drawntuanbao

1ImprovementofarapiddiagnosticapplicationofmonoclonalantibodiesagainstavianinfluenzaH7subtypevirususingEuropiumnanoparticlesSeon-JuYeo1,DuongTuanBao1,Ga-EunSeo2,CucThiBui1,DoThiHoangKim1,NguyenThiVietAnh1,TrinhThiThuyTien1,NguyenThiPhuongLinh1,Hae-JinSohn2,Chom-KyuChong3,Ho-JoonShin2&HyunPark1Thedevelopmentofasensitiveandrapiddiagnostictestisneededforearlydetectionofavianinfluenza(AI)H7subtype.
Inthisstudy,novelmonoclonalantibodies(mAbs)againstinfluenzaAH7N9recombinanthemagglutinin(rHA)1weredevelopedandappliedtoaEuropiumnanoparticle–basedrapidfluorescentimmunochromatographicstriptest(FICT)toimprovethesensitivityoftherapiddiagnosticsystem.
Twoantibodies(2F4and6D7)exhibitedH7subtypespecificityinadot-FICTassaybyoptimizationoftheconjugateandthepHofthelysisbuffer.
ThesubtypespecificitywasconfirmedbyanimmunofluorescenceassayandWesternblotanalysis.
ThelimitofdetectionoftheFICTemployingnovelmAbs31ng/mLforH7N9rHA1and40hemagglutinationunits/mLforH7subtypevirus.
Sensitivitywasimproved25-foldusingEuropiumasconfirmedbycomparisonofcolloidalgold-basedrapiddiagnostickitusingthe2F4and6D7mAbs.
RecurrenceofhighlypathogenicavianinfluenzaA(HPAI)virusH7subtypeinhumansandpoultrycontinuestobeaseriousconcerntopublichealth1.
Priorto2003,outbreaksofavianinfluenza(H7N1andH7N3)occurredinpoultryinEurope.
Sincethen,human-to-humantransmissionHPAIvirus(H7N7)hasbeenreported.
Anout-breakintheNetherlandsinfected89people2–6.
In2013,anoutbreakofH7N7resultedinthecullingofmorethan1millionchickens7.
Threeof200workersinvolvedintheculldevelopedconjunctivitisdespitestrictinfectioncontrolproceduresthatwereinplace.
AnotherHPAIvirusH7subtypethatinfectshumansisavianinfluenzaA(H7N9).
ThefirstH7N9outbreakoccurredinChinain2013.
Mostrecently,anoutbreakresultedinabout400humancasesinChina,HongKongandTaiwan,withamortalityrateof27–36%8–10.
AirbornetransmissionofthesubtypesH7N1,H7N7,andH7N9hasbeenreported11–13.
H7subtypevirusinfectionisverycontagiousinpoultryandhumans,andisanimmediatethreattopublichealth14.
AvianinfluenzaAvirusinfectioninhumanscannotbetypicallydiagnosedbyclinicalsignsandsymptomsalone.
Laboratorytestingisrequired.
AguidelineoftheUnitedStatesCentersforDiseaseControlandPrevention(CDC)recommendsmoleculardetectionmethodslikereal-timereversetranscription-polymerasechainreaction(rRT-PCR)forthelaboratorydiagnosisofinfluenzainfectionsandhemagglutinin(HA)subtypeidentificationispreferred15.
Althoughrapidpoint-of-caredetectiontests(POCT)arenotgenerallyperformedforavianinflu-enzabecausethelowsensitivitytodetectAIvirus,severalstudieshavesoughttodevelopH7subtype-sensitive16orspecific17POCT;suchtestswouldbeconvenienttouseon-siteduringoutbreaks.
But,thedevelopmentofH7-specificPOCThasbeenhinderedbythehighlaborcoststodevelopthespecificantibodytestandthe1ZoonosisResearchCenter,DepartmentofInfectionBiology,SchoolofMedicine,WonkwangUniversity,460,Iksan-daero,Iksan,54538,RepublicofKorea.
2DepartmentofMicrobiology,AjouUniversitySchoolofmedicine,andDepartmentofBiomedicalScience,GraduateSchoolofAjouUniversity,Suwon,RepublicofKorea.
3GenBodyInc,No.
206,BiotechBusinessIC,DanKookUniversity,San-29,Anseo-dong,Dongnam-gu,Cheonan,RepublicofKorea.
Seon-JuYeoandDuongTuanBaocontributedequallytothiswork.
CorrespondenceandrequestsformaterialsshouldbeaddressedtoH.
-J.
S.
(email:hjshin@ajou.
ac.
kr)orH.
P.
(email:hyunpk@wku.
ac.
kr)Received:1February2017Accepted:7July2017Published:xxxxxxxxOPEN2difficultyinidentifyinganantibodythatissufficientlystableandmaintainsitsantigenrecognitionfunctionalityinnon-optimalenvironments18.
Inthisstudy,twonovelH7subtype-specificmonoclonalantibodies(mAbs)weredevelopedandcombinedwithafluorescencemolecule.
ThemAbswereadaptedtoPOCTtodeveloparapidfluorescentimmunochroma-tographicstriptest(FICT)assay.
Theassaywascapableofproducingresultsin15minutes.
TheFICTassayisattractiveasarapiddiagnostictestbecauseofitsgreatlyimprovedsensitivityforinfluenzaRDT19–22.
Europiumisawidely-usedandefficientfluorescencematerial,whichhasbeenincorporatedinrapiddiagnostictests23,24.
Thesetestsrequiredelicatebiosensorequipment,suchasmicrochipandmicroplatereader.
Untilnow,nostudyhassoughttoapplytheEuropiumtoH7subtype-specificrapiddiagnostictest.
ThepresentstudyintroducesnovelH7subtype(H7N1andH7N7)-specificmAbsanddemonstratestheirsuccessinimprovingtheperformanceofaFICTassaythatutilizesEuropiumnanoparticles.
ResultsCharacterizationofthemAbs.
AfterimmunizationofmicewithH7N9rHA1antigen,10hybridomacelllinesproducingmAbwereestablished.
Theantibodyamountsrangedfrom0.
5to2ODaccordingtoanELISAforH7N9rHA1antigen(Fig.
1A).
IndirectELISAwithall10mAbswasconductedforfourinfluenzaAsubtypevirus(H1N1,H5N3,H7N1,andH7N7)at1,000HAU/mL.
Reactivityoffourclones(2H1,2C3,6B2,and6B9)toviruswasrelativelylowerthanotherclones,althoughtheyshowedpositivesignalsinrecombinantantigen-mediatedindirectELISA(Fig.
1B).
SixmAbs(6B7,2F4,6A9,6F3,6G5,and6D7)reactedwiththeH7N1andH7N7subtypesandwithH1N1virus.
OnemAb(6G5)reactedwithallsubtypes.
Aspositivecontrol,anti-influenzaAnucleoprotein(NP)(3G6)wasusedforconfirmationoftheequalamountofeachsubtypevirus;anODofabout2.
5wasproducedinthepresenceof1,000HAunits(HAU)/mL.
Adot-FICTassaywasusedtoselectthepairofH7subtype-specificmAbs,mimickingthestriptest(Fig.
2A).
Thebroaddetection(6G5)to1,000HAU/mLofeachviruscompelleditschoiceasthedetectionelement.
FiveothermAbs(6A9,6D7,6F3,6B7,and2F4)withstrongreactionto1,000HAU/mLofvirusweredottedonthestrip.
The6D7mAbdemonstratedthestrongestreactivitywithH7subtypeswith6G5.
The6F3and2F4mAbs,whichhadstrongreactivitytovirus,hardlyreactedwith6G5,implyingasharedepitope.
Asthestrongestreac-tivitywasobservedby6D7forH7subtypes,reactivitywithothermAbswasexaminedtoascertainspecificity(Fig.
2B).
FurthertestingshowedthatthreemAbs(6G5,6F3,and6B7)cross-reactedwithH1N1inadditiontoH7subtypes.
However,2F4showedthepotentialH7subtypespecificitywith6D7,showingstrongreactivitytoH7N1andH7N7virus.
The6A8mAbfailedtorecognizeH7N1virus.
The2F4and6D7mAbswereselectedforsubsequentexperiments.
Tofurtherconfirmthespecificity,theviralreactivityofthemAbswasanalyzedbyWesternblotassayandimmunofluorescenceassay(Fig.
3).
Hybridomacellswereexpandedinascitesandthepurifiedantibodywascon-firmedbySDS-PAGE.
Thegelsdisplayedtwodominantbandsat50kDa(heavychain)and25kDa(lightchain)(Fig.
3A).
ToassesstheabilityofthesemAbstorecognizethelinearepitope,1,000HAU/laneofviruswasusedforWesternblotanalysis.
The2F4mAbreactedtobothH7N1andH7N7subtypes(Fig.
3B).
The6D7mAbdidnotreactwithlinearizedantigen,implyingthatthisantibodywouldcontributetothespecificreactivitythroughconformationalstructureoftargetantigen.
Intheimmunofluorescenceassay,the2F4and6D7mAbsbothwerereactiveagainsttheH7N1andH7N7virussubtypesandnotagainsttheothersubtypetested(Fig.
3C).
Theposi-tivesignalofanti-influenzaNP(3G6)showedthepresenceofvirusinthetwoassays.
Figure1.
DevelopmentofH7subtype-specificantibodies.
ThefusionswereperformedusingmousespleencellsinoculatedwithH7N9virusrHA1.
Tenhybridomaswereproduced.
Thesecretedantibodiesfromeachhybridomaweretestedforrecombinantantigen(H7N9HA1(A)anddifferentinfluenzasubtypevirus(B)byindirectELISA.
Pre-immune,serumfromahealthymouse;P.
C.
,positivecontrolantibody(anti-influenzaAnucleoproteinantibody).
3OptimizationofFICTassay.
Figure4AschematicallyillustratestheEuropiumconjugate-basedFICTassay.
TheteststripforFICThasaconjugatepadforconjugateandasamplepadforapplicationofsampleonanitro-cellulosemembrane.
Anti-influenzaH7subtype-specificantibody(6D7)andanti-mouseIgGAbswerecoatedonthetestline(TL)andthecontrolline(CL)ofthenitrocellulosemembrane,respectively.
Toperformthediagnos-ticassay,theconjugateswereloadedontheconjugationpadinadvance.
Virusinlysisbufferwasappliedtothesamplepad.
Intheabsenceofvirus,theconjugatesfailedtoreactwithmAbintheTL.
However,inthepresenceofvirus,conjugatewasonTLline.
AttheCL,anti-mouseIgGrecognizedtheantibodyonconjugate.
Aftertheapplicationofsampletothestrip,thefluorescenceintensitywasdigitalizedbythelight-emittingdiode-basedportablestripreaderin15min.
TheoptimalperformanceoftheFICTassaywasobtainedusing1,000HAU/mLofH1NvirusandH7N7virus.
Thedot-FICTdidnotshowedcross-reactivitywiththeAbswhenH1N1waspresentat1,000HAU/mL(Fig.
4B).
Nonspecificreactionofbioconjugatewasexploredbyusingdifferentamountsofbovineserumalbumin(BSA,0.
3125–5%)inconjugatestoragebuffer.
BSAat5%wasabletodecreasethecross-reactionofH1N.
Thisconcen-trationwaschosenforthebioconjugate,eventhoughH1N1cross-reactivitypersisted.
InsteadoftheincreasingBSAfurther,wetesteddifferentpHs(8–12)oflysisbuffer.
Thecross-reactionofH1N1wasdecreasedwithreactivityofH7N7virus(Fig.
4C).
Theremainingcross-reactionofH1N1wasnotsignificantlydifferentatpH12butthereactivityofH7N1wasdecreasedatthatpH.
Therefore,wechosethelysisbufferofpH11.
Theresultsindicatethatlysisbufferplaysakeyroleintheantigen-Abenvironmentinadditiontohelpingthemigrationofsamplesonanitrocellulosemembranewithoutnon-specificreaction.
FICTassayperformance.
SerialdilutionsofH7N9HA1andH5N1HA1(15–500ng/mLwereusedtodeterminethelimitofdetection(LOD)oftheFICTassay,basedonthelimitofblank(LOB),asdescribedpre-viously25.
TheFICTdisplayedH7N9HA1reactivitywithgoodcorrelation(r2=0.
9826),butnoreactivitywithH5N1rHA1(Fig.
5).
LODforH7N9rHA1was31ng/mL.
TheproposeddiagnosticsystemtargetedtheHA1H7subtype.
Toconfirmthevirusspecificity,serialdilutions(20–640HAU/mL)oftheH1N1,H5N3,H7N1,andH7N7subtypesweretestedinFICTusinga2F4(conjugate)/6D7(strip)Abpair.
TheproposedassayshowedgoodlinearregressionforH7N1virus(r2=0.
9743)andH7N7virus(r2=0.
9897).
However,itcouldnotrecognizetheothertwosubtypestested(Fig.
6A).
LODwas40HAU/mLoftwoH7subtypes.
Therefore,theassayhadH7subtypespecificity.
Figure2.
Monoclonalantibody(mAb)reactivitieswithdifferentH7virusstrainsandsubtypesinthedot-FICTassay.
Dot-FICTassayswereusedtoexaminethespecificityofthemAbsagainstdifferentAIvirussubtypesanddiverseH7isolates.
6G5waschosenasdetectionelementandwasconjugatedwithEuropium.
Fivecloneswereusedfordottingonstripanddifferentvirussubtypeswereappliedtothedot-FICTassay(A).
6D7waschosenfordottingonstripandotherantibodieswereconjugatedwithEuropiumanddot-FICTassaywasperformed(B).
Rawdataforfluorescentimageofdottingareshownintherightpanelofeachgraph.
4TheRNAcopynumberoftheLOD(40HAU/mL)ofFICTwasdetermined(Fig.
6B).
Afterpreparingtwo-folddilutionsfromof10HAU/mLto80plaqueformingunits(PFU)/mLoftwoH7subtypeviruses,75μLofsamplewasusedforRNAextraction.
ElutedRNAwasusedforrRT-PCRwiththestandardusingplasmiddilution.
Togenerateacalibrationcurve,serially-dilutedRNAstandardwasused.
AstandardcurvewasdrawntoshowthestartingcopynumberofthestandardRNAvsthecyclethreshold(Ct).
RNAcopynumber/μLwasdeterminedbyplasmiddilutionasdescribedpreviously26.
TheplotofastandardcurveofCtvaluesagainstthelogarithmicdilu-tionsproducedar2value>0.
997andtheslopecorrespondedtoefficiencyintherange87%,whichwasclosetotheoptimizedprotocol.
The40HAU/mLofH7N1andH7N7virusshowedaCtvalueof30and31,respectively.
TheFICTRNAcopynumberLODwas2.
03*103ofRNAforH7N1and1.
59*103forH7N7.
TheimprovedperformanceoftheFICTassaywashighlightedbycomparisonofRDTusingcolloidalgoldnanoparticles.
The2F4/6DFAbRDTshowedapositivesignalat1000HAU/mLofH7subtypevirus,butnotwiththeothersubtypestested(Fig.
7A).
ThisindicatedthatthefluorescentdyeimprovedtheperformanceofRDTby25-fold.
TheconventionalinfluenzaANPRDTdisplayedaLODof320,160,80,and80HAU/mLforH1N1,H5N3,H7N1,andH7N7virus,respectively(Fig.
7B).
TheH7subtype-specificFICTassayincorporatingEuropiumnanoparticlesmarkedlyimprovedtheperformanceoftheRDTassay,surpassingtheperformanceoftheRDTdetectionofnucleoprotein,ahighlyexpressedviralantigen,comparedtoHA.
DiscussionForadecade,manystudieshavesoughttodevelopH7subtype-specificrapiddiagnosticimmunoassays.
ThreeELISA-basedH7subtype-specificsystemshavebeendeveloped27–29.
Recently,H7subtype-specificAbswerereportedtohavevaccineefficacy30,31.
TheLODoftheH7rRT-PCRassayvariesdependingonprimer,probe,andH7subtypestrain.
TworeportsachievedaLODof103–104copiesofH7RNA32,33.
Usingcurrentlyavailableprobeandprimers,Ctvaluesof35–37,correspondingto20and200copiesperreaction,havebeenreportedastheLODofrRT-PCRofH7N1virus34.
Figure3.
CharacterizationofH7subtype-specificantibodies.
Both2F4and6D7antibodieswereproducedinascitesandpurifiedbyProteinGandSDS-PAGEwasconductedtoidentifytheheavy-andlightchainofeachantibody(A).
FourinfluenzaAsubtypeviruseswereloadedonaSDS-PAGEgelandWesternblottingwasusedtoanalyzeeachantibody(B).
Intheimmunofluorescenceassay,cellswereindependentlyinfectedwiththevirussubtypesfor12h,andthecellswerefixedandincubatedwith2F4and6D7antibodies.
Afterwashing,fluorescencewasdetectedbyaFITC-conjugatedsecondaryantibody(C).
M,marker;Mock,novirusinfection.
5Untilnow,onlytwostudieshavereportedonaH7subtype-specificdiagnosticsystemusingnovelmAbs.
Inonestudy,oneHAunitofH7wasdetectedusinganELISAsystem35.
TheotherstudydevelopedaH7subtype-specificRDTandreportedtheLODH7-specificityvariedwidelyfromCtsof26–31byrRT-PCR,dependingontheisolatesandstrainofH7N917.
Inourstudy,theLODoftheFICTassayachievedthelatter,withaCtof30forH7N1and31forH7N7byrRT-PCR.
Therefore,ourproposedFICTassaythatutilizesfluorescentEuropiumperformsbetterintermofaverageofCtsvalues.
TheoutstandingimprovementofthisFICTassaywashighlightedbycomparisonofRDTusingthecolloidalgoldnanoparticle-conjugatedmAbs(2F4/6D7).
ThisRDTwasabletodetectonly1,000HAU/mLofH7subtypeviruswhenweappliedthepairoftheseAbs.
NPisawell-conservedantigen,whichhasaroleinencapsidationofviralRNAandwhichisusedtoiden-tifythepresenceofinfluenzaAorBvirusinRDTsystemsbecauseofthepresenceofmultiplecopies36.
Thesurface-orientedglycoproteinHAantigenisacomponentofasubtype-specificprotein.
PresentresultsfromWesternblotanalysisandimmunofluorescenceassayindicatedHAexpressioninAIviruswasdetectedatlowlevelcomparedtothatofNP.
Therefore,theRDTLODof1,000HAU/mLachievedusing2F4/6D7couldbepartiallyduetotherelativelylowexpressionofHA.
ThefindingisreasonablecomparedtoNPbecauseacommercialinfluenzaA/BkittargetingNPhasaLODof80–320HAU/mLallfoursubtypevirus(Fig.
7B).
Additionally,theimprovementofperformanceofFICTemployingEuropiumwasconfirmedbyFICTtarget-ingNP(FigureS1).
WhenwetestedtheEuropium-conjugatewithanti-influenzaNPinFICT,LODofRDT(LOD:80HAU/mL)wasimprovedby8-fold(LOD:10HAU/mL)inEuropium-employingFICTassay.
Therefore,ourstudysupportedthatthelowsensitivityofRDTcouldbeimprovedbyemployingthefluorescencematerial.
TheFigure4.
OptimizationofFICTassay.
SchematicdiagramrapidfluorescencediagnosticsystememployingEuropium-conjugatedH7subtypespecificantibodywasshown(A).
6D7mAbwascoatedinthetestline(TL)onNCmembrane.
ViruswaspretreatedwithlysisbufferandEuropium-conjugatedantibody(2F4)wasdroppedontotheconjugatepadonthestrip.
Subsequently,samplewasappliedtothesamplepadandafter15min,thestripwasusedtodetectthefluorescentlightonthestripthroughthelightemittingdiode(LED)-builtportablefluorescentstripreader.
IntheLEDmodule,theexcitationlight(355nm)wasfilteredbytheemissionfilter(612nm).
ToreducenonspecificreactionsoftheFICTassayagainstH1N1virus,differentconcentrationofBSAwastestedasablockingagenttogeneratethebioconjugate(B).
LysisbufferwastestedindifferentpHfrom8to12(C).
Allexperimentswereconductedintriplicate.
Thedataareshownasmean±SD.
Rawfluorescencepeaksfromthetestline(TL)andcontrolline(CL)inFICTareshowninthebottompanel.
6mainchallengeinthepresentstudywastofindamethodthatwouldbesuitablyabletoimprovetheabilityoftherapiddiagnosticassay.
WeachievedthisgoalusingEuropiumcontainingnanoparticles.
AlthoughcouplingoffluorescentdyetoantibodyhasagoodrecordofimprovingRDT,thishasnothith-ertobeenexploredusingEuropium,eventhoughEuropiumispopularfluorescentmaterial.
ThismayhavebeenbecauseconjugatingEuropiumnanoparticlestoantibodycanfunctionallyinactivatetheantibody37.
ThecurrentlyoptimizedFICTcanbeeasilyexploitedtoapplyEuropiuminRDTsforinfluenzavirusandotherdiseasesthatrequireimprovedRDTsensitivity.
Virusinfectioncouldbediagnosedbyimmunofluorescenceassayusingfluorescentmicroscope.
However,fluorescencemicroscopyrequirestheintensivehighpowerlightsourceincludingxenon,mercuryorlaser38.
ThislightsourcelimitsthefluorescencemicroscopytobecomeaPOCT.
Recently,modernsmartphonesareequippedwithalightweight,high-performancecamera/imagingmodule,becomingthepreferredtoolforuseinmanyPOCapplications39,40.
Therefore,theproposedfluorescentdiagnosticsystemcancontributetotheadvanceofthePOCTdiagnosticplatformconnectedwithmodernsmartphone-baseddiagnosticapplication.
BSAisawell-knownblockerusedtosuppressnon-specificreactionsinanimmunoassay41.
pHcancausestructuralchangeofHAofinfluenzavirus42.
Therefore,wetesteddifferentpHstofindamoresuitablecondi-tionofourFICTassayemploying6D7,whichrecognizesconformationalstructureoftheHA1epitopeofH7subtypes.
H1N1cross-reactionwassuppressedbyincreasingpH,indicatingthatanon-physiologicpHcouldbeefficientindiscriminatingHAsubtypes.
Antibody-antigenforceisaweakinteractionandthetypeoftheweakFigure5.
DetectionlimitofFICTassayfortargetantigen.
FICTemployingEuropium-conjugatedantibodiesweretestedforthelimitofdetection(LOD)againstH7N9rHA1(A).
Thedata(n=3)areshownasmean±SD.
Linearregressionisshownwiththedottedline.
ThearrowindicatestheantigenconcentrationofLOD.
Rawfluorescencepeaksfromthetestline(TL)andcontrolline(CL)inFICTareshowninthebottompanel.
7interactioniscomposedofVanderWaalsforce,hydrogenbond,hydrophobicinteraction,andion-dipole43.
Weakinteractionisinvolvedintheepitope-paratopebindingandafewaminoacidsiscomprisedinthespecificbind-ing44.
Manyfactorsinfluenceantigen-antibodyreactionandoneofthefactorsaffectingantigen-antibodyreac-tionispH43.
Therefore,weconsiderthatmodificationofpHmaybeagoodwaytofine-tunetheconformationalantigen-antibodyreaction.
TheEuropium-basedassayincludesstrip(cost:<$2USD/teststrip)andLEDdevice(MedisensorInc.
,Daegu,SouthKorea,cost:~$20,000).
Astestkitisgenerallysoldatapriceof20USD/testkit45,currentEuropium-basedstripitselfhasacompetition.
Althoughtheproposedassayrequiresanexpensivereader,itstillhasadvantagebecausedevicecanbesharedinonelocalareaandreusable.
WetestedthepotentialofperformanceinFICTwithclinicalsamplesbyspikingvirusandtheproposeddiagnosticmethodisconsideredtobeusefulasclinicalapplicationwithhumannasopharyngealspecimen(FigureS2).
OurstudyislimitedbythelackofdataofhumanH7N9-infectedpatients,althougharecombinantantigenofH7N9HA1couldallowdetectionofH7N9virus.
ThedetectionsensitivityandspecificityofthisassayneedstobeevaluatedwhenH7N9patientsamplesareavailable.
Inconclusion,novelH7subtype-specificmAbsweredevelopedandappliedtoarapidfluorescentdiagnosticsystemthatincorporatedEuropiumnanoparticles.
ThesystemdisplayedH7subtype-specificityandenhancedH7subtypevirusdetectionsensitivity.
MethodsReagents.
EuropiumnanoparticleswerepurchasedfromBangsLaboratoriesInc.
(Fishers,IN,USA).
AliphaticaminelatexBeads(100nmdiameter)werepurchasedfromLifeTechnology(Carlsbad,CA,USA).
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride(EDC)andN-hydroxysulfosuccinimideFigure6.
AssessmentofFICTperformancebyrRT-PCR.
Seriallydilutedvirus(10–620HAU/mL)wasmeasuredbyFICTassay.
ThequantitativerangeofFICTassaywasdeterminedbylinearregressionusingtheGraphPadPrism5.
0software,asshownbythedottedline(A).
TheblackarrowindicatesthevirustiterofLODofFICTassay.
Thedata(n=3)areshownasmean±SD.
Rawfluorescencepeaksfromthetestline(TL)andcontrolline(CL)inFICTareshowninrightpanel.
FICTassaywascomparedwithrRT-PCR(B).
Thelinearrelationshipbetweenthethresholdcycle(Ct)andRNAcopynumberwasshown.
Afterpreparingtwo-folddilutionsfromof10HAU/mLto80PFU/mLoftwoH7subtypeviruses,samplewassubjectedtoRNAextractionandusedforrRT-PCR.
BottompanelshowsthePCRproductofdifferentRNAforstandardandeachdilutedvirusfrom10–80HAU/mL.
TheredarrowindicatesthepointthatcorrespondstotheRNAcopynumbersandvirustiter.
NTC,notemplatecontrol.
8sodiumsalt(Sulfo-NHS)werepurchasedfromThermoScientific(Waltham,MA,USA).
AllotherchemicalswerepurchasedfromSigma-Aldrich(St.
Louis,MO,USA)withoutfurtherpurification.
Recombinanthemagglu-tinin1(rHA1)ofH7N9(A/Anhui/1/2013)andrHA1ofH5N1(A/Vietnam/1/2003)werepurchasedfromSinoBiologicalInc.
(Beijing,China).
Anti-influenzaAnucleoprotein(NP)(Clone3G6)wasprovidedbyProfessorHo-JoonShin,AjouUniversity,Suwon,RepublicofKorea.
Polyclonalgoatanti-mouseIgGwaspurchasedfromSigma-Aldrich.
Cellandvirus.
Madin–DarbyCanineKidney(MDCK,NBL-2,ATCCCCL-34TM)cellswaspurchasedfromAmericanTypeCultureCollection(ATCC,Manassas,VA,USA).
H1N1waspurchasedfromKoreaCenterforDiseaseControlandPrevention.
AIvirus(H5N3,H7N1,andH7N7)werekindlyprovidedfromProfessorHaanWooSung,KangwonNationalUniversity,Chuncheon,RepublicofKorea.
Theegg-culturedvirusstockwastitratedbyHAassayandplaqueassayswereperformedaspreviouslydescribed22,46.
ProductionandcharacterizationofmAbs.
H7N9(A/Anhui/1/2013)recombinantHA1(rHA1)(50μg/100μL)wasmixedwithanequalvolumeofFreund'scompleteadjuvant(Sigma-Aldrich)andinjectedintraperitoneallyintoa6-week-oldfemaleBALYSISBUFFER/cmice(DaehanBio-Link,Eumseong,Korea).
BoostsconsistedoftheH7N9rHA1protein(25μg/100μL)mixedwithanequalvolumeofFreund'sincompleteadjuvantbiweekly.
Afterthethirdboost,adjuvant-freeH7N9rHA1(5μg/100μL)wasinjectedintravenously.
Thecellfusiontechniquewaspreviouslydescribed47.
Inbrief,afterconfirmationoftheAbinmouseserum,splenocyteswereextractedfromaselectedimmunemouseandfusedwithmyelomacell(F/0cellline)ataratioof1:5to1:1in50%polyethyleneglycolandthenseededineachwellofa96-wellcultureplate.
HybridomacellswereselectedbysubcultureinHAT(hypoxanthine,aminopterin,andthymidine)andHT(hypoxanthineandthymidine)mediaina5%CO2incubatorat37°Cfor2weeks.
Aftercoloniesappearedineachwell,Abtitersweredeterminedbyanenzyme-linkedimmunosorbentassay(ELISA).
HybridomacellsproducingmAbwereselectedbyELISAandculturedwithacompleteDMEMina24-wellcultureplate.
Forscaled-upmAbFigure7.
LimitofdetectionofconventionalRDT.
The2F4mAbconjugatedwithcolloidalgoldnanoparticle(AuNP)wascapturedontheTL(6D7mAb)iftherewasvirusduringlateralflowreaction.
After20min,thestripwasreadwiththenakedeyeforabinarydecision.
Twonon-H7subtypeviruses(H1N1andH5N3)wereappliedinhightiter(1,000HAU/mL)andfortwoH7subtypevirus(H7N1andH7N7),500and1,000HAU/mLofeachviruswereappliedtoRDT(A).
ConventionalinfluenzaARDTtargetinginfluenzaAnucleoprotein(NP)wastestedwiththetwo-foldseriallydilutedfoursubtypevirusfrom40to80HAU/mL(B).
ThearrowindicatestheLOD.
9production,mAb-producingcellswereintraperitoneallyinjectedintoa10-week-oldfemaleBALYSISBUFFER/cmouse.
Twoweekslater,mouseasciteswerecollectedandcentrifugedat5,000*gfor15min.
ThepurifiedmAbfromtheasciteswasobtainedusingaproteinAagarosecolumn(AmershamBiosciences,Uppsala,Sweden)andidentifiedbyWesternblotting.
ForexaminationofthemAbisotypes,anImmunotypeTMmousemonoclonalanti-bodyisotypingkit(Sigma-Aldrich)wasused.
IndirectELISAassay.
IndirectELISAwasperformedasdescribepreviously46.
Briefly,thepurifiedantigenorvirusweredilutedin50mMbicarbonate/carbonatecoatingbuffer(pH9.
6)and75Lofsamplewerepreparedandusedtocoatwellsofa96-wellmicrotitreplatewerepreparedandusedtocoatwellsofa96-wellmicrotitreplate(Greiner,Germany)37°Cfor2h.
Theplatewaswashedwith200μLofPBS,0.
1%Tween20(PBS-T,pH7.
4)andthenblockedwith5%non-fatdrymilkat37°Cfor2h.
Toreacttheantibody,incaseofrecombinantantigen-mediatedELISA,pre-immuneseraderivedfromhealthymice(0.
5μLofsera/100μL/well)andmAb(1μg/100μL/well)wereaddedtoeachwell,respectively.
Incaseofvirusdetection,mAbs(2μg/100μL/well)wereaddedtoeachwellandpositivecontrol(anti-influenzaANP,2μg/100μL/well)toconvincetheeachsubtypevirusperwellwereaddedtoeachwellandincubatedat37°Ctodetectantigens.
After1h,secondaryAbintheformofhorseradishperoxidase(HRP)-conjugatedrabbitanti-mouseIgG(Abcam,Cambridge,UK)wasaddedtoeachwellaccordingtothemanufacture'sprotocol.
StringentwashingwithPBS-Twasperformedfivetimestoremovenonspecificbidingand100μLof3,3′,5,5′-tetramethylbenzidine(Sigma-Aldrich)substratesolutionwasadded.
Westernblotanalysis.
Thefoursubtypevirus(H5N,H1N1,H7N1,andH7N7)weresubjectedto12%SDS-PAGEandarunninggelat100Vfor2h.
Thegelwassoakedintransferbuffer,andtheresolvedproteinsweretransferredtoapolyvinylidenedifluoridemembrane.
Themembranewasblockedwith5%non-fatmilkfor2hat37°Candeachantibodydilutedtoaconcentrationof10μg/mLwasaddedfor1h.
TheHRP-conjugatedantibodywasusedfordetectionaccordingtothemanufacturer'sprotocol.
Anti-influenzaNP(Clone3G6)wasusedforloadingcontrolofeachvirus.
Finally,ClarityWesternECLsubstrate(Bio-Rad,Hercules,CA,USA)wasusedtovisualizethebandusingtheChemiDocMPSystem(Bio-Rad).
IFA.
IFAwasperformedasdescribedpreviously48.
Thefirstday,MDCKcellswerecoatingonglasscoverslipspositionedinwellsofa6wellplateandincubatedat37°Covernightinanatmosphereof5%CO2.
Thenextday,cellswereinfectedatamultiplicityofinfectionof0.
5inDMEMcontaining1%antibiotic,0.
3%BSAand1μg/mLtrypsinfrombovinepancreasfor12h,37°C,and5%CO2.
Usingfresh4%paraformaldehydeinPBS(pH6.
9),cellswerefixedfor15minandafterwashingthreetimeswith0.
1%Tween20inPBS,asolutionof0.
1%TritonX-100wasaddedtoeachwellforpermeabilization.
Subsequently,cellswereblockedwith5%BSAand0.
1%Tween20inPBSfor2hatroomtemperature.
Todetectvirus,mAbs(2F4and6D7)diluted1:5000inblockingbuffer(0.
5μg/mL)wereused.
Subsequently,fluoresceinisothiocyanate(FITC)-conjugatedrabbitanti-mouseIgGsecondaryantibodywasincubatedfor1hatroomtemperature.
Finally,eachcoverslipwasmountedonaslideincludingmountingmediumand4′,6-diamidino-2-phenylindole(DAPI;VectorLaboratories,Burlingame,CA,USA).
Imagesoffluorescentcellswereacquiredwithafluorescencemicroscope(Olympus,Tokyo,Japan)usinga400*objective.
Anti-influenzaNP(3G6)wasusedaspositivecontrolantibody.
ConjugationofEuropiumnanoparticle.
Briefly,antibodywascovalentlyconjugatedtoEuropiumbyawell-establishedprocedurefromBangsLaboratories.
Briefly,10LEuropium(0.
2m,1%w/t)wasaddedto500L0.
1MTris-HCl(pH7.
0)andincubatedfor1hat25°Cinthepresenceof0.
13mMcarbodiimide(EDC)and10mMSulfo-N-hydroxysulfosuccinimide(Sulfo-NHS).
EDCandSulfo-NHSsurpluswasremovedbycen-trifugationat27,237*gfor5min.
TheactivatedEuropiumwasmixedwith30L10MAb(2F4)in500L0.
1Msodiumphosphate(pH8.
0)andallowedtoreactfor2hat30°C.
Aftercentrifugationat27,237*gfor5min,theEuropium-conjugatedantibodywascollected,washedwith2mMPBS(pH8.
0),resuspendedin100Lstoragebuffer(1%BSAinPBS)andstoredat4°C.
Lateralflowteststripsforfluorescentimmunochromatographictest(FICT).
Theteststripsusedconsistedoffourcomponents:sampleapplicationpad,conjugatepad,nitrocellulose(NC)membraneandabsorbentpad.
Thetestline(TL)ofthestripwaspreparedbydispensingadesiredvolumeof2.
5mg/mLmousemonoclonal(anti-influenzaH7subtype-specificmAb;6D7)and0.
5mg/mlpolyclonalgoatanti-mouseIgGonthecontrolline(CL).
Thediagnosticstripwastestedafterdryingthemembraneat30°Cfor2days.
Toselectthepairantibody,adot-FICTassaywasused.
Briefly,Ab(0.
1g/L)wasdottedonaNCmembranelackingaTLanddriedat30°Cfor2days.
ToperformtheFICTassay,2LofEuropium-conjugated2F4Abfromastocksolutionwasdroppedontoconjugatepadandamixtureof75Lofsampleswith75Loflysisbuffer(100mMTris-HCl,pH9.
0,0.
025MEDTA,0.
5%sodiumdeoxycholate,and1%TritonX-100)weredroppedontosamplepadfor15minutes.
Theresultsofteststripswerereadwithaportablefluorescentstripreaderatexcitationandemissionwavelengthsofat355nmand612nm,respectively(Medisensor,Daegu,SouthKorea)49.
BothTLandCLsignalsweremeasuredandtheTL/CLratiowascalculatedautomatically.
Real-timeRT-PCR(rRT-PCR).
rRT-PCRwasperformedusingaQuantitectProbeRT-PCRKit(QIAGEN,Hilden,Germany)todeterminethecyclethreshold(Ct)valuesusingaCFX96Real-TimePCRDetectionSystem(Bio-Rad,Hercules,CA).
TheH7primers,probes,andRT-PCRconditionweredescribedpreviously34.
Forstand-ardofRNAcopynumber,thetemplatewasgeneratedinplasmidpGEM-TEasy(Promega,Madison,WI,USA)includinga132basepair(bp)HA1insert.
InvitrotranscriptionofHA1RNAusedaRiboMax(Promega)kittodeterminetheRNAcopynumberforthelimitofdetectionofFICT.
Thestandardcurvewascalculatedautomati-callybyplottingtheCtvaluesagainsteachstandardofknownRNAcopynumberandbyextrapolatingthelinearregressionlineofthiscurve.
ThePCRproductswereanalyzedinagarosegels(2%).
10Colloidalgold-basedRDTassay.
ToevaluatetheperformanceofFICTassay,colloidalgoldnanopar-ticlesconjugatedtoAbwaspreparedaspreviouslydescribed50.
After0.
02%chloroauricacid(HAuCl4·4H2O)(Sigma-Aldrich)wasbroughttoaboil,0.
2%sodiumcitratewasaddedwithconstantstirring.
Afterthemixturechangedtoawine-reddishcolor,thesolutionwasboiledforadditional5minandthenstirringfor10minwithoutboiling.
Next,1mgofthemAb(2F4)wasconjugatedwiththepreparedcolloidalgoldparticles(100mL).
ThemAb-goldconjugatewasprecipitatedbycentrifugation(27,237*g)andresuspendedinPBScontaining0.
1%ofBSAtoanopticaldensityat450nm(OD450)of10.
TheTLofthestripwaspreparedbydispensingadesiredvol-umeof2.
5mg/mLmousemAb(6D7)and0.
5mg/mlIgGmouseontheCL.
SamelysisbufferwasusedforRDT.
AcommercialinfluenzavirusA/BRDT(StandardDiagnostics,Yongin,SouthKorea)wasusedforcomparison.
Sampleswereappliedfollowingthemanufacturer'sinstructions.
Ethicalstatement.
ThestudywasapprovedbytheInstitutionalReviewBoardoftheWonkwangUniversityHospital(ApprovalNo.
WKUH201607-HRBR-078)andallmethodswerecarriedoutinaccordancewithrelevantguidelinesandregulations.
Statistics.
Themean±standarddeviation(SD)wascalculatedandalldatawereplottedusingGraphPadPrism5.
0(Graphpad,LaJolla,CA,USA).
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AcknowledgementsThisresearchwassupportedbytheBio&MedicalTechnologyDevelopmentProgramoftheNationalResearchfoundation(NRF)fundedbytheKoreangovernment,MSIP(2014M3A9E2064699)andbythePriorityResearchCentersProgramthroughtheNationalResearchFoundationofKorea(NRF),fundedbytheMinistryofEducation(NRF-2015R1A6A1A03032236).
AuthorContributionsS.
J.
andD.
T.
B.
developeddot-FICTandperformedFICTassaytofindthesuitablepairofmAbsundertheguidanceofH.
P.
G.
E.
andH.
J.
SohndevelopedmAbsunderH.
J.
Shin.
B.
T.
,N.
T.
V.
,T.
T.
,andN.
T.
P.
performedFICTimmunoassayforoptimization.
S.
J.
andD.
T.
H.
conductedrRT-PCRandanalyzeddata.
C.
K.
developedtheAu-NP-basedR.
D.
T.
withnovelmAbs.
S.
J.
,H.
J.
Shin,andH.
P.
wrotethepaperwithinputsfromallotherauthors.
AdditionalInformationSupplementaryinformationaccompaniesthispaperatdoi:10.
1038/s41598-017-08328-9CompetingInterests:Theauthorsdeclarethattheyhavenocompetinginterests.
Publisher'snote:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.
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