An indirect competitive enzyme-linked

AnalyticaChimicaActa688 (2011) 84–89

ContentslistsavailableatScienceDirect

AnalyticaChimica

Acta

journalhomepage:http://wendang.chazidian.com/locate/ac

a

Anindirectcompetitiveenzyme-linkedimmunosorbentassayfordeterminationofnor?oxacininwatersusingaspeci?cpolyclonalantibody

JianlanCuia,b,KunZhanga,b,QiuxinHuanga,b,YiyiYua,b,XianzhiPenga,?

ab

StatekeyLaboratoryofOrganicGeochemistry,GuangzhouInstituteofGeochemistry,ChineseAcademyofSciences,511KehuaSt.,510640Guangzhou,Guangdong,ChinaGraduateUniversityofChineseAcademyofSciences,100049Beijing,China

articleinfoabstract

Aspeci?cpolyclonalanti-nor?oxacinantibodywasobtained,andasensitiveindirectcompetitiveenzyme-linkedimmunosorbentassay(icELISA)wasdevelopedfordeterminingtraceamountsofnor-?oxacininvariouswaters.Goodlinearitywasachievedintherangefrom0.1to10?gL?1.TheaverageIC50valuewasdeterminedtobe2.2?gL?1andthelimitofdetectionwas0.016?gL?1atasignal-to-noiseratioof3inphosphate-bufferedsalinebuffer.Recoveriesofnor?oxacinatvariousspikinglevelsrangedfrom74to105%ingroundwater,surfacewater,treatedanduntreatedwastewatersamples,withrelativestandarddeviationsof3–5%.Theassaywasappliedfordeterminingnor?oxacininmunicipalwastewater,surfacewater,andgroundwatercollectedinametropolisofChina.Rawwastewatersampleswereonlysubmittedto?ltrationandpHadjustmentwhiletheotherwatersampleswerepre-concentratedbysolidphaseextractionpriortotheicELISAassay.GoodagreementoftheresultsobtainedbytheicELISAandliquidchromatographytandemmassspectrometryfurthercon?rmedthereliabilityandaccuracyoftheicELISAforrapiddetectionofnor?oxacininwaters.

© 2010 Elsevier B.V. All rights reserved.

Articlehistory:

Received12August2010Receivedinrevisedform18December2010

Accepted20December2010

Available online 28 December 2010Keywords:Nor?oxacin

Polyclonalantibody

Indirectcompetitiveenzyme-linkedimmunosorbentassay

LiquidchromatographytandemmassspectrometryWatersamples

1.Introduction

Nor?oxacinisa?uoroquinoloneantibacterialagentthatisextensivelyusedinbothhumanandveterinarymedicine.Inrecentyears,nor?oxacinrankedasthesecondmostprescribed?uoroquinoloneantibacterialjustnexttolevo?oxacininChina.Nor?oxacin,o?oxacinandcipro?oxacinhavebeensofarthemostfrequentlydetected?uoroquinolonesintheenvironmentassum-marizedbyKümmerer[1].Itiswellknownthatantibacterialsintheenvironmentmaypromotethedevelopmentofantibac-terialresistanceinorganisms[2,3].Thenon-targettoxicityofantibacterials,includingnor?oxacin,hasalsobeendocumentedinliterature[4,5].Althoughacutetoxicityofnor?oxacinattheenvironmentallyrelevantlevelshasnotbeenreported,environ-mentalmonitoringforlowlevelofnor?oxacinintheenvironmentisindispensableconsideringtheunknownpotentialecologicalrisks.

Anumberofworkshavebeenconductedtodetermine?uo-roquinolonesinvariousenvironmentalmatricesincludingwater,mainlybasedonhighperformanceliquidchromatography(LC)sep-

?Correspondingauthor.Tel.:+862085290009;fax:+862085290706.E-mailaddress:pengx@http://wendang.chazidian.com(X.Peng).0003-2670/$–seefrontmatter© 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2010.12.030

arationcoupledtovariousdetectorsincludingmassspectrometry[6–8],tandemmassspectrometry(MS/MS)[9–13],?uorescencedetection[14–17]andultraviolet(UV)detection[18,19].However,thesetechnologiesgenerallyrequiredcomplicatedsampleprepa-rationandexpensiveequipment.

Enzyme-linkedimmunosorbentassay(ELISA)hasbeenexten-sivelyusedforscreeningofveterinarydrugresiduesincluding?uoroquinolonesinfoodandanimaltissues(e.g.,muscle,kid-ney,liver,serum,milk,andegg)duetoitssensitivity,speci?city,rapidity,andsimplicity[20–22].Nevertheless,usingELISAfordeterminationof?uoroquinolonesandotherpharmaceuticalsinwatersampleshasstillbeenrarelyreported[23].

Nor?oxacinhasalsobeenwidelydetectedinwastewaterandriverwaterinChina[24].Theobjectiveofthisstudyistodeveloparapid,reliable,andsensitivemethodfordetectingnor?oxacininwater.Aspeci?cpolyclonalantibodyagainstnor?oxacinwasachievedandasensitiveindirectcompetitiveELISA(icELISA)methodwassubsequentlydevelopedthroughoptimizingconcen-trationofcoatingantigen,dilutionofantibody,incubationtime,etc.Themethodwasappliedtodeterminenor?oxacinintreatedwastewater,municipalwastewater,urbanriverwater,lakewater,andgroundwatersampledfromGuangzhou,ametropolisinSouthChina.TheicELISAresultswerefurthercomparedwiththosebyLC–MS/MSanalysis.

J.Cuietal./AnalyticaChimicaActa688 (2011) 84–8985

2.Experiment

2.1.Chemicalsandmaterials

N-(3-Dimethylaminopropyl)-N??-ethylcarbodiimidehydrochlo-ride(EDC.HCl),ovalbumin(OVA),bovineserumalbumin(BSA),N,N-hydroxysuccinimide(NHS),nor?oxacin(NFX),o?oxacin,cipro?oxacin,enro?oxacin,pe?oxacinmesylatedihydrate,enoxacin,marbo?oxacin,lome?oxacinhydrochlorideaswellasbothFreund’scompleteadjuvant(cFA)andincompleteadjuvant(iFA)werepurchasedfromSigma–AldrichCompany(St.Louis,MO,USA).Goatanti-rabbitIgG-horseradishperoxidaseconjugate(GaRIgG-POD)waspurchasedfromPierce(Rockford,IL,USA).O-Phenylenediamine(OPD)waspurchasedfromSolarbio(Beijing,China).N,N-Dimethylformamide(DMF),Tween-20andhydrogenperoxide(H2O2,30%)werepurchasedfromDonghongChemicalCompany(Guangzhou,GD,China).

BlockingbufferwaspurchasedfromKem-En-TecDiagnos-ticsCompany(Taastrup,Denmark)andwasusedasaccepted.Otherbufferswerepreparedwithultra-purewater.Thecoat-ingbufferwas0.05mmolL?1sodiumcarbonatebuffer(pH9.6).Phosphate-bufferedsaline(PBS,pH7.4)consistedof1.47mmolL?1KH2PO4,8.48mmolL?1Na2HPO4·12H2O,2.70mmolL?1KCland137mmolL?1NaCl.Thewashingbuffer(PBST)wasaPBSbufferwith0.05%(v/v)Tween-20.Thesubstratebuffer(pH5.0)was0.1molL?1phosphate/citratebuffer.Thehaltingsolutionwas2molL?1H2SO4.Thesubstratesolutionwaspreparedbydissolving10mgofOPDin25mLofsubstratebuffer.TenmicrolitersofH2O2wasaddedintothesubstratesolution3minpriortouse.2.2.Instrumentation

icELISAwascarriedoutin96-wellmicrotitreplates(Nunc,Roskilde,Denmark).Immunoassayabsorbancewasreadbyamul-tiscanMK3microplatereader(ThermoFisherScienti?c,Vantaa,Finland).ThemicroplatewaswashedusingaThermoFisherSci-enti?c4MK2wellwasher.AHeliosAlphaspectrometer(ThermoSpectronic,Cambridge,England)wasusedtocollectUVdata.2.3.Synthesisofimmunogenandcoatingantigen

AnN-hydroxysuccinimideactiveestermethod[25]wasusedforthesynthesisofimmunogenNFX-BSAandcoatingantigenNFX-OVA.Brie?y,forthepreparationofNFX-BSA,NFX(9.6mg),NHS(17.25mg),andEDCHCl(57.5mg)weresequentiallyaddedinto3mLofDMF.Themixturesolutionwasmagneticallystirred(420rpm)for24hatroomtemperatureindarkness.ThemixturewasthenaddedtoBSAsolutionthatwaspreparedbydissolving750mgofBSAin8mLofPBSbuffer.Thereactionmixturewasstirredforanother3hfollowedbydialysis(molecularweightcut-off10,000Da)againstPBSbufferfor3days.ThePBSbufferwasrenewed3timesperdaytoremovetheuncoupledfreehapten.TheobtainedNFX-BSAimmunogenwasfreezedriedandstoredat?20?C.ThecoatingantigenNFX-OVAwaspreparedinasimilarmanner.UVabsorbancespectrumwasusedtocon?rmtheconju-gation.

2.4.Antiserumproduction

TwofemaleNewZealandwhiterabbitsweresubcutaneouslyimmunizedwiththeimmunogenNFX-BSAatmultiplesites.Theinitialimmunizationwasperformedbyinjecting1.5mgofNFX-BSAdissolvedin0.5mLofsterilizedphysiologicalsalinesolution(PSS)andemulsi?edwith0.5mLofcFA.Fiveboosterimmuniza-tionswereconductedbyinjecting0.75mgofNFX-BSAin0.5mLofPSSand0.5mLofiFAat17-dayintervals.Thesixthbooster(0.75mg

ofNFX-BSAin1mLofPSS)wasdone10dayslater.Fromthethirdboosteronward,serumtitersweredeterminedbyELISAsevendaysaftereachimmunizationtomonitorthequalityoftheantiserafromtheimmunizedrabbits.Antiserumwasobtainedbycentrifugingthebloodat10,010×gfor10minfollowingclottingfor1hat4?C.

Sevendaysafterthelastbooster,50mLofbloodwascollectedfromthejugularveinofeachrabbit.Theantiserumwasisolatedbycentrifugingthebloodandwasfurtherpuri?edwithanIgGpuri-?edkit(Millipore,Billerica,MA,USA).Theobtainedantiserumwasfreezedriedandstoredat?20?C.Theserumfromtherabbitthatshowedthehighesttiterandsensitivitywasusedinthiswork.2.5.IndirectcompetitiveELISA

OnehundredmicrolitersofNFX-OVAsolution(1.6?gL?1incoatingbuffer)wascoatedineachwellofa96-wellmicrotitreplate.Theplatewasincubatedfor100minat37?Candwaswashedwith350?LofPBSTforfourtimesusingthewellwasher.Kem-En-Tecsyntheticblockingbuffer(250?L)wasthenaddedintoeachwellandtheplatewasincubatedfor8minatroomtemperature.Aftertheplatewaswashedasdescribedbefore,50?Lofdilutedantibody(1:128000inPBS)andnor?oxacinstandardsolutionsorsamplesweresubsequentlyadded,leadingtothe?nalincubationvolumeof100?Lperwell.Followingadditionalincubationfor1hat37?C,theplatewaswashedagain.GaRIgG-PODwasthenadded(1:10000inPBS;100?Lperwell)andtheplatewasincubatedat37?Cfor1h.Forcolordevelopment,100?Lofsubstratesolu-tionwasaddedintoeachwell.Sixminuteslater,thereactionwasstoppedbyadding50?Lofhaltingsolution(2molL?1H2SO4)intoeachwell.Theabsorbancewasmeasuredbythemicroplatereaderat492nm.Theresultswerepresentedasinhibition=B/B0,whereBandB0wereabsorbanceofthewellswithandwithoutcompetitor,respectively.AcalibrationcurvewasconstructedintheformofB/B0versusLogCatnor?oxacinconcentrationsof0.01,0.1,0.5,1,5,10,and100?gL?1.Concentrationsofnor?oxacininsamplesassayedinthesameplatecouldthenbedeterminedusingthestandardcurve.2.6.Sensitivityandspeci?cityoftheassay

IC50value(inhibitorconcentrationatB/B0ratioof0.5)andcross-reactivity(CR)weredeterminedtoevaluatethesensitivityandspeci?http://wendang.chazidian.competitiveimmunoassayswereper-formedusingsevenother?uoroquinolonecompoundsincludingcipro?oxacin,enro?oxacin,enoxacin,lome?oxacin,marbo?oxacin,o?oxacinandpe?oxacin.Thesecompoundswerechosenbasicallybecausetheyarestructurallyrelatedtonor?oxacin(Table1).Inaddition,someofthem,suchascipro?oxacinando?oxacinhavealsobeenwidelydetectedintheenvironmentasnor?oxacin[1].Thetested?uoroquinolones(0.01–10,000?gL?1)weredeployedtotheicELISAprocedureasdescribedabovefornor?oxacin.TheCRvalueswerethencalculatedasCR(%)=(IC50-norfolxacin/IC50-testedcompound)×100.

2.7.Samplecollectionandpreparation

WatersampleswerecollectedfromGuangzhou,ametropolisinSouthChina.Threerawwastewatersamplesandonetreatedwastewatersamplewerecollectedfromasewagetreatmentplantthatservesapopulationofaround2.5million.Surfacewatersam-pleswerecollectedfromthePearlRiverandanurbanlake.Agroundwatersamplewascollectedfromawell.Allthesampleswerestoredinamberglassbottleswithoutheadspaceandplacedonicepacksduringtransporttothelaboratory.

Sampleswere?lteredthrough0.7?mglass?ber?lter(What-man,Maidstone,England).RawwastewatersampleswereonlyadjustedtopH7.4priortoicELISAassay.Surfacewater,treated

86J.Cuietal./AnalyticaChimicaActa688 (2011) 84–89Table1

IC50valuesandcross-reactivityoftheantibodytowardnor?oxacinandotherselected?uoroquinolones.

CompoundStructureIC50(?gL?1)Cross-reactivity(%)

F

Nor?oxacin42O2.2100

8N1

F

O?oxacin76548132OHO>5000<0.1

NO

F

Cipro?oxacin2>5000<0.18N1

O

F

Enro?oxacin42O>5000<0.18N1

O

F

Pe?oxacin2O8N13700.6

O

F

Enoxacin42O>5000<0.1

N

8N1

F

Marbo?oxacin7658132OHO>5000<0.1

NNO

F

Lome?oxacin2O>5000<0.11wastewater,andgroundwatersampleswereconcentratedusing

asolidphaseextraction(SPE)procedurethathadbeendetailed

elsewhere[17].Brie?y,100mLofthe?lteredsamplewasadded

withEDTAat0.5gL?1,adjustedtopH4.2,andloadedontoanHLB

cartridge(Waters,Milliford,MA,USA)atabout5mLmin?1.Thecar-tridgehadbeenpreconditionedsuccessivelywith5mLofmethanoland5mLofultra-purewater.Aftersamplepassage,thecartridgewaswashedwith2mLof10%methanolsolutionandvacuumdriedfor10min.Thecartridgewasthenelutedwith5mLofmethanol.Theeluentwasbroughttodrynessunderagentle?owofhigh

J.Cuietal./AnalyticaChimicaActa688 (2011) 84–8987

puritynitrogen.Theextractswerere-dissolvedintoappropriatevolumesofPBSbuffer,leadingtoenrichmentfactorsof5–10forsurfacewaterandtreatedwastewaterand100forgroundwater.2.8.LC–MS/MSanalysis

Chemicalanalysisofnor?oxacinwasalsoundertakenusingLC–MS/MS.Analiquotofthe?lteredsample(100mLofwastew-aterand400mLoftheotherwatersamples)wasspikedwith50ngofcipro?oxacin-d8astheinternalstandardfollowedbyenrichmentwithSPEasdescribedabove.Theextractwas?nallyreconstitutedin1mLofmethanoland?lteredthrougha0.22?msyringe?lter(Anpel,Shanghai,China).LC–MS/MSanalysiswasperformedonanAgilentLC1200systemcoupledtoanAgilent6410triplequadrupoleMSwithelectrosprayionizationinposi-tivemode(Agilent,PaloAlto,CA,USA).AnAgilentZorbaxEclipseXRDC18column(150mm×3mm,3.5?mparticlesize)wasusedata?owrateof0.25mLmin?1at25?C.A4.0mmguardcolumn(Phenomenex,Torrance,CA,USA)containingthesamesorbentwaspre-connected.Themobilephasewaswater:methanol(40:60,v/v)with5mmolL?1ammoniumacetateand0.2%formicacid.Theretentiontimeofnor?oxacinwas3.39min.

TheoptimalMSparametersweredetailedelsewhere[26].TheMStemperaturewas100?C.Nitrogenwasusedasdrygaswitha?owrateof10Lmin?1andtemperatureat350?C.Nitrogenwasalsousedasthecollisiongas.Thecapillaryvoltagewassetat4500Vandthenebulizerpressurewas275.8kPa.Protonatedmolecularionm/z320.1wassetastheprecursorionfornor?oxacin.Theproductionm/z302.1correspondingto[M+H-H2O]+exhibitedthehigh-estintensity,followedbym/z276.0correspondingto[M+H-CO2].Dataacquisitionwasconductedinmultiplereactionmonitoringmodeusingprecursor–productionpairsm/z320.1>302.1andm/z320.1>276.0asthequanti?erandquali?er,respectively.Adwelltimeof50mswassetforeachiontransitiontomaximizethesen-sitivity.InstrumentcontrolanddataacquisitionweremanagedbyMassHunterWorkstation.Thelimitsofquanti?cationfornor-?oxacinwere0.100?gL?1inrawwastewaterand0.020?gL?1inriverwater.2.9.Recoverytests

Recoverytestswerecarriedoutbyspikingnor?oxacinintoPBSbuffer(pH7.4),groundwater,surfacewater,treatedanduntreatedwastewateratvariouslevelsfrom0.05to10?gL?1todeterminetheef?ciencyoftheicELISAassay.Samplesspikedat0.05and0.1?gL?1weresubjectedtoanSPEprocedurewithanenrichmentfactorof5priortoicELISA,whereassamplesforti?edathigherconcentrations(0.2,0.5,1,5,and10?gL?1)wereassayeddirectly.Recoverieswerecalculatedasthefollowing:Recovery(%)=

Css?Cus

s

×100(1)

whereCssandCusareconcentrationsmeasuredinthespikedsampleandunspikedsamples,respectivelyandCsisthespikedconcentration.

3.Resultsanddiscussion

3.1.Productionandcharacterizationoftheantibody

Toobtainaspeci?canti-nor?oxacinantibody,itisessentialtodevelopaneffectiveimmunoassay.Asasmallmolecule,nor?oxacin(molecularmass=319.3)hastobeconjugatedwithacarrierproteintoelicittheimmuneresponseofananimaltoproducetheanti-nor?oxacinantibody.Fortunately,theactivecarboxylicacidgrouppresentedatposition3inthemoleculeofnor?oxacin(Table1

)

Fig.1.UVspectraofNFX,NFX-BSAandBSA.

enablesdirectconjugationtoacarrierprotein[27].BSAandOVAareamongthemostcommonlyusedproteincarriers[28].AnimmunogenNFX-BSAandacoatingantigenNFX-OVAweresyn-thesizedusingtheN-hydroxysuccinimideactiveestermethod[25].UVspectrumwasemployedtomonitortheeffectivenessofconju-gationreaction[28].TheabsorbanceforNFX-BSAshowsared-shiftmaximumat286nmcomparedwiththe272nmmaximumfornor?oxacinand273nmmaximumforBSA(Fig.1),indicatingthesuccessfulconjugationbetweenNFXandBSA.ThecoatingantigenNFX-OVAgivesaUVspectrumsimilartothatofNFX-BSA.TheUVspectralanalysisofthehapten–proteinconjugatesindicatedthatNFXwasincorporatedontoBSAandOVAatabout3.2and2.2molpermolofprotein,respectively.

Titeroftheantiserum,de?nedasthereciprocalofthedilutionthatresultedinanabsorbancevaluetwicethatoftheblank(serumfromthesamerabbitbeforeimmunization),increasedaccordingtoimmunizationtimes.The?nalpuri?edantibodyhasatiterof>1,024,000.

3.2.OptimizationoftheicELISA

Todevelopaspeci?candsensitiveELISA,assayconditions,suchasconcentrationofthecoatingantigenNFX-OVA,dilutionsoftheantibodyandGaRIgG-POD,selectionoftheblockingreagent,incu-bationtimeandtemperaturewereoptimized.Criteriausedtoassesstheoptimizationincludedmaximumabsorbance(B0),linearrange,IC50value,andlimitofdetection(LOD).

Accordingly,concentrationsofcoatingantigenNFX-OVAfrom0.004to10?gL?1incombinationwithdilutionsofantibodyNFX-BSAfrom1:500to1:1024000anddilutionsofGaRIgG-PODfrom1:500to1:20000weretestedusingacheckerboardprocedure[23].TheoptimalcombinationoftheimmunoreagentswasacoatingantigenNFX-OVAconcentrationat0.16?gmL?1withdilutionsof1:128000fortheantibodyand1:10000forGaRIgG-POD,producingamaximumabsorbanceofaround1intheabsenceofananalyte.

Theeffectivenessoftheincubationforboththecoatinganti-genandtheimmunoreactionwasexaminedaccordingtothelinearrange,IC50value,andlimitofdetection(LOD).Differentincuba-tionconditionsforthecoatingantigen(overnightat4?C,60minand100minat37?C)weretested.Nosigni?cantdifferencewasobservedinincubationeffectivenesswhentheplateswerecoatedovernightat4?Corfor100minat37?C.However,ahighIC150value(>20?gL?)wasfoundwhentheplateswerecoatedat37?Cfor60min.Therefore,theplateswere?nallycoatedfor100minat37?C.Immunoreactionperiodsof30min,60minand120minwere

88J.Cuietal./AnalyticaChimicaActa

688 (2011) 84–89

Fig.2.Optimizedcalibrationcurveofnor?oxacingeneratedinPBSbuffer.Standarddeviations(n=16)areindicatedaserrorbars.

testedat37?C.Theresultshowedthat60minofimmunoreactiongavethebestperformance,whichgeneratedthelowestIC50.valueandLODandthemaximumspanoflinearrange.

Theblockingbuffershouldbecarefullyselectedbecauseitisusedtopreventnon-speci?cabsorbance[29].Twoblockingreagents(OVAandcasein)preparedwithPBSataconcentrationof1%andaKem-En-Tecsyntheticblockingbufferweretestedfortheirblockingcapacity.TheKem-En-Tecsyntheticblockingbuffergeneratedthelowestbackgroundsignal(<5%)andthereforewasselectedastheblockingbufferinthisstudy.3.3.Methodperformance

3.3.1.Stabilityandsensitivity

ThestabilityoftheassaywastestedbyrunningtheicELISApro-ceduresfor16individualtimesoveratwo-monthperiod.Fig.2showstheaveragecalibrationcurvegeneratedfrom16assays.Therelativestandarddeviation(RSD)ofthemeasuredabsorbanceforsixteenreplicatesateachstandardconcentrationwasfrom1.1to2.5%,indicatingthestabilityoftheicELISA.

IC50valueisakeycriterionforevaluatingthesensitivityofELISA.Inthisstudy,thecalibrationcurvewasconstructedfrom0.01to100?gL?1,withalinearrangefrom0.1to10?gL?1(r2=0.99,Fig.2).TheaverageIC50valuewas2.2?gL?1andtheLODatasignal-to-noiseratioof3was0.016?gL?1,suggestingthattheestablishedicELISAwashighlysensitive.

3.3.2.Speci?city

TheCRvaluesofo?oxacin,enro?oxacin,cipro?oxacin,enoxacin,marbo?oxacinandlome?oxacinwerealllessthan0.1%.CRvalueofpe?oxacinwasslightlyhigher,whichwas0.6%(Table1).Theseresultsindicatedthespeci?cityofthegeneratedantibody.

Previousresearchhasrevealedthatthesubstituentslocatedinquinoloneringsmayin?uencethecross-reactivityofanti-body[27,30].All?uoroquinolonespossessanitrogen-containing,six-memberedheterocyclicaromaticring,acarboxylicgroupatposition3,aketonegroupatposition4anda?uorinesubstituentatposition6(Table1).Substituentsatpositions1,7and8differbycompound.Huetetal.[21]suggestedthatsubstituentatposition7isthegoverningstructuralfactordeterminingthebindingaf?nityof?uoroquinolonemoleculestoanti-nor?oxacinantibodyandanti-sara?oxacinantibody.Thesubstituentatposition8inthestructureofgati?oxacinwasalsobelievedtobeacontributiontothehighspeci?cityofanti-gati?oxacinantibody[28].Theimportanceof

Table2

Recoveryofnor?oxacindeterminedbyicELISAinspikedwatersamples.Samples

Spiking

Recovery(%)

Relativeconcentrationstandard(?gL?1)deviation(%)PBSbuffer(n=4)

0.05a8240.1a8540.5906193451085101019Groundwater0.05a845(n=4)

0.1a844Treatedwastewater0.05a865(n=4)

0.1a845Wastewater(n=4)0.29640.51054Surfacewater0.1a853(n=8)

0.05a8630.27730.5

74

4

a

SamplewasenrichedbysolidphaseextractionpriortoicELISA.

substitutionatposition1in?uoroquinolonestructuresforanti-bodybindinghasalsobeenstressedpreviously[27,30].Thepooraf?nity(<0.1%)oftheproducedanti-nor?oxacinantibodytowardo?oxacin,enro?oxacinandmarbo?oxacinseemsreasonableduetodifferentsubstituentsatpositions1and7inthestructuresofthesecompoundsfromthoseofnor?oxacin.Enoxacinandlome-?oxacinhaveanethylgroupatposition1asnor?oxacin,however,enoxacinhasanitrogensubstituentandlome?oxacinhasa?uo-rinesubstituentatposition8.Amolecularmodelandanelectronicpropertyofglobalminimumenergyconformationsforenoxacinandlome?oxacinhavecon?rmedthatchangeatposition8couldleadtosigni?cantchangesinmoleculeshapeandelectrostaticfea-tures[27].ThismayexplaintheirlowCRvalues(<0.1%).However,itisdif?culttoexplainthelowrecognitionofcipro?oxacin(<0.1%)andpe?oxacin(0.6%)bytheantibodybecausebothcompoundsarequitestructurallysimilartonor?oxacin.Theonlydifferencebetweenthemolecularstructuresofcipro?oxacinandnor?oxacinisthesubstituentatposition1,whichisacyclopropylgroupforcipro?oxacinbutanethylgroupfornor?oxacin.Pe?oxacinandnor?oxacindifferintheirstructuresbythesubstituentatposition7(Table1).Oneofthethreebroad-spectrumantibodiesproducedbyWangetal.[27]usingthenor?oxacinhaptenalsoshowedpooraf?nitytope?oxacin(<0.01%)butmoderateaf?n-itytocipro?oxacin(24%).Overall,thelowIC50value(2.2?gL?1)andpoorcross-reactivityagainstother?uoroquinolonesdemon-stratethesensitivityandselectivityofthedevelopedassayforthedeterminationofnor?oxacin.

3.3.3.Accuracy

Recoveriesofnor?oxacinfromspikedPBSbufferatconcentra-tionsof0.5,1,5,and10?gL?1rangedfrom90to108%,withRSDsof4–9%(n=4).Satisfactoryrecoverieswereachievedfornor?oxacinfromwastewater(96–105%)andsurfacewater(74–77%)spikedat0.2and0.5?gL?1withgoodreproducibility(RSD≤4%).Goodandconstantrecoveries(82–86%)wereobtainedatlowspikinglev-els(0.05and0.1?gL?1)fromvariouswaterswhenthesamplesweretreatedwithSPEpriortotheicELISA(Table2).Therecover-iesfromspikedsurfacewatersamplesat0.05and0.1?gL?1wereevenslightlyhigherthanthoseat0.2and0.5?gL?1,probablyduetothereductionofmatrixinterferencebytheSPEprocedure.3.4.Analysisofwatersamples

TheicELISAassayhasbeenappliedtodeterminenor?oxaciningroundwater,surfacewater(riverwaterandlakewater),treated