1A. EF-CPMVas prepared in borate buffer (50 mM), pH 9.0, and exposHS-Alexa dye, at a ratio of∼3600 fluorophores to 1 viral parle, for 2–3 h at room temperature and then overnight at 4◦C. Theample was dialyzed, using 50 kDa MWCO Spectra/Pore® dial-sis bags (Spectrum Laboratories Inc.; Rancho Domiguezgainst 50 mM phosphate buffer (PB) pH 7.4 (Sigma–Alrichouis, MO) to remove unreacted dye molecules.

The IgG proteins (chicken IgG and/or mouse IgG; JackmmunoResearch; West Grove, PA) were prepared in 50B plus 0.15 M NaCl pH 7.2 (PBS) at a concentration

ig. 1. (A) Schematic showing the procedure for modifying EF-CPMVmmunoassay, with Alexa-EF-CPMV-IgG, taken using the NRL array bios

)

Glass microscope slides were prepared as describedusly with covalently attached NeutrAvidin and were assemith patterning poly(dimethylsiloxane) (PDMS) (Nusil Silicoechnology; Carpintera, CA) flow cells (described inGoldent al., in press; Feldstein et al., 1999). For the direct immunoaays the channels were exposed to either biotinylated rnti-chicken IgG or biotinylated goat anti-mouse IgG (Jack

mmunoResearch; West Grove, PA), 10�g/ml in PBS + 0.05%ween-20 (PBST), overnight at 4◦C (columns [1]–[6];Fig. 1B).he antibody functionalized slides were then assembled inDMS flow cells, whose channels run perpendicular toolumns of patterned antibodies (rows [1*]–[6*];Fig. 1B), andxposed for 1 h at RT to 5�g/ml Alexa-EF-CPMV function

lexa fluorescent dye and chicken and mouse IgG. (B) CCD image ofr.

1670 K.E. Sapsford et al. / Biosensors and Bioelectronics 21 (2006) 1668–1673

alized with either chicken IgG, mouse IgG or both, preparedin PBST + 0.1% BSA (PBSTB). The channels were then rinsedwith 1 ml of PBSTB, the PDMS flow cell removed, the slidedried and imaged with the NRL array biosensor. The biosen-sor, equipped with a diode laser (633 nm) and a charge coupleddevice (CCD) camera, has been described previously (Feldsteinet al., 1999).

2.3. Functionalization of EF-CPMV with dye andantibodies

Three antibodies were chosen for this study, against Staphylo-coccus aureus enterotoxin B (SEB) [polyclonal rabbit and sheepanti-SEB and SEB toxin from Toxin Technology Inc.; Sara-sota, FL], botulinum toxin [polyclonal rabbit anti-Bot. toxin andbotulinum toxoid A supplied by the U.S. Department of DefenseCritical Reagents Program] andCampylobacter jejuni [poly-clonal rabbit anti-Camp. jejuni supplied by Biodesign Interna-tional; Saca, ME]. The bacteria,Camp. jejuni (ATCC35918) wasgrown by Dr. Avraham Rasooly (NIH) and used under BiosafetyII conditions which required the cells to be killed with azide priorto shipment to the NRL (Sapsford et al., 2004). EF-CPMV wasfirst labeled with Alexa dye and then GMBS activated antibod-ies using the procedure described in Section2.1 for the IgGproteins.

2.4. Sandwich immunoassays withAlexa-EF-CPMV-antibody

In the case of sandwich immunoassays, NeutrAvidin slides,assembled with the patterning PDMS flow cells, were exposed toeither biotinylated rabbit anti-SEB, biotinylated rabbit anti-Bot.toxin or biotinylated rabbit anti-Camp. jejuni, 10�g/ml in PBST,overnight at 4◦C (columns [1]–[6];Fig. 2A). The assay PDMSflow cells were then attached, and the slides were exposed toeither PBSTB or the target analytes; 50 ng/ml SEB, 1�g/ml Bot.toxoid A or 1× 105 cfu/ml Camp. jejuni for 30 min at RT (rows[1*]–[6*]; Fig. 2A). The channels were then rinsed and exposedfor 1 h at RT to 20�g/ml of the Alexa-EF-CPMV functional-ized with the appropriate antibody (rows [1*]–[6*];Fig. 2A),prepared in PBSTB. The slides were prepared and imaged asdescribed in Section2.2.

2.5. Functionalization of DM-CPMV with dye and SEBantibodies

In this modified scheme, 1 mg/ml sheep-anti-SEB (250�l)functionalized with GMBS was mixed with 200�g of DM-CPMV in a final volume of 2 ml and incubated for 36 h.The sample was then concentrated to a final volume of500�l using a 100 k MWCO centrifugal filter (Microsep unit,

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ig. 2. Sandwich assays with Alexa-EF-CPMV-antibody. (A) CCD image of sanor blank and positive regions of the slide:Camp. jejuni (columns [5] and [6]; rowsows [3*] and [4*] dotted box) and SEB (columns [3] and [4]; rows [1*] and [2*]

dwich immunoassays using EF-CPMV complex as the tracer. (B) Plot of net intensity[5*] and [6*] dashed box), botulinum toxoid A (Bot. Tox) (columns [1] and [2];solid box). Note that the average standard deviation in the data is∼20%,n = 2.

K.E. Sapsford et al. / Biosensors and Bioelectronics 21 (2006) 1668–1673 1671

MWCO = 100 kDa; from VWR International; West Chester,PA). Excess anti-SEB was removed by size exclusion chro-matography using a SuperoseTM 6 column (Amersham Bio-sciences: 18 cm long, 1 cm diameter, flow rate 0.5 ml/min)equilibrated with 100 mM PB pH 7.0. DM-CPMV-containingfractions (determined by UV–vis spectroscopy;λmax= 260 nm)were pooled and concentrated, as before, to a final volume of400�l. The DM-CPMV-anti-SEB sample was incubated on icefor 5 min prior to the addition of 100�l of DMSO followed byaddition of 50�l of 10�g/�l of AlexaFluor® 647-C2-maleimidein DMSO (Molecular Probes Inc.; Eugene, OR). The mixturewas incubated at room temperature in the dark for 24 h at RTand 16 h at 4◦C. The Alexa-DM-CPMV-anti-SEB sample wasthen purified using the SuperoseTM 6 column (size exclusion)and eluted with 50 mM (PB) pH 7.0 buffer, twice, to ensurecomplete removal of unreacted dye.

2.6. SEB sandwich immunoassays withAlexa-DM-CPMV-anti-SEB

NeutrAvidin slides were assembled with the patterningPDMS flow cells (12-channel) and the channels exposed to

biotinylated rabbit anti-chicken 10�g/ml, biotinylated rabbitanti-SEB 10–0.01�g/ml, and PBST overnight at 4◦C (columns[1]–[12]; Fig. 3A and B). The assay PDMS flow cells werethen attached, and the slides exposed to varying concentrationsof SEB (100–0.01 ng/ml) for 20 min at RT (rows [1*]–[6*];Fig. 3A and B). The channels were then rinsed and exposed for1 h at RT to either 10�g/ml of the Alexa-DM-CPMV-anti-SEB(rows [1*]–[6*]; Fig. 3A) or 0.3�g/ml Alexa-anti-SEB (rows[1*]–[6*]; Fig. 3B), prepared in 100 mM phosphate buffer (PB).The slides were prepared and imaged as described in Section2.2.

3. Results and discussion

Initial experiments to optimize protein–virus labelinginvolved functionalizing EF-CPMV with dye and IgG protein.A total of three modifications of the Alexa-EF-CPMV were pre-pared: one modified with chicken IgG, one with mouse IgGand the final with both chicken and mouse IgG, as outlined inFig. 1A. This scheme takes advantage of the native lysines andinserted cysteines present on the CPMV to controllably func-tionize its surface. The final EF-CPMV, modified with Alexadye and both chicken and mouse IgG, was characterized by

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ig. 3. Sandwich assays with Alexa-DM-CPMV-anti-SEB. (A) CCD image ofhe tracer. (B) CCD image of SEB sandwich immunoassays using Alexa-annti-SEB was varied from 10 to 0.01�g/ml in the columns, while the SEB was vaEB concentration exposed to the slide for the virus complex and antibody aoncentration of the capture antibody exposed to the surface for the virus comtandard deviation in the data is∼10%,n = 2.

SEB sandwich immunoassays using the Alexa-DM-CPMV-anti-SEB complex asti-SEB as the tracer. In images 3A and 3B the concentration of biotinylated rabbitried from 100 to 0.01 ng/ml in the rows [1*]–[6*]. (C) Plot of net intensity versuss tracer; at a biotinylated rabbit anti-SEB of 1�g/ml. (D) Plot of net intensity versusplex and antibody as tracer; at a SEB concentration of 100 ng/ml. Note that the average

1672 K.E. Sapsford et al. / Biosensors and Bioelectronics 21 (2006) 1668–1673

UV–vis spectroscopy giving the final ratio of Alexa 647:EF-CPMV as 13.7:1. Functionalization of the virus with dye andIgG was confirmed with a direct immunoassay using a waveg-uide modified with rabbit anti-chicken IgG and goat anti-mouseIgG modified waveguide.Fig. 1B clearly demonstrates that theAlexa-EF-CPMV-chicken IgG was only captured by the rabbit-anti-chicken IgG and likewise the Alexa-EF-CPMV-mouse IgGby goat anti-mouse IgG, as demonstrated by the increased sig-nal intensity observed in these regions from the dual dye andIgG labeled virus. The lower intensity of the mouse regionsas opposed to the chicken regions of the slide is due to thelower affinity of the goat anti-mouse IgG for mouse IgG rel-ative to the rabbit anti-chicken IgG for chicken IgG, and hasalso been demonstrated in direct assays run with the Alexa-labeled IgGs (data not shown). The Alexa-EF-CPMV modifiedwith both the chicken and mouse IgG, as expected, was capturedby both the rabbit anti-chicken IgG and the goat anti-mouseIgG (Fig. 1B) with little effect on the final intensity reachedrelative to Alexa-EF-CPMV modified with a single proteinspecies. This demonstrates that the dye-labeled virus, function-alized with up to two different species of IgG can be used as atracer.

The next step was to immobilize functional antibodies to thesurface of a dye-labeled virus and use the resulting complex as atracer in sandwich immunoassays. For these studies Alexa-EF-CPMV was functionalized with antibodies to either SEB, bot.t dyp cat-a s ara dureT -E s oft anti-b mpls nti-B tedr idew nr hisi solidb esw n inL atesa tibodi r sido g tha r eacAA allyt )a d [6]r ita d-i g itt allyb bbita abbi

anti-SEB (data not shown), suggesting it is an antibody issueand not an effect of the CPMV.

These studies clearly demonstrate that once immobilized tothe surface of the virus, the antibodies remain functional and bindto their specific analyte. In direct mole-to-mole comparisonsbetween standard Alexa-labeled antibodies and the Alexa-EF-CPMV-antibody complexes used as tracers in immunoassays,however, we found that the plain antibodies gave a lower limitof detection (LOD). This is likely a combined effect of thefairly low improvement in the dye-to-virus ratio obtained (Alexa647:EF-CPMV) of 13.7:1, relative to the 4.5:1 for the dye-to-plain antibody, and the overall larger size of the antibody–viruscomplex compared to the antibody alone.

One way to improve the LOD obtained by the Alexa-EF-CPMV-antibody complex was to further increase the overalldye-to-virus ratio. In order to achieve this we developed an alter-native scheme, which used a double CPMV mutant (DM-CPMV:228/2102), containing 120 surface thiols, to increase the num-ber of dyes-per-virus. In this scheme the DM-CPMV is firstmodified with the GMBS functionalized antibody, in this casesheep-anti-SEB, and then the Alexa-dye. However, unlike theinitial scheme (Fig. 1A), both the antibody and the Alexa dyetarget the surface thiol groups. The final ratio of Alexa 647:DM-CPMV, characterized by UV–vis spectroscopy, was calculatedto be 60:1, a factor of 4.4 improvement over the initial scheme.

Sandwich immunoassays were run to confirm successfulm Ba in theL lee m-p wichit ur-f turea nsityi lutiond useda nali tion( EB( EBc alento as an mbero

ti-S the0 theA en-t -m isr ODc ndardc uldi nti-S pa-r ole

oxin or Camp. jejuni. Experiments carried out using antibore-labeled with Cy3 (Amersham Biosciences Corp.; Pisway, NJ) fluorescent dye suggest that typically 3–5 proteinttached to the virus surface using this conjugation procehe final CCD image is shown inFig. 2A; clearly, the AlexaF-CPMV-antibody complex is only captured in the region

he slide functionalized first with the appropriate captureody and second exposed to the correct analyte. For exaignal intensity is only observed for the Alexa-EF-CPMV-Aot. toxin in the columns [1] and [2] exposed to biotinyla

abbit anti-Bot. toxin and then only in row [3*] where the slas first exposed to 1�g/ml botulinum toxoid A and not i

ow [4*] where the analyte is not present (dotted box). Ts also true for the other sandwich immunoassays, SEB (ox) andCamp. jejuni (dashed box), carried out. The imagere analyzed using a custom software application writteabWindows/CVI (National Instruments); the program cremask consisting of data squares (where the capture an

s patterned) and background rectangles located on eithef a data square. The net intensities obtained by subtractinverage background signal from the average data signal folexa-EF-CPMV-antibody complex are shown inFig. 2B. Thelexa-EF-CPMV-anti-SEB was found to bind non-specific

o the biotinylated rabbit anti-Camp. jejuni columns ([5] and [6]s observed by the low intensity observed in columns [5] anows [1*] and [2*], Fig. 2A, but not to the biotinylated rabbnti-Bot. toxin columns ([1] and [2]). This non-specific bin

ng occurs in the presence and absence of SEB suggestinhe Alexa-EF-CPMV-anti-SEB complex that is non-specificinding and not the SEB. The non-specific binding of ranti-SEB has previously been observed for Alexa-labeled r

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odification of the DM-CPMV with both dye and anti-SEntibodies and also to investigate potential improvementsOD. The final CCD images,Fig. 3A and B, compare moquivalent amounts of the Alexa-DM-CPMV-anti-SEB colex to the Alexa-anti-SEB when used as tracers in sand

mmunoassays for SEB, respectively. As seen fromFig. 3A,he Alexa-DM-CPMV-anti-SEB complex only binds to the sace of the waveguide in the regions with both the capnti-SEB antibody and the SEB analyte. The signal inte

s found to decrease as the concentration of the SEB in soecreases. The same holds true when Alexa-anti-SEB iss a tracer,Fig. 3B. The images were analyzed and the sig

ntensity plotted either as a function of the SEB concentraFig. 3C) or the concentration of the biotinylated rabbit anti-SFig. 3D). Clearly in both cases the Alexa-DM-CPMV-anti-Somplex produces a stronger signal than the mole equivf Alexa-anti-SEB demonstrating the advantage of CPMVanoscaffold to couple active biomolecules and a larger nuf reporter dye molecules on the same capsid.

The current LOD (1 ng/ml) for the Alexa-DM-CPMV-anEB complex used in this study is not as sensitive as.1 ng/ml obtained for standard immunoassays run withrray Biosensor format, using typical Alexa-anti-SEB conc

rations of 10�g/ml (Sapsford et al., 2005). Clearly further optiization of the Alexa-DM-CPMV-anti-SEB immunoassay

equired to determine if the virus complex can improve the Lompared to Alexa-anti-SEB immunoassays run under staonditions. Optimization, which is currently ongoing, wonclude scaling up the production of the Alexa-DM-CPMV-aEB complex to produce higher final concentrations, com

able to the typical Alexa-anti-SEB concentrations, on a m

K.E. Sapsford et al. / Biosensors and Bioelectronics 21 (2006) 1668–1673 1673

basis, used in assays (Sapsford et al., 2005). Also investigatingthe effects of flow conditions on the Alexa-DM-CPMV-anti-SEB immunoassay could shorten the assay time to the stan-dard 15–20 min. These antibody-labeled virus complexes havea number of other potential applications including, in partic-ular, antibody directed in vivo diagnostics or therapeutics (Linet al., 2005). Antibody driven organization of the virus particles,as demonstrated byStrable et al. (2004)using oligonucleotides,may have interesting applications such as porous templates inthe production of unique optical (Falkner et al., 2005) or func-tional materials, such as antibody-based filters to remove toxicmaterials from the environment.

4. Conclusion

In summary, we have demonstrated that it is possible tofunctionalize the surface of two CPMV mutants with both afluorescent dye and up to two different IgG proteins or func-tional antibodies. Such modified virus species have a numberof potential applications including tracers in biosensors capableof multi-analyte recognition, building blocks to the productionof complex nanoassemblies for the development of nanoscaledevices in medical diagnostics and treatment, or analyte reac-tive/sensitive materials.

Acknowledgment

wse oset ernm

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