A New immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti- U3RNP antibody detection in systemic...

MISSING IMAGE

Material Information

Title:
A New immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti- U3RNP antibody detection in systemic sclerosis
Series Title:
Arthritis Research & Therapy
Physical Description:
Book
Language:
English
Creator:
Ceribelli, Angela
Satoh, Minoru
Chan, Edward K. L.
Publisher:
BioMed Central
Publication Date:

Notes

Abstract:
Introduction: Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies. Methods: Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard. Results: By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP. Conclusions: Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
Resource Identifier:
doi - 10.1186/ar3858
System ID:
AA00012629:00001


This item is only available as the following downloads:


Full Text

PAGE 1

RESEARCHARTICLE OpenAccessAnewimmunoprecipitation-realtime quantitativePCRassayforanti-Th/ToandantiU3RNPantibodydetectioninsystemicsclerosisAngelaCeribelli1,MinoruSatoh2andEdwardKLChan1*AbstractIntroduction: Classicanti-nucleolarantibodiesanti-Th/ToandU3ribonucleoprotein(-U3RNP)canhelpinthe diagnosis,predictionoforganinvolvementandprognosisinsystemicsclerosis(SSc);however,novalidated commercialassayisavailable.WeaimedatestablishinganovelquantitativerealtimePCR(qPCR)methodto detecttheseantibodies. Methods: Standardimmunoprecipitation(IP)wasperformedusingK562cellextractandRNAcomponentswere extracted.cDNAwasreversetranscribedfromRNAcomponentsandThRNAandU3RNAweredetectedbyqPCR usingcustomprimers.Cyclethreshold(Ct)valueswerecomparedinatitrationexperimenttodeterminetheassay efficacy.Thenewassaywasevaluatedbytesting22anti-Th/Toand12anti-U3RNPpositivesamplesinadditionto 88controls,andtheresultswerecomparedwithIPasagoldstandard. Results: Bytestingserial1:8dilutionsofcelllysateasthesubstrateintheIPstep,RNAextractedafterIP,andits derivedcDNA,lineardoseresponsecurveswerenotedforbothanti-Th/Toand-U3RNP.Witheverydilution,Ct valueschangedapproximatelythreeasexpected,reflectingtheeight-folddifferenceofcDNA.TheCtdifference betweenpositiveandnegativesampleswas8to13,whichwassimilarthroughoutthedilutions.Inthespecificity analysis,theCtvaluesofpositivesampleswereclearlydifferentfromthenegativegroupsandtheresultsbyqPCR hadanearperfectcorrelationwithIP. Conclusions: OurnewmethodreadilydetectsthesetwoclinicallyimportantantibodiesinSSc.Makingtestsfor anti-Th/Toand-U3RNPantibodieswidelyavailabletocliniciansshouldbehelpfulinthediagnosisandfollow-upof SScpatients.IntroductionScleroderma(SystemicSclerosis,SSc)isasystemicautoimmunediseasecharacterizedbyfibrosis,vascular changes,andtheproductionofautoantibodies.The mostcommonantibodiesassociatedwithSScareanticentromere(ACA),-topoisomeraseI(topoI)and-RNA polymeraseIII(RNAPIII)antibodies,approximately20% each[1-5].Anti-topoIandACAhavebeenusedfor about30yearsfordiagnosticpurposes,whileanti-RNAPIIIELISAhasbeenaddedtoroutinescreeningonly recently[6-8].SScpatientscanbeclassifiedintotwo majorsubsets:limited(lcSSc)anddiffuse(dcSSc) cutaneousvariants.ThedcSScisfrequentlyassociated withanti-topoI,-RNAPIII, or-U3RNP,whilelcSScis associatedwithACAandanti-Th/Toantibodies[1,9]. TheseautoantibodiesarefairlyspecificforSScandcan bedetectedevenbeforediagnosis.Theyareassociated withuniqueclinicalfeaturesandareusefulinpredicting clinicalmanifestationsofSSc[1,10-12]. Anti-Th/Toand-U3RNPareanti-nucleolarantibodies thathavebeenknownformorethan25years.Despite theirclinicalimportance,theseSScautoantibodieshave notbeenutilizedclinicallybecauseoftheunavailability ofantibodytesting[7,13].Urea-polyacrylamidegelelectrophoresis(PAGE)analysisoftheRNAcomponentsin immunoprecipitates,eitherbysilverstainingorbyusing32P-labelingofcells,isthe standardmethod,butitis performedonlyinasmallnumberofresearch *Correspondence:echan@ufl.edu1DepartmentofOralBiology,UniversityofFlorida,P.O.Box100424,1395 CenterDrive,Gainesville,FL32610-0424,USA FulllistofauthorinformationisavailableattheendofthearticleCeribelli etal ArthritisResearch&Therapy 2012, 14 :R128 http://arthritis-research.com/content/14/3/R128 2012Ceribellietal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

PAGE 2

laboratories.Nocommercialwidely-availablevalidated immunoassaykithasbeenproducedsofar[14].The aimofourstudyistoestablishanewmethodtodetect anti-Th/Toand-U3RNPantibodiesbasedonquantitativePCR(qPCR)detectionoftheRNAcomponentsof theribonucleoproteinautoantigens.MaterialsandmethodsImmunoprecipitationandquantitativePCRImmunoprecipitation(IP)wasperformedusingK562 celllysateandassociatedRNAwasextractedusingphenol/chloroform/isoamylalcohol(25:24:1)asdescribed [13,15].RNApelletswereresuspendedin30lRNAgradewater.cDNAwasobtainedfromeachRNAsample(10l)byreversetranscription(RT)usingRTMasterMix(HighCapacitycDNARTkit,Applied BiosystemsInc.,ABI,FosterCity,CA,USA).ThethermalcyclerfortheRTsettingwas:10minutesat25C, 120minutesat37C,5secondsat85C.Quantitative PCR(qPCR)wasperformedusingtheTaqManFast UniversalPCRMasterMix(ABI).ForThRNA(7-2 RNA,RMRP)detection, Hs03298751_s1 primerbyABI wasused,whiletheprimerforU3RNA(SNORD3A, IntegratedDNATechnologies,Coralville,IA,USA)has thefollowingsequence: Probe5 -/56-FAM/CCAAGCAAC/ZEN/GCCAGAAAGCCG/3IABkFQ/-3 ; Primer1 (FOR.)5 -TGTAGAGCACCGAAAACCAC-3 ; Primer2 (REV.)5 -TCCCTCTCACTCCCCAATAC-3 .qPCRwas performedinduplicateusingtheStepOnecycler(ABI) for40cycles,andresultswereevaluatedbycyclethreshold(Ct)values. Insomeexperiments,La-depletedcellextractwasalso usedtoexaminetheeffectsofLadepletioninalimited numberofsamples(n=24).Anextractfrom25106K562cellswasabsorbedwith0.5mlofanti-LaimmunoglobulinG-cyanogenbromid e(IgG-CNBr)-activated Sepharose4BbeadstodepleteLa[16].SerumsamplesTheprotocolofthisstudywasapprovedbytheInstitutionalReviewBoard(IRB).Thisstudymeetsandisin compliancewithallethicalstandardsinmedicineand informedconsentwasobtainedfromallpatientsaccordingtotheDeclarationofHelsinki.Forthetitration experiment,oneeachofanti-Th/To,-U3RNPandnormalhumanserum(NHS)wastested,withserialeightfolddilutions(from1to1:4096)ofcelllysateassubstrateintheIPstep,RNAextractedafterIP,andits derivedcDNA.Tovalidatethenewassay,22anti-Th/ To,12anti-U3RNP-positivesamples,58SScseranegativeforanti-Th/Toand-U 3RNPantibodies(20antiTopoI,18anti-RNAPIII,15ACA,5anti-PM-Scl),12 anti-La/SSB,3anti-trimethylguanosine(TMG)[17],and 15NHS,wereanalyzed.Thespecificityoftheseserawas confirmedbyIPandurea-PAGEanalysisasdescribed [13].Ourdataarerepresentativeoftwoormoreindependentexperiments.PCRwasanalyzedinduplicate andimmunoprecipitationwasalsotestedinduplicatein someexperiments.Thetypicalreproducibilitywasvery highshowingadifferencewithinoneCtvalue.Inthe newassaydescribedhere,nohousekeepinggenemRNA wouldbepresentconstantly,whichisdifferentfromthe standardqPCRusedtoexaminethelevelsofmRNAfor thegeneofinterestintotalRNAortotalmRNA.Nevertheless,thelevelof18SRNAwasalsoexaminedasa potentialinternalcontrolfornonspecificbinding(data notshown),buttheresultsshowedvariationinitslevels sothatitwasnotconsideredsuitableasaninternalcontrolinourassay.StatisticalanalysisDatabetweengroupswerecomparedbyKruskal-Wallis andDunn smultiplecomparisontestsusingPrism5.0 forMacintosh(GraphPadSoftware,Inc).Thesameprogramwasappliedtoobtainthereceiveroperatingcharacteristic(ROC)curves,comparingtheanti-Th/Toor -U3RNPpositivegroupversusthenegativegroupto determinethecut-off.Statisticalsignificancewas acceptedat P <0.05.ResultsAutoantibodydetectionusingqPCRfortheRNA componentoftheribonucleoproteinautoantigen complexThesensitivityandlinearityoftheassaywasevaluated forthedetectionoftheThandU3RNAcomponents, usingserial1:8dilutions(1-1:4096)ofcelllysate,RNA purifiedafterIP(bothst artedfromextractsof107cells persample,butdatashownarebasedontheuseof10 outof30lofRNA,equivalentto3.306cells)and cDNAobtainedafterRT(theequivalentofcDNAmade fromRNAderivedfrom3.3106cells)(Figure1).Ct valuesforthetitrationusingseriallydilutedcelllysate were8.3(extractfrom107cells,equivalenttoundiluted or 1 inFigure1A)to22.7(extractfrom2.4103cells, correspondingto1:4096inFigure1A)forThRNAand 13.4to28.9forU3RNA(notshown),usingtheprototypesera.Lineardoseresponsecurveswerenotedfor celllysate,RNA,andcDNAdilutions,andthedifference betweenpositivesamplesandNHSwasclearevenat 1:4096dilution,whichcorrespondedtoacellextract from2.4103K562cells(Figure1A).Ctvalueschangedbyapproximatelythreeforevery1:8dilution, reflectingtheeight-folddifferenceandthehighefficiencyoftheqRT-PCRasexpected(Figure1A).Similar titrationofRNAandcDNA,showninFigure1Band 1D,demonstratedthesamelinearrelationshipineach stepoftheqRT-PCR.IPusingthestandard8lofCeribelli etal ArthritisResearch&Therapy 2012, 14 :R128 http://arthritis-research.com/content/14/3/R128 Page2of6

PAGE 3

humanserumversus1lwasalsocomparedinthe titrationexperiment,toexaminetheeffectsontheCt values.ThedifferenceinCtvalueswasapproximately three,furtherdemonstratingthehighsensitivityofthe newmethodalsowith1lofserum. ThesensitivityofdetectionofThorU3RNAbythe qPCRmethodwascomparedwiththatofthestandard urea-PAGE-silverstaining methodbytestingthesame amountofseriallydilutedThRNA(Figure1BversusC) orU3RNA(notshown).Whenusingserialdilutionsof RNA,theqPCRassayclearlydetectedasignalatthe highestdilutionof1:4096(Figure1B)whereasThRNA wasbarelyvisibleonlyupto1:16dilutionbysilverstaining(Figure1C).Thus,qPCRwasatleast256-foldmore sensitivethansilverstaining.ForU3RNAdetection, qPCRwasclearlypositiveatadilutionof1:4096,similarlyto7-2RNA,whereasU3RNAwasdetectedonlyup toa1:8dilutionbysilverstaining(datanotshown),indicatingthattheqPCRassaywasatleast512-foldmore sensitive.Basedonthesedata,cellextractsfromonemillioncells,whichcorrespondto1/10ththeamountofcell lysatenormallyusedforIP,wereusedforscreeningof seraforanti-Th/To,-U3RNP,andotherantibodies.SpecificityofIP-qPCRassayappliedtoclinicalsamplesThemeanCtvaluewas19.81(standarddeviation(SD) 1.45)foranti-U3RNPand15.27(SD0.73)foranti-Th/ Tosamples(Figure2A,B).Thesevalueswereclearly separatedfromthoseofthecontrolgroups,withadifferenceofapproximatelyeightCtvaluesforantiU3RNPandapproximately13Ctvaluesforanti-Th/To ( P <0.05)(Figure2A,B).TheCtvaluesofanti-TMG seraweresimilartothoseofanti-U3RNPpositivesera (Figure2A).Thiswasexpectedsinceanti-TMGantibodiesrecognizethecapstructureonU1-U5RNA,includingU3RNA[17].Despitema intainingstatistical significance,theCtdifferencebetweenanti-Thand-La sampleswassmallerthantheCtdifferencebetween anti-Thandothernegativecontrols(Figure2C).This couldbeexplainedbasedontheinteractionandcoimmunoprecipitationofThRNAbyLaantigens,as shownpreviously[18].Forthisreason,anti-Laserawere alsotestedintheIP-qPCRassayusingLa-depletedK562 celllysate.Ctvaluesincreasedbyabouttwoandbecame comparabletoothernegativegroupswhenLa-depleted celllysatewasusedinplaceofwholecelllysate(data notshown).ThisindicatedthatthelowCtvaluesof anti-Laseraintheanti-ThqPCRassaywereduetocoIPoftheThRNAbytheLacomplex,andnotbycoexistinganti-Thantibodies.Whentestinganti-U3RNP samples,5/12anti-LapositivesampleshadlowCt values,whichfellbelowthecut-off(Figure2A).This mightbeexplainedbytheobservationthatLacould bindandstabilizeprecursorsoftheU3smallnucleolar Figure1 Highsensitivityandlineardetectionofanti-Th/ToantibodiesintheIP-qPCRassaydemonstratedbytitrationanalyses .One prototypeanti-Th/TosampleandNHSwereanalyzed,andCtvaluesplottedagainstserialdilutionofcelllysateusedassubstrateforIP( A ),RNA extractedfromIP( B ),orthederivedcDNA( D ).Dilutionsstartwithlysatefrom107K562cellspersample. C.ThesensitivityofournewIP-qPCR methodiscomparedwithRNAdilutiondetectedbysilverstaining,whichallowstheidentificationofthetwoRNAbands(8-2RNA,7-2RNA) onlyupto1:16dilution.Ct,cyclethreshold;IP-qPCR,immunoprecipitation-quantitativepolymerasechainreaction;NHS,normalhumanserum. Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R128 http://arthritis-research.com/content/14/3/R128 Page3of6

PAGE 4

RNAtopreventdegradatio n[19].Thus,U3RNAcould beweaklydetectedintheanti-Laimmunoprecipitate. ConsideringIPwithsilver stainingasthegoldstandardforthedetectionofanti-Thand-U3RNPantibodies,thesensitivitybyo urnewIP-qPCRmethodwas 100%forboth(Table1).Assa yspecificityforanti-Th and-U3RNPinSScpatientswas100%and98.9%, respectively(Table1).For anti-U3RNPdetection,one anti-Th/Toserumwasrepeatedlypositiveforan unknownreason.DiscussionTheimportanceofthedevelopmentofaconvenient assayforanti-Th/Toand-U3RNPantibodiescannotbe underestimatedbecausethesetwoclassicanti-nucleolar autoantibodiesareassociat edwithparticularSScvariantsandclinicalfeatures[1].Infact,anti-Th/Toantibodiesarefrequentlydetect edinlcSScpatientswith pericarditisandmildlunginvolvement[13,20],while anti-U3RNPantibodiesarea ssociatedwithseverepulmonary,renalandmusculardisease,mainlyinAfricanAmericandcSScpatients[1,21].Thus,theaimofour studywastoestablishanewmethodthatcanbeused widelyandeasilytodetectanti-Th/Toand-U3RNP antibodiestoreplacecomplicatedRNAanalysisbygel electrophoresis. OurresultsclearlyshowthatqPCRishighlyefficient andreliabletodetecttheRNAcomponentsoftheSSc nucleolarautoantigens,whendataarecomparedwith thegoldstandardIP.Advantagesofthisnewmethod includeeasyapplicationwithouttechnicallydemanding RNAanalysisbyurea-PAGEandwithouttheuseof toxicchemicals(forexample,acrylamide,silverstaining) or32Pradioisotopes.TheresultsobtainedbyourIPqPCRassayareconsistentandreproducible,andare Figure2 Specificdetectionofanti-Th/ToandU3RNPantibodiesbyIP-qPCRassay A,B .SerawithantibodiestoTh/To(n=22),U3RNP(n =12),TMG(n=3),La(n=12),topoI(n=20),RNAPIII(n=18),ACA(n=15),PM-Scl(n=5),andnormalhumansera(NHS,n=15)were testedbytheIP-qPCRmethod.Datapointsandmedianbararerepresentativeofatleasttwoindependentexperiments. P <0.05byKruskalWallisandDunn smultiplecomparisontestswaspresentamongallthegroups,withtheexceptionoftheanti-U3RNP/anti-TMGgroups( P =ns). Thecut-offwasdeterminedbyROCcurveandpositiverangeisshownasshadedareas.ACA,anti-centromereantibodies;IP-qPCR, immunoprecipitation-quantitativepolymerasechainreaction;NHS,normalhumanserum;RNAPIII,RNApolymeraseIII;ROCcurve,receiver operatingcharacteristiccurve;TMG,trimethylguanosine;topoI,topoisomeraseI. Table1SensitivityandspecificityofournewIP-qPCR method.AutoantibodyspecificityanAnti-Th/To byIP-qPCR Anti-U3RNP byIP-qPCR Scleroderma Th/To22100%4.5%(1/22) U3RNP 120% 100% TopoI 200% 0% RNAPIII 180% 0% ACA 150% 0% PM-Scl 50% 0% Others TMG 30% 100% La 120%42%(5/12) NHS 150% 0%Resultsshowthehighsensitivityandspecificityofthenewassay,forthe detectionofanti-Th/Toand-U3RNPantibodies,comparedwithsilverstaining asgoldstandard.aAntibodyspecificityvalidatedbyIP,urea-PAGE,andsilver staining.ACA,anti-centromereantibodies;IP,immunoprecipitation;NHS, normalhumanserum;RNAPIII,RNApolymeraseIII;TMG,trimethylguanosine; topoI,topoisomeraseI.Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R128 http://arthritis-research.com/content/14/3/R128 Page4of6

PAGE 5

expressedassemi-quantitativedata,whilethesilver staininggelonlydetectsthepresenceorabsenceofspecificbands.Thehighsensitivityofournewmethodis demonstratedbyrequiringcellextractfromonlyone millioncellsand1lofserumtoobtainasignalthatis atleast128-to512-foldmoresensitivethanstandard IP-silverstainingmethod.Anotherimportantadvantage, whenconsideringitsuseinalargelaboratorysetting,is thehighnumberofsamplesthatthenewassaycanhandleatatime.Infact,a96-wellplatecanbeusedfor qPCR,whileonly10to16samplescanbetestedina standardgelforRNAanalysisbyurea-PAGE. AminorlimitationoftheIP-qPCRassayisthelackof informationonotherRNAsimmunoprecipitatedbythe sameserumsample.Examplesareco-IPof7-2and8-2 RNAbyLaantigen[18,22],andU3RNAbyrareautoimmuneserathatrecognizetheTMGcapstructureof U1-U5RNA[17].Inpractice,itwillbereasonableto applythisqPCRmethodforanti-Th/ToandU3RNP antibodiesonlytoSScpatientswhoarenegativefor anti-topoI,RNAPIII,andACA,sincethecoexistenceof multipleSScautoantibodiesisuncommon[1].Also, nucleolarimmunofluorescencestaininginantinuclear antibody(ANA)screeningwillbehelpfultodecide whichpatientsshouldbetestedbythisIP-qPCR method,sincethemajorityofanti-Th/To-andU3RNPpositivepatientsshowclearnucleolarstaining[13,23],in contrasttoanti-RNAPI/IIIserawhichoftendonot showanucleolarpattern[7,9].Itshouldbenotedthat thespecificityoftheIP-qPCRassayisnearly100%in SScsamplesandthatnon-SScserathatareexpectedto be,orpotentiallyare,falsepositives(suchasanti-La, anti-TMG)wereincludedinthestudytoclarifythe potentiallimitationsoftheassay.TheSScserawetested werenotrandomlyselected,however,theproportionof eachspecificityisnotfarfromarandomlyselectedSSc cohort,anditshouldgiveusareasonableideaofthe specificityoftheassayingeneralSScpatients.Thus,the positiveresultsobtainedbythenewIP-qPCRassayin thepresenceofrareantibodiesinSSc,suchasanti-La andanti-TMG,donotrepresentaclinicallysignificant problem. Thenewmethodcanbereadilyappliedtodetect otherautoantibodies,suchasautoantibodiestoaminoacyl-tRNAsynthetasesandsi gnalrecognitionparticle (SRP)inpolymyositis/dermatomyositispatients,justby usingappropriatespecificprimersfortheRNAspecies ofinterest.WiththeIP-qPCRmethod,eachtRNAspecificitycanbereadilydefinedwithoutthecomplicated aminoacylationassay[24].Al thoughclinicalmanifestationsofmanyanti-synthetaseantibodiesappearsimilar andtheyareknownasthe anti-synthetasesyndrome ,it ispossiblethatfinespecificitystillhasclinicalsignificanceastherearesomesuggestionsofdifferentclinical manifestationsinpatientswithdifferentanti-synthetase antibodies[15,25,26].ConclusionsWehaveestablishedanewmethodtodetectautoantibodiestoTh/ToandU3RNPbasedonqPCRdetection oftheRNAcomponentsoftheautoantigens.Highspecificityandsensitivitycomparedwiththegoldstandard IPwereconfirmed.Thisnewassaywillbeusefulfor detectionofautoantibodiestoanyRNA-proteincomplexautoantigenandmayallowclinicalutilizationof severalautoantibodiesthathavebeenmainlydetected byIPandnotutilizedwidelyinthepast.Abbreviations ACA:anti-centromereantibodies;ANA:anti-nuclearantibodies;cDNA: complementaryDNA;Ct:cyclethreshold;dcSSc:diffusecutaneousSSc; ELISA:enzyme-linkedimmunosorbentassay;IP:immunoprecipitation;lcSSc: limitedcutaneousSSc;NHS:normalhumanserum;qPCR:quantitative polymerasechainreaction;RNAPIII:RNApolymeraseIII;RTreverse transcription;SD:standarddeviation;SRP:signalrecognitionparticle;SSc: scleroderma:systemicsclerosis;TMG:trimethylguanosine;topoI: topoisomeraseI;U3RNP:U3ribonucleoprotein;urea-PAGE:ureapolyacrylamidegelelectrophoresis. Acknowledgements WethankDr.W.H.Reeves,Dr.E.S.Sobel,Dr.M.R.BubbfromtheDivisionof RheumatologyandClinicalImmunology,DepartmentofMedicine,University ofFlorida(Gainesville,Fl,USA)andDr.F.Franceschini,Dr.I.Cavazzanaand Prof.A.TincanifromtheRheumatologyUnit,SpedaliCivili(Brescia,Italy)for providingsomeofthesamples.WealsothankMr.PaulR.DominguezGutierrezforhelpingwithprimerdesignandinitialtechnicalassistancewith RTandqPCR.SupportedinpartbyagrantfromtheLupusResearch InstituteandtheNationalInstitutesofHealthgrantAI47859.Publicationof thisarticlewasfundedinpartbytheUniversityofFloridaOpen-Access PublishingFund. Authordetails1DepartmentofOralBiology,UniversityofFlorida,P.O.Box100424,1395 CenterDrive,Gainesville,FL32610-0424,USA.2DivisionofRheumatology andClinicalImmunology,DepartmentofMedicine,andDepartmentof Pathology,Immunology,andLaboratoryMedicine,UniversityofFlorida,P.O. Box100221,1600SWArcherRd,Gainesville,FL32610-0221,USA. Authors contributions ACandMScarriedouttheimmunoassays.MSandEKLCdesignedthestudy. MSperformedthestatisticalanalysis.AC,MSandEKLCdraftedthe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Competinginterests Patentapplication: AutoantibodytoRNA-proteincomplexdetectedby quantitativePCR .U.S.PCTInternationalApplicationNo.PCT/US11/60789. FilingDate:November15,2011.Inventors:EdwardK.L.Chan,MinoruSatoh, andAngelaCeribelli. Received:24January2012Revised:5May2012 Accepted:29May2012Published:29May2012 References1.SteenVD: Autoantibodiesinsystemicsclerosis. SeminArthritisRheum 2005, 35 :35-42. 2.vonMuhlenCA,TanEM: Autoantibodiesinthediagnosisofsystemic rheumaticdiseases. SeminArthritisRheum 1995, 24 :323-358. 3.HamaguchiY: Autoantibodyprofilesinsystemicsclerosis:predictive valueforclinicalevaluationandprognosis. JDermatol 2010, 37 :42-53.Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R128 http://arthritis-research.com/content/14/3/R128 Page5of6

PAGE 6

4.KoenigM,DieudeM,SenecalJL: Predictivevalueofantinuclear autoantibodies:Thelessonsofthesystemicsclerosisautoantibodies. AutoimmunRev 2008, 7 :588-593. 5.GrasseggerA,Pohla-GuboG,FrauscherM,HintnerH: Autoantibodiesin systemicsclerosis(scleroderma):cluesforclinicalevaluation,prognosis andpathogenesis. WienMedWochenschr 2008, 158 :19-28. 6.KuwanaM,OkanoY,PandeyJP,SilverRM,FertigN,MedsgerTAJr: Enzyme-linkedimmunosorbentassayfordetectionofanti-RNA polymeraseIIIantibody:analyticalaccuracyandclinicalassociationsin systemicsclerosis. ArthritisRheum 2005, 52 :2425-2432. 7.SatohM,Vazquez-DelMercadoM,ChanEKL: Clinicalinterpretationof antinuclearantibodytestsinsystemicrheumaticdiseases. Mod Rheumatol 2009, 19 :219-228. 8.SatohT,IshikawaO,IhnH,EndoH,KawaguchiY,SasakiT,GotoD, TakahashiK,TakahashiH,MisakiY,MimoriT,MuroY,YazawaN,SatoS, TakeharaK,KuwanaM: Clinicalusefulnessofanti-RNApolymeraseIII antibodymeasurementbyenzyme-linkedimmunosorbentassay. Rheumatology(Oxford) 2009, 48 :1570-1574. 9.SatohM,ChanEK,SobelES,KimpelDL,YamasakiY,NarainS,MansoorR, ReevesWH: Clinicalimplicationofautoantibodiesinpatientswith systemicrheumaticdiseases. ExpertRevClinImmunol 2007, 3 :721-738. 10.ReveilleJD,SolomonDH: Evidence-basedguidelinesfortheuseof immunologictests:anticentromere,Scl-70,andnucleolarantibodies. ArthritisRheum 2003, 49 :399-412. 11.MitriGM,LucasM,FertigN,SteenVD,MedsgerTAJr: Acomparison betweenanti-Th/To-andanticentromereantibody-positivesystemic sclerosispatientswithlimitedcutaneousinvolvement. ArthritisRheum 2003, 48 :203-209. 12.HarveyGR,ButtsS,RandsAL,PatelY,McHughNJ: Clinicalandserological associationswithanti-RNApolymeraseantibodiesinsystemicsclerosis. ClinExpImmunol 1999, 117 :395-402. 13.CeribelliA,CavazzanaI,FranceschiniF,AiroP,TincaniA,CattaneoR, PauleyBA,ChanEK,SatohM: Anti-Th/Toarecommonantinucleolar autoantibodiesinItalianpatientswithscleroderma. JRheumatol 2010, 37 :2071-2075. 14.VillaltaD,MorozziG,TampoiaM,AlpiniC,BruscaI,SalgaroloV,PapischW, BizzaroN: Antibodiestofibrillarin,PM-SclandRNApolymeraseIII detectedbyELISAassaysinpatientswithsystemicsclerosis. ClinChim Acta 2010, 411 :710-713. 15.YamasakiY,YamadaH,NozakiT,AkaogiJ,NicholsC,LyonsR,LoyAC, ChanEK,ReevesWH,SatohM: Unusuallyhighfrequencyof autoantibodiestoPL-7associatedwithmildermusclediseasein Japanesepatientswithpolymyositis/dermatomyositis. ArthritisRheum 2006, 54 :2004-2009. 16.SatohM,AkizukiM,YamagataH,NakayamaS,HommaM: Restricted heterogeneityandchangingspectrotypesinautoantibodiestoLa/SS-B. Autoimmunity 1996, 24 :229-236. 17.OkanoY,MedsgerTAJr: Novelhumanautoantibodiesreactivewith5 terminaltrimethylguanosinecapstructuresofUsmallnuclearRNA. J Immunol 1992, 149 :1093-1098. 18.OkanoY,MedsgerTAJr: AutoantibodytoThribonucleoprotein(nucleolar 7-2RNAproteinparticle)inpatientswithsystemicsclerosis. Arthritis Rheum 1990, 33 :1822-1828. 19.KufelJ,AllmangC,ChanfreauG,PetfalskiE,LafontaineDL,TollerveyD: PrecursorstotheU3smallnucleolarRNAlacksmallnucleolarRNP proteinsbutarestabilizedbyLabinding. MolCellBiol 2000, 20 :5415-5424. 20.OkanoY,SteenVD,MedsgerTAJr: AutoantibodyreactivewithRNA polymeraseIIIinsystemicsclerosis. AnnInternMed 1993, 119 :1005-1013. 21.AggarwalR,LucasM,FertigN,OddisCV,MedsgerTAJr: Anti-U3RNP autoantibodiesinsystemicsclerosis. ArthritisRheum 2009, 60 :1112-1118. 22.HashimotoC,SteitzJA: Sequentialassociationofnucleolar7-2RNAwith twodifferentautoantigens. JBiolChem 1983, 258 :1379-1382. 23.KrzyszczakME,LiY,RossSJ,CeribelliA,ChanEK,BubbMR,SobelES, ReevesWH,SatohM: Genderandethnicitydifferencesintheprevalence ofscleroderma-relatedautoantibodies. ClinRheumatol 2011, 30 :1333-1339. 24.TargoffIN,ArnettFC,ReichlinM: Antibodytothreonyl-transferRNA synthetaseinmyositissera. ArthritisRheum 1988, 31 :515-524. 25.KalluriM,SahnSA,OddisCV,GharibSL,Christopher-StineL,DanoffSK, Casciola-RosenL,HongG,DellaripaPF,HighlandKB: ClinicalprofileofantiPL-12autoantibody.Cohortstudyandreviewoftheliterature. Chest 2009, 135 :1550-1556. 26.HirakataM,SuwaA,TakadaT,SatoS,NagaiS,GenthE,SongYW,MimoriT, TargoffIN: Clinicalandimmunogeneticfeaturesofpatientswith autoantibodiestoasparaginyl-transferRNAsynthetase. ArthritisRheum 2007, 56 :1295-1303.doi:10.1186/ar3858 Citethisarticleas: Ceribelli etal .: Anewimmunoprecipitation-realtime quantitativePCRassayforanti-Th/Toandanti-U3RNPantibody detectioninsystemicsclerosis. ArthritisResearch&Therapy 2012 14 :R128. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Ceribelli etal ArthritisResearch&Therapy 2012, 14 :R128 http://arthritis-research.com/content/14/3/R128 Page6of6


xml version 1.0 encoding utf-8 standalone no
mets ID sort-mets_mets OBJID sword-mets LABEL DSpace SWORD Item PROFILE METS SIP Profile xmlns http:www.loc.govMETS
xmlns:xlink http:www.w3.org1999xlink xmlns:xsi http:www.w3.org2001XMLSchema-instance
xsi:schemaLocation http:www.loc.govstandardsmetsmets.xsd
metsHdr CREATEDATE 2012-06-19T12:16:32
agent ROLE CUSTODIAN TYPE ORGANIZATION
name BioMed Central
dmdSec sword-mets-dmd-1 GROUPID sword-mets-dmd-1_group-1
mdWrap SWAP Metadata MDTYPE OTHER OTHERMDTYPE EPDCX MIMETYPE textxml
xmlData
epdcx:descriptionSet xmlns:epdcx http:purl.orgeprintepdcx2006-11-16 xmlns:MIOJAVI
http:purl.orgeprintepdcxxsd2006-11-16epdcx.xsd
epdcx:description epdcx:resourceId sword-mets-epdcx-1
epdcx:statement epdcx:propertyURI http:purl.orgdcelements1.1type epdcx:valueURI http:purl.orgeprintentityTypeScholarlyWork
http:purl.orgdcelements1.1title
epdcx:valueString A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis
http:purl.orgdctermsabstract
Abstract
Introduction
Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.
Methods
Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.
Results
By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.
Conclusions
Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.
http:purl.orgdcelements1.1creator
Ceribelli, Angela
Satoh, Minoru
Chan, Edward KL
http:purl.orgeprinttermsisExpressedAs epdcx:valueRef sword-mets-expr-1
http:purl.orgeprintentityTypeExpression
http:purl.orgdcelements1.1language epdcx:vesURI http:purl.orgdctermsRFC3066
en
http:purl.orgeprinttermsType
http:purl.orgeprinttypeJournalArticle
http:purl.orgdctermsavailable
epdcx:sesURI http:purl.orgdctermsW3CDTF 2012-05-29
http:purl.orgdcelements1.1publisher
BioMed Central Ltd
http:purl.orgeprinttermsstatus http:purl.orgeprinttermsStatus
http:purl.orgeprintstatusPeerReviewed
http:purl.orgeprinttermscopyrightHolder
Angela Ceribelli et al.; licensee BioMed Central Ltd.
http:purl.orgdctermslicense
http://creativecommons.org/licenses/by/2.0
http:purl.orgdctermsaccessRights http:purl.orgeprinttermsAccessRights
http:purl.orgeprintaccessRightsOpenAccess
http:purl.orgeprinttermsbibliographicCitation
Arthritis Research & Therapy. 2012 May 29;14(3):R128
http:purl.orgdcelements1.1identifier
http:purl.orgdctermsURI http://dx.doi.org/10.1186/ar3858
fileSec
fileGrp sword-mets-fgrp-1 USE CONTENT
file sword-mets-fgid-0 sword-mets-file-1
FLocat LOCTYPE URL xlink:href ar3858.xml
sword-mets-fgid-1 sword-mets-file-2 applicationpdf
ar3858.pdf
structMap sword-mets-struct-1 structure LOGICAL
div sword-mets-div-1 DMDID Object
sword-mets-div-2 File
fptr FILEID
sword-mets-div-3


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui ar3858
ji 1478-6354
fm
dochead Research article
bibl
title p A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis
aug
au id A1 snm Ceribellifnm Angelainsr iid I1 email dott.ceribelli@libero.it
A2 SatohMinoruI2 minoru.satoh@medicine.ufl.edu
A3 ca yes Chanmi KLEdwardechan@ufl.edu
insg
ins Department of Oral Biology, University of Florida, P.O. Box 100424, 1395 Center Drive, Gainesville, FL 32610-0424, USA
Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, P.O. Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610-0221, USA
source Arthritis Research & Therapy
issn 1478-6354
pubdate 2012
volume 14
issue 3
fpage R128
url http://arthritis-research.com/content/14/3/R128
xrefbib pubidlist pubid idtype doi 10.1186/ar3858pmpid 22643159
history rec date day 24month 1year 2012revrec 552012acc 2952012pub 2952012
cpyrt 2012collab Ceribelli et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.
Methods
Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.
Results
By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.
Conclusions
Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.
bdy
Introduction
Scleroderma (Systemic Sclerosis, SSc) is a systemic autoimmune disease characterized by fibrosis, vascular changes, and the production of autoantibodies. The most common antibodies associated with SSc are anti-centromere (ACA), -topoisomerase I (topo I) and -RNA polymerase III (RNAPIII) antibodies, approximately 20% each abbrgrp abbr bid B1 1B2 2B3 3B4 4B5 5. Anti-topo I and ACA have been used for about 30 years for diagnostic purposes, while anti-RNAPIII ELISA has been added to routine screening only recently B6 6B7 7B8 8. SSc patients can be classified into two major subsets: limited (lcSSc) and diffuse (dcSSc) cutaneous variants. The dcSSc is frequently associated with anti-topo I, -RNAPIII, or -U3RNP, while lcSSc is associated with ACA and anti-Th/To antibodies 1B9 9. These autoantibodies are fairly specific for SSc and can be detected even before diagnosis. They are associated with unique clinical features and are useful in predicting clinical manifestations of SSc 1B10 10B11 11B12 12.
Anti-Th/To and -U3RNP are anti-nucleolar antibodies that have been known for more than 25 years. Despite their clinical importance, these SSc autoantibodies have not been utilized clinically because of the unavailability of antibody testing 7B13 13. Urea-polyacrylamide gel electrophoresis (PAGE) analysis of the RNA components in immunoprecipitates, either by silver staining or by using sup 32P-labeling of cells, is the standard method, but it is performed only in a small number of research laboratories. No commercial widely-available validated immunoassay kit has been produced so far B14 14. The aim of our study is to establish a new method to detect anti-Th/To and -U3RNP antibodies based on quantitative PCR (qPCR) detection of the RNA components of the ribonucleoprotein autoantigens.
Materials and methods
Immunoprecipitation and quantitative PCR
Immunoprecipitation (IP) was performed using K562 cell lysate and associated RNA was extracted using phenol/chloroform/isoamyl alcohol (25:24:1) as described 13B15 15. RNA pellets were resuspended in 30 µl RNA-grade water. cDNA was obtained from each RNA sample (10 µl) by reverse transcription (RT) using RT Master Mix (High Capacity cDNA RT kit, Applied Biosystems Inc., ABI, Foster City, CA, USA). The thermal cycler for the RT setting was: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C. Quantitative PCR (qPCR) was performed using the TaqMan Fast Universal PCR Master Mix (ABI). For Th RNA (7-2 RNA, RMRP) detection, 'Hs03298751_s1' primer by ABI was used, while the primer for U3 RNA (SNORD3A, Integrated DNA Technologies, Coralville, IA, USA) has the following sequence: ul Probe 5'-/56-FAM/CCAAGCAAC/ZEN/GCCAGAAAGCCG/3IABkFQ/-3'; Primer 1 (FOR.) 5'-TGTAGAGCACCGAAAACCAC-3'; Primer 2 (REV.) 5'-TCCCTCTCACTCCCCAATAC-3'. qPCR was performed in duplicate using the StepOne cycler (ABI) for 40 cycles, and results were evaluated by cycle threshold (Ct) values.
In some experiments, La-depleted cell extract was also used to examine the effects of La depletion in a limited number of samples (n = 24). An extract from 25 × 106 K562 cells was absorbed with 0.5 ml of anti-La immunoglobulin G-cyanogen bromide (IgG-CNBr)-activated Sepharose 4B beads to deplete La B16 16.
Serum samples
The protocol of this study was approved by the Institutional Review Board (IRB). This study meets and is in compliance with all ethical standards in medicine and informed consent was obtained from all patients according to the Declaration of Helsinki. For the titration experiment, one each of anti-Th/To, -U3RNP and normal human serum (NHS) was tested, with serial eight-fold dilutions (from 1 to 1:4096) of cell lysate as substrate in the IP step, RNA extracted after IP, and its derived cDNA. To validate the new assay, 22 anti-Th/To, 12 anti-U3RNP-positive samples, 58 SSc sera negative for anti-Th/To and -U3RNP antibodies (20 anti-Topo I, 18 anti-RNAPIII, 15 ACA, 5 anti-PM-Scl), 12 anti-La/SSB, 3 anti-trimethylguanosine (TMG) B17 17, and 15 NHS, were analyzed. The specificity of these sera was confirmed by IP and urea-PAGE analysis as described 13. Our data are representative of two or more independent experiments. PCR was analyzed in duplicate and immunoprecipitation was also tested in duplicate in some experiments. The typical reproducibility was very high showing a difference within one Ct value. In the new assay described here, no housekeeping gene mRNA would be present constantly, which is different from the standard qPCR used to examine the levels of mRNA for the gene of interest in total RNA or total mRNA. Nevertheless, the level of 18S RNA was also examined as a potential internal control for nonspecific binding (data not shown), but the results showed variation in its levels so that it was not considered suitable as an internal control in our assay.
Statistical analysis
Data between groups were compared by Kruskal-Wallis and Dunn's multiple comparison tests using Prism 5.0 for Macintosh (GraphPad Software, Inc). The same program was applied to obtain the receiver operating characteristic (ROC) curves, comparing the anti-Th/To or -U3RNP positive group versus the negative group to determine the cut-off. Statistical significance was accepted at it P <0.05.
Results
Autoantibody detection using qPCR for the RNA component of the ribonucleoprotein autoantigen complex
The sensitivity and linearity of the assay was evaluated for the detection of the Th and U3 RNA components, using serial 1:8 dilutions (1-1:4096) of cell lysate, RNA purified after IP (both started from extracts of 107 cells per sample, but data shown are based on the use of 10 out of 30 µl of RNA, equivalent to 3.3 × 106 cells) and cDNA obtained after RT (the equivalent of cDNA made from RNA derived from 3.3 × 106 cells) (Figure figr fid F1 1). Ct values for the titration using serially diluted cell lysate were 8.3 (extract from 107 cells, equivalent to undiluted or '1' in Figure 1A) to 22.7 (extract from 2.4 × 103 cells, corresponding to 1:4096 in Figure 1A) for Th RNA and 13.4 to 28.9 for U3 RNA (not shown), using the prototype sera. Linear dose response curves were noted for cell lysate, RNA, and cDNA dilutions, and the difference between positive samples and NHS was clear even at 1:4096 dilution, which corresponded to a cell extract from 2.4 × 103 K562 cells (Figure 1A). Ct values changed by approximately three for every 1:8 dilution, reflecting the eight-fold difference and the high efficiency of the qRT-PCR as expected (Figure 1A). Similar titration of RNA and cDNA, shown in Figure 1B and 1D, demonstrated the same linear relationship in each step of the qRT-PCR. IP using the standard 8 µl of human serum versus 1 µl was also compared in the titration experiment, to examine the effects on the Ct values. The difference in Ct values was approximately three, further demonstrating the high sensitivity of the new method also with 1 µl of serum.
fig Figure 1caption High sensitivity and linear detection of anti-Th/To antibodies in the IP-qPCR assay demonstrated by titration analysestext
b High sensitivity and linear detection of anti-Th/To antibodies in the IP-qPCR assay demonstrated by titration analyses. One prototype anti-Th/To sample and NHS were analyzed, and Ct values plotted against serial dilution of cell lysate used as substrate for IP (A), RNA extracted from IP (B), or the derived cDNA (D). Dilutions start with lysate from 107 K562 cells per sample. C. The sensitivity of our new IP-qPCR method is compared with RNA dilution detected by silver staining, which allows the identification of the two RNA bands (8-2 RNA, 7-2 RNA) only up to 1:16 dilution. Ct, cycle threshold; IP-qPCR, immunoprecipitation-quantitative polymerase chain reaction; NHS, normal human serum.
graphic file ar3858-1 hint_layout double
The sensitivity of detection of Th or U3 RNA by the qPCR method was compared with that of the standard urea-PAGE-silver staining method by testing the same amount of serially diluted Th RNA (Figure 1B versus C) or U3 RNA (not shown). When using serial dilutions of RNA, the qPCR assay clearly detected a signal at the highest dilution of 1:4096 (Figure 1B) whereas Th RNA was barely visible only up to 1:16 dilution by silver staining (Figure 1C). Thus, qPCR was at least 256-fold more sensitive than silver staining. For U3 RNA detection, qPCR was clearly positive at a dilution of 1:4096, similarly to 7-2 RNA, whereas U3 RNA was detected only up to a 1:8 dilution by silver staining (data not shown), indicating that the qPCR assay was at least 512-fold more sensitive. Based on these data, cell extracts from one million cells, which correspond to 1/10th the amount of cell lysate normally used for IP, were used for screening of sera for anti-Th/To, -U3RNP, and other antibodies.
Specificity of IP-qPCR assay applied to clinical samples
The mean Ct value was 19.81 (standard deviation (SD) 1.45) for anti-U3RNP and 15.27 (SD 0.73) for anti-Th/To samples (Figure F2 2A, B). These values were clearly separated from those of the control groups, with a difference of approximately eight Ct values for anti-U3RNP and approximately 13 Ct values for anti-Th/To (P <0.05) (Figure 2A, B). The Ct values of anti-TMG sera were similar to those of anti-U3RNP positive sera (Figure 2A). This was expected since anti-TMG antibodies recognize the cap structure on U1-U5RNA, including U3RNA 17. Despite maintaining statistical significance, the Ct difference between anti-Th and -La samples was smaller than the Ct difference between anti-Th and other negative controls (Figure 2C). This could be explained based on the interaction and co-immunoprecipitation of Th RNA by La antigens, as shown previously B18 18. For this reason, anti-La sera were also tested in the IP-qPCR assay using La-depleted K562 cell lysate. Ct values increased by about two and became comparable to other negative groups when La-depleted cell lysate was used in place of whole cell lysate (data not shown). This indicated that the low Ct values of anti-La sera in the anti-Th qPCR assay were due to co-IP of the Th RNA by the La complex, and not by coexisting anti-Th antibodies. When testing anti-U3RNP samples, 5/12 anti-La positive samples had low Ct values, which fell below the cut-off (Figure 2A). This might be explained by the observation that La could bind and stabilize precursors of the U3 small nucleolar RNA to prevent degradation B19 19. Thus, U3RNA could be weakly detected in the anti-La immunoprecipitate.
Figure 2Specific detection of anti-Th/To and U3RNP antibodies by IP-qPCR assay
Specific detection of anti-Th/To and U3RNP antibodies by IP-qPCR assay. A, B. Sera with antibodies to Th/To (n = 22), U3RNP (n = 12), TMG (n = 3), La (n = 12), topo I (n = 20), RNAPIII (n = 18), ACA (n = 15), PM-Scl (n = 5), and normal human sera (NHS, n= 15) were tested by the IP-qPCR method. Data points and median bar are representative of at least two independent experiments. P < 0.05 by Kruskal-Wallis and Dunn's multiple comparison tests was present among all the groups, with the exception of the anti-U3RNP/anti-TMG groups (P = ns). The cut-off was determined by ROC curve and positive range is shown as shaded areas. ACA, anti-centromere antibodies; IP-qPCR, immunoprecipitation-quantitative polymerase chain reaction; NHS, normal human serum; RNAPIII, RNA polymerase III; ROC curve, receiver operating characteristic curve; TMG, trimethylguanosine; topo I, topoisomerase I.
ar3858-2
Considering IP with silver staining as the gold standard for the detection of anti-Th and -U3RNP antibodies, the sensitivity by our new IP-qPCR method was 100% for both (Table tblr tid T1 1). Assay specificity for anti-Th and -U3RNP in SSc patients was 100% and 98.9%, respectively (Table 1). For anti-U3RNP detection, one anti-Th/To serum was repeatedly positive for an unknown reason.
tbl Table 1Sensitivity and specificity of our new IP-qPCR method.tblbdy cols 4
r
c left
Autoantibody specificity
a
center
n
Anti-Th/To
by IP-qPCR
Anti-U3RNP
by IP-qPCR
cspan
hr
Scleroderma
Th/To
22
100%
4.5% (1/22)
U3RNP
12
0%
100%
Topo I
20
0%
0%
RNAPIII
18
0%
0%
ACA
15
0%
0%
PM-Scl
5
0%
0%
Others
TMG
3
0%
100%
La
12
0%
42% (5/12)
NHS
15
0%
0%
tblfn
Results show the high sensitivity and specificity of the new assay, for the detection of anti-Th/To and -U3RNP antibodies, compared with silver staining as gold standard. aAntibody specificity validated by IP, urea-PAGE, and silver staining. ACA, anti-centromere antibodies; IP, immunoprecipitation; NHS, normal human serum; RNAPIII, RNA polymerase III; TMG, trimethylguanosine; topo I, topoisomerase I.
Discussion
The importance of the development of a convenient assay for anti-Th/To and -U3RNP antibodies cannot be underestimated because these two classic anti-nucleolar autoantibodies are associated with particular SSc variants and clinical features 1. In fact, anti-Th/To antibodies are frequently detected in lcSSc patients with pericarditis and mild lung involvement 13B20 20, while anti-U3RNP antibodies are associated with severe pulmonary, renal and muscular disease, mainly in African-American dcSSc patients 1B21 21. Thus, the aim of our study was to establish a new method that can be used widely and easily to detect anti-Th/To and -U3RNP antibodies to replace complicated RNA analysis by gel electrophoresis.
Our results clearly show that qPCR is highly efficient and reliable to detect the RNA components of the SSc nucleolar autoantigens, when data are compared with the gold standard IP. Advantages of this new method include easy application without technically demanding RNA analysis by urea-PAGE and without the use of toxic chemicals (for example, acrylamide, silver staining) or 32P radioisotopes. The results obtained by our IP-qPCR assay are consistent and reproducible, and are expressed as semi-quantitative data, while the silver staining gel only detects the presence or absence of specific bands. The high sensitivity of our new method is demonstrated by requiring cell extract from only one million cells and 1 µl of serum to obtain a signal that is at least 128- to 512-fold more sensitive than standard IP-silver staining method. Another important advantage, when considering its use in a large laboratory setting, is the high number of samples that the new assay can handle at a time. In fact, a 96-well plate can be used for qPCR, while only 10 to 16 samples can be tested in a standard gel for RNA analysis by urea-PAGE.
A minor limitation of the IP-qPCR assay is the lack of information on other RNAs immunoprecipitated by the same serum sample. Examples are co-IP of 7-2 and 8-2 RNA by La antigen 18B22 22, and U3 RNA by rare autoimmune sera that recognize the TMG cap structure of U1-U5RNA 17. In practice, it will be reasonable to apply this qPCR method for anti-Th/To and U3RNP antibodies only to SSc patients who are negative for anti-topo I, RNAPIII, and ACA, since the coexistence of multiple SSc autoantibodies is uncommon 1. Also, nucleolar immunofluorescence staining in antinuclear antibody (ANA) screening will be helpful to decide which patients should be tested by this IP-qPCR method, since the majority of anti-Th/To- and U3RNP-positive patients show clear nucleolar staining 13B23 23, in contrast to anti-RNAPI/III sera which often do not show a nucleolar pattern 79. It should be noted that the specificity of the IP-qPCR assay is nearly 100% in SSc samples and that non-SSc sera that are expected to be, or potentially are, false positives (such as anti-La, anti-TMG) were included in the study to clarify the potential limitations of the assay. The SSc sera we tested were not randomly selected, however, the proportion of each specificity is not far from a randomly selected SSc cohort, and it should give us a reasonable idea of the specificity of the assay in general SSc patients. Thus, the positive results obtained by the new IP-qPCR assay in the presence of rare antibodies in SSc, such as anti-La and anti-TMG, do not represent a clinically significant problem.
The new method can be readily applied to detect other autoantibodies, such as autoantibodies to aminoacyl-tRNA synthetases and signal recognition particle (SRP) in polymyositis/dermatomyositis patients, just by using appropriate specific primers for the RNA species of interest. With the IP-qPCR method, each tRNA specificity can be readily defined without the complicated aminoacylation assay B24 24. Although clinical manifestations of many anti-synthetase antibodies appear similar and they are known as the 'anti-synthetase syndrome', it is possible that fine specificity still has clinical significance as there are some suggestions of different clinical manifestations in patients with different anti-synthetase antibodies 15B25 25B26 26.
Conclusions
We have established a new method to detect autoantibodies to Th/To and U3RNP based on qPCR detection of the RNA components of the autoantigens. High specificity and sensitivity compared with the gold standard IP were confirmed. This new assay will be useful for detection of autoantibodies to any RNA-protein complex autoantigen and may allow clinical utilization of several autoantibodies that have been mainly detected by IP and not utilized widely in the past.
Abbreviations
ACA: anti-centromere antibodies; ANA: anti-nuclear antibodies; cDNA: complementary DNA; Ct: cycle threshold; dcSSc: diffuse cutaneous SSc; ELISA: enzyme-linked immunosorbent assay; IP: immunoprecipitation; lcSSc: limited cutaneous SSc; NHS: normal human serum; qPCR: quantitative polymerase chain reaction; RNAPIII: RNA polymerase III; RT reverse transcription; SD: standard deviation; SRP: signal recognition particle; SSc: scleroderma: systemic sclerosis; TMG: trimethylguanosine; topo I: topoisomerase I; U3 RNP: U3 ribonucleoprotein; urea-PAGE: urea-polyacrylamide gel electrophoresis.
Competing interests
Patent application: 'Autoantibody to RNA-protein complex detected by quantitative PCR'. U.S. PCT International Application No. PCT/US11/60789. Filing Date: November 15, 2011. Inventors: Edward K. L. Chan, Minoru Satoh, and Angela Ceribelli.
Authors' contributions
AC and MS carried out the immunoassays. MS and EKLC designed the study. MS performed the statistical analysis. AC, MS and EKLC drafted the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
We thank Dr. W.H. Reeves, Dr. E.S. Sobel, Dr. M.R. Bubb from the Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida (Gainesville, Fl, USA) and Dr. F. Franceschini, Dr. I. Cavazzana and Prof. A. Tincani from the Rheumatology Unit, Spedali Civili (Brescia, Italy) for providing some of the samples. We also thank Mr. Paul R. Dominguez-Gutierrez for helping with primer design and initial technical assistance with RT and qPCR. Supported in part by a grant from the Lupus Research Institute and the National Institutes of Health grant AI47859. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.
refgrp Autoantibodies in systemic sclerosisSteenVDSemin Arthritis Rheum20053535lpage 4210.1016/j.semarthrit.2005.03.005link fulltext 16084222Autoantibodies in the diagnosis of systemic rheumatic diseasesvon MuhlenCATanEMSemin Arthritis Rheum19952432335810.1016/S0049-0172(95)80004-27604300Autoantibody profiles in systemic sclerosis: predictive value for clinical evaluation and prognosisHamaguchiYJ Dermatol201037425320175839Predictive value of antinuclear autoantibodies: The lessons of the systemic sclerosis autoantibodiesKoenigMDieudeMSenecalJLAutoimmun Rev2008758859310.1016/j.autrev.2008.06.01018617021Autoantibodies in systemic sclerosis (scleroderma): clues for clinical evaluation, prognosis and pathogenesisGrasseggerAPohla-GuboGFrauscherMHintnerHWien Med Wochenschr2008158192810.1007/s10354-007-0451-518286246Enzyme-linked immunosorbent assay for detection of anti-RNA polymerase III antibody: analytical accuracy and clinical associations in systemic sclerosisKuwanaMOkanoYPandeyJPSilverRMFertigNMedsgerTAsuf JrArthritis Rheum2005522425243210.1002/art.2123216052583Clinical interpretation of antinuclear antibody tests in systemic rheumatic diseasesSatohMVazquez-Del MercadoMChanEKLMod Rheumatol20091921922810.1007/s10165-009-0155-3pmcid 287609519277826Clinical usefulness of anti-RNA polymerase III antibody measurement by enzyme-linked immunosorbent assaySatohTIshikawaOIhnHEndoHKawaguchiYSasakiTGotoDTakahashiKTakahashiHMisakiYMimoriTMuroYYazawaNSatoSTakeharaKKuwanaMRheumatology (Oxford)2009481570157410.1093/rheumatology/kep290Clinical implication of autoantibodies in patients with systemic rheumatic diseasesSatohMChanEKSobelESKimpelDLYamasakiYNarainSMansoorRReevesWHExpert Rev Clin Immunol2007372173810.1586/1744666X.3.5.72120477023Evidence-based guidelines for the use of immunologic tests: anticentromere, Scl-70, and nucleolar antibodiesReveilleJDSolomonDHArthritis Rheum20034939941210.1002/art.1111312794797A comparison between anti-Th/To- and anticentromere antibody-positive systemic sclerosis patients with limited cutaneous involvementMitriGMLucasMFertigNSteenVDMedsgerTAJrArthritis Rheum20034820320910.1002/art.1076012528120Clinical and serological associations with anti-RNA polymerase antibodies in systemic sclerosisHarveyGRButtsSRandsALPatelYMcHughNJClin Exp Immunol199911739540210.1046/j.1365-2249.1999.00964.x190533810444276Anti-Th/To are common antinucleolar autoantibodies in Italian patients with sclerodermaCeribelliACavazzanaIFranceschiniFAiroPTincaniACattaneoRPauleyBAChanEKSatohMJ Rheumatol2010372071207510.3899/jrheum.10031620682663Antibodies to fibrillarin, PM-Scl and RNA polymerase III detected by ELISA assays in patients with systemic sclerosisVillaltaDMorozziGTampoiaMAlpiniCBruscaISalgaroloVPapischWBizzaroNClin Chim Acta201041171071310.1016/j.cca.2010.01.03720138166Unusually high frequency of autoantibodies to PL-7 associated with milder muscle disease in Japanese patients with polymyositis/dermatomyositisYamasakiYYamadaHNozakiTAkaogiJNicholsCLyonsRLoyACChanEKReevesWHSatohMArthritis Rheum2006542004200910.1002/art.2188316732549Restricted heterogeneity and changing spectrotypes in autoantibodies to La/SS-BSatohMAkizukiMYamagataHNakayamaSHommaMAutoimmunity19962422923610.3109/089169396089947159147581Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNAOkanoYMedsgerTAJrJ Immunol1992149109310981634764Autoantibody to Th ribonucleoprotein (nucleolar 7-2 RNA protein particle) in patients with systemic sclerosisOkanoYMedsgerTAJrArthritis Rheum1990331822182810.1002/art.17803312101701994Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La bindingKufelJAllmangCChanfreauGPetfalskiELafontaineDLTollerveyDMol Cell Biol2000205415542410.1128/MCB.20.15.5415-5424.20008599310891482Autoantibody reactive with RNA polymerase III in systemic sclerosisOkanoYSteenVDMedsgerTAJrAnn Intern Med1993119100510138214977Anti-U3 RNP autoantibodies in systemic sclerosisAggarwalRLucasMFertigNOddisCVMedsgerTAJrArthritis Rheum2009601112111810.1002/art.2440919333934Sequential association of nucleolar 7-2 RNA with two different autoantigensHashimotoCSteitzJAJ Biol Chem1983258137913826185483Gender and ethnicity differences in the prevalence of scleroderma-related autoantibodiesKrzyszczakMELiYRossSJCeribelliAChanEKBubbMRSobelESReevesWHSatohMClin Rheumatol2011301333133910.1007/s10067-011-1751-021523365Antibody to threonyl-transfer RNA synthetase in myositis seraTargoffINArnettFCReichlinMArthritis Rheum19883151552410.1002/art.17803104083128989Clinical profile of anti-PL-12 autoantibody. Cohort study and review of the literatureKalluriMSahnSAOddisCVGharibSLChristopher-StineLDanoffSKCasciola-RosenLHongGDellaripaPFHighlandKBChest20091351550155610.1378/chest.08-223319225060Clinical and immunogenetic features of patients with autoantibodies to asparaginyl-transfer RNA synthetaseHirakataMSuwaATakadaTSatoSNagaiSGenthESongYWMimoriTTargoffINArthritis Rheum2007561295130310.1002/art.2250617393393


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui ar3858
ji 1478-6354
fm
dochead Research article
bibl
title p A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis
aug
au id A1 snm Ceribellifnm Angelainsr iid I1 email dott.ceribelli@libero.it
A2 SatohMinoruI2 minoru.satoh@medicine.ufl.edu
A3 ca yes Chanmi KLEdwardechan@ufl.edu
insg
ins Department of Oral Biology, University of Florida, P.O. Box 100424, 1395 Center Drive, Gainesville, FL 32610-0424, USA
Division of Rheumatology and Clinical Immunology, Department of Medicine, and Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, P.O. Box 100221, 1600 SW Archer Rd, Gainesville, FL 32610-0221, USA
source Arthritis Research & Therapy
issn 1478-6354
pubdate 2012
volume 14
issue 3
fpage R128
url http://arthritis-research.com/content/14/3/R128
xrefbib pubidlist pubid idtype doi 10.1186/ar3858pmpid 22643159
history rec date day 24month 1year 2012revrec 552012acc 2952012pub 2952012
cpyrt 2012collab Ceribelli et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.
Methods
Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.
Results
By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.
Conclusions
Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.
bdy
Introduction
Scleroderma (Systemic Sclerosis, SSc) is a systemic autoimmune disease characterized by fibrosis, vascular changes, and the production of autoantibodies. The most common antibodies associated with SSc are anti-centromere (ACA), -topoisomerase I (topo I) and -RNA polymerase III (RNAPIII) antibodies, approximately 20% each abbrgrp abbr bid B1 1B2 2B3 3B4 4B5 5. Anti-topo I and ACA have been used for about 30 years for diagnostic purposes, while anti-RNAPIII ELISA has been added to routine screening only recently B6 6B7 7B8 8. SSc patients can be classified into two major subsets: limited (lcSSc) and diffuse (dcSSc) cutaneous variants. The dcSSc is frequently associated with anti-topo I, -RNAPIII, or -U3RNP, while lcSSc is associated with ACA and anti-Th/To antibodies 1B9 9. These autoantibodies are fairly specific for SSc and can be detected even before diagnosis. They are associated with unique clinical features and are useful in predicting clinical manifestations of SSc 1B10 10B11 11B12 12.
Anti-Th/To and -U3RNP are anti-nucleolar antibodies that have been known for more than 25 years. Despite their clinical importance, these SSc autoantibodies have not been utilized clinically because of the unavailability of antibody testing 7B13 13. Urea-polyacrylamide gel electrophoresis (PAGE) analysis of the RNA components in immunoprecipitates, either by silver staining or by using sup 32P-labeling of cells, is the standard method, but it is performed only in a small number of research laboratories. No commercial widely-available validated immunoassay kit has been produced so far B14 14. The aim of our study is to establish a new method to detect anti-Th/To and -U3RNP antibodies based on quantitative PCR (qPCR) detection of the RNA components of the ribonucleoprotein autoantigens.
Materials and methods
Immunoprecipitation and quantitative PCR
Immunoprecipitation (IP) was performed using K562 cell lysate and associated RNA was extracted using phenol/chloroform/isoamyl alcohol (25:24:1) as described 13B15 15. RNA pellets were resuspended in 30 µl RNA-grade water. cDNA was obtained from each RNA sample (10 µl) by reverse transcription (RT) using RT Master Mix (High Capacity cDNA RT kit, Applied Biosystems Inc., ABI, Foster City, CA, USA). The thermal cycler for the RT setting was: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C. Quantitative PCR (qPCR) was performed using the TaqMan Fast Universal PCR Master Mix (ABI). For Th RNA (7-2 RNA, RMRP) detection, 'Hs03298751_s1' primer by ABI was used, while the primer for U3 RNA (SNORD3A, Integrated DNA Technologies, Coralville, IA, USA) has the following sequence: ul Probe 5'-/56-FAM/CCAAGCAAC/ZEN/GCCAGAAAGCCG/3IABkFQ/-3'; Primer 1 (FOR.) 5'-TGTAGAGCACCGAAAACCAC-3'; Primer 2 (REV.) 5'-TCCCTCTCACTCCCCAATAC-3'. qPCR was performed in duplicate using the StepOne cycler (ABI) for 40 cycles, and results were evaluated by cycle threshold (Ct) values.
In some experiments, La-depleted cell extract was also used to examine the effects of La depletion in a limited number of samples (n = 24). An extract from 25 × 106 K562 cells was absorbed with 0.5 ml of anti-La immunoglobulin G-cyanogen bromide (IgG-CNBr)-activated Sepharose 4B beads to deplete La B16 16.
Serum samples
The protocol of this study was approved by the Institutional Review Board (IRB). This study meets and is in compliance with all ethical standards in medicine and informed consent was obtained from all patients according to the Declaration of Helsinki. For the titration experiment, one each of anti-Th/To, -U3RNP and normal human serum (NHS) was tested, with serial eight-fold dilutions (from 1 to 1:4096) of cell lysate as substrate in the IP step, RNA extracted after IP, and its derived cDNA. To validate the new assay, 22 anti-Th/To, 12 anti-U3RNP-positive samples, 58 SSc sera negative for anti-Th/To and -U3RNP antibodies (20 anti-Topo I, 18 anti-RNAPIII, 15 ACA, 5 anti-PM-Scl), 12 anti-La/SSB, 3 anti-trimethylguanosine (TMG) B17 17, and 15 NHS, were analyzed. The specificity of these sera was confirmed by IP and urea-PAGE analysis as described 13. Our data are representative of two or more independent experiments. PCR was analyzed in duplicate and immunoprecipitation was also tested in duplicate in some experiments. The typical reproducibility was very high showing a difference within one Ct value. In the new assay described here, no housekeeping gene mRNA would be present constantly, which is different from the standard qPCR used to examine the levels of mRNA for the gene of interest in total RNA or total mRNA. Nevertheless, the level of 18S RNA was also examined as a potential internal control for nonspecific binding (data not shown), but the results showed variation in its levels so that it was not considered suitable as an internal control in our assay.
Statistical analysis
Data between groups were compared by Kruskal-Wallis and Dunn's multiple comparison tests using Prism 5.0 for Macintosh (GraphPad Software, Inc). The same program was applied to obtain the receiver operating characteristic (ROC) curves, comparing the anti-Th/To or -U3RNP positive group versus the negative group to determine the cut-off. Statistical significance was accepted at it P <0.05.
Results
Autoantibody detection using qPCR for the RNA component of the ribonucleoprotein autoantigen complex
The sensitivity and linearity of the assay was evaluated for the detection of the Th and U3 RNA components, using serial 1:8 dilutions (1-1:4096) of cell lysate, RNA purified after IP (both started from extracts of 107 cells per sample, but data shown are based on the use of 10 out of 30 µl of RNA, equivalent to 3.3 × 106 cells) and cDNA obtained after RT (the equivalent of cDNA made from RNA derived from 3.3 × 106 cells) (Figure figr fid F1 1). Ct values for the titration using serially diluted cell lysate were 8.3 (extract from 107 cells, equivalent to undiluted or '1' in Figure 1A) to 22.7 (extract from 2.4 × 103 cells, corresponding to 1:4096 in Figure 1A) for Th RNA and 13.4 to 28.9 for U3 RNA (not shown), using the prototype sera. Linear dose response curves were noted for cell lysate, RNA, and cDNA dilutions, and the difference between positive samples and NHS was clear even at 1:4096 dilution, which corresponded to a cell extract from 2.4 × 103 K562 cells (Figure 1A). Ct values changed by approximately three for every 1:8 dilution, reflecting the eight-fold difference and the high efficiency of the qRT-PCR as expected (Figure 1A). Similar titration of RNA and cDNA, shown in Figure 1B and 1D, demonstrated the same linear relationship in each step of the qRT-PCR. IP using the standard 8 µl of human serum versus 1 µl was also compared in the titration experiment, to examine the effects on the Ct values. The difference in Ct values was approximately three, further demonstrating the high sensitivity of the new method also with 1 µl of serum.
fig Figure 1caption High sensitivity and linear detection of anti-Th/To antibodies in the IP-qPCR assay demonstrated by titration analysestext
b High sensitivity and linear detection of anti-Th/To antibodies in the IP-qPCR assay demonstrated by titration analyses. One prototype anti-Th/To sample and NHS were analyzed, and Ct values plotted against serial dilution of cell lysate used as substrate for IP (A), RNA extracted from IP (B), or the derived cDNA (D). Dilutions start with lysate from 107 K562 cells per sample. C. The sensitivity of our new IP-qPCR method is compared with RNA dilution detected by silver staining, which allows the identification of the two RNA bands (8-2 RNA, 7-2 RNA) only up to 1:16 dilution. Ct, cycle threshold; IP-qPCR, immunoprecipitation-quantitative polymerase chain reaction; NHS, normal human serum.
graphic file ar3858-1 hint_layout double
The sensitivity of detection of Th or U3 RNA by the qPCR method was compared with that of the standard urea-PAGE-silver staining method by testing the same amount of serially diluted Th RNA (Figure 1B versus C) or U3 RNA (not shown). When using serial dilutions of RNA, the qPCR assay clearly detected a signal at the highest dilution of 1:4096 (Figure 1B) whereas Th RNA was barely visible only up to 1:16 dilution by silver staining (Figure 1C). Thus, qPCR was at least 256-fold more sensitive than silver staining. For U3 RNA detection, qPCR was clearly positive at a dilution of 1:4096, similarly to 7-2 RNA, whereas U3 RNA was detected only up to a 1:8 dilution by silver staining (data not shown), indicating that the qPCR assay was at least 512-fold more sensitive. Based on these data, cell extracts from one million cells, which correspond to 1/10th the amount of cell lysate normally used for IP, were used for screening of sera for anti-Th/To, -U3RNP, and other antibodies.
Specificity of IP-qPCR assay applied to clinical samples
The mean Ct value was 19.81 (standard deviation (SD) 1.45) for anti-U3RNP and 15.27 (SD 0.73) for anti-Th/To samples (Figure F2 2A, B). These values were clearly separated from those of the control groups, with a difference of approximately eight Ct values for anti-U3RNP and approximately 13 Ct values for anti-Th/To (P <0.05) (Figure 2A, B). The Ct values of anti-TMG sera were similar to those of anti-U3RNP positive sera (Figure 2A). This was expected since anti-TMG antibodies recognize the cap structure on U1-U5RNA, including U3RNA 17. Despite maintaining statistical significance, the Ct difference between anti-Th and -La samples was smaller than the Ct difference between anti-Th and other negative controls (Figure 2C). This could be explained based on the interaction and co-immunoprecipitation of Th RNA by La antigens, as shown previously B18 18. For this reason, anti-La sera were also tested in the IP-qPCR assay using La-depleted K562 cell lysate. Ct values increased by about two and became comparable to other negative groups when La-depleted cell lysate was used in place of whole cell lysate (data not shown). This indicated that the low Ct values of anti-La sera in the anti-Th qPCR assay were due to co-IP of the Th RNA by the La complex, and not by coexisting anti-Th antibodies. When testing anti-U3RNP samples, 5/12 anti-La positive samples had low Ct values, which fell below the cut-off (Figure 2A). This might be explained by the observation that La could bind and stabilize precursors of the U3 small nucleolar RNA to prevent degradation B19 19. Thus, U3RNA could be weakly detected in the anti-La immunoprecipitate.
Figure 2Specific detection of anti-Th/To and U3RNP antibodies by IP-qPCR assay
Specific detection of anti-Th/To and U3RNP antibodies by IP-qPCR assay. A, B. Sera with antibodies to Th/To (n = 22), U3RNP (n = 12), TMG (n = 3), La (n = 12), topo I (n = 20), RNAPIII (n = 18), ACA (n = 15), PM-Scl (n = 5), and normal human sera (NHS, n= 15) were tested by the IP-qPCR method. Data points and median bar are representative of at least two independent experiments. P < 0.05 by Kruskal-Wallis and Dunn's multiple comparison tests was present among all the groups, with the exception of the anti-U3RNP/anti-TMG groups (P = ns). The cut-off was determined by ROC curve and positive range is shown as shaded areas. ACA, anti-centromere antibodies; IP-qPCR, immunoprecipitation-quantitative polymerase chain reaction; NHS, normal human serum; RNAPIII, RNA polymerase III; ROC curve, receiver operating characteristic curve; TMG, trimethylguanosine; topo I, topoisomerase I.
ar3858-2
Considering IP with silver staining as the gold standard for the detection of anti-Th and -U3RNP antibodies, the sensitivity by our new IP-qPCR method was 100% for both (Table tblr tid T1 1). Assay specificity for anti-Th and -U3RNP in SSc patients was 100% and 98.9%, respectively (Table 1). For anti-U3RNP detection, one anti-Th/To serum was repeatedly positive for an unknown reason.
tbl Table 1Sensitivity and specificity of our new IP-qPCR method.tblbdy cols 4
r
c left
Autoantibody specificity
a
center
n
Anti-Th/To
by IP-qPCR
Anti-U3RNP
by IP-qPCR
cspan
hr
Scleroderma
Th/To
22
100%
4.5% (1/22)
U3RNP
12
0%
100%
Topo I
20
0%
0%
RNAPIII
18
0%
0%
ACA
15
0%
0%
PM-Scl
5
0%
0%
Others
TMG
3
0%
100%
La
12
0%
42% (5/12)
NHS
15
0%
0%
tblfn
Results show the high sensitivity and specificity of the new assay, for the detection of anti-Th/To and -U3RNP antibodies, compared with silver staining as gold standard. aAntibody specificity validated by IP, urea-PAGE, and silver staining. ACA, anti-centromere antibodies; IP, immunoprecipitation; NHS, normal human serum; RNAPIII, RNA polymerase III; TMG, trimethylguanosine; topo I, topoisomerase I.
Discussion
The importance of the development of a convenient assay for anti-Th/To and -U3RNP antibodies cannot be underestimated because these two classic anti-nucleolar autoantibodies are associated with particular SSc variants and clinical features 1. In fact, anti-Th/To antibodies are frequently detected in lcSSc patients with pericarditis and mild lung involvement 13B20 20, while anti-U3RNP antibodies are associated with severe pulmonary, renal and muscular disease, mainly in African-American dcSSc patients 1B21 21. Thus, the aim of our study was to establish a new method that can be used widely and easily to detect anti-Th/To and -U3RNP antibodies to replace complicated RNA analysis by gel electrophoresis.
Our results clearly show that qPCR is highly efficient and reliable to detect the RNA components of the SSc nucleolar autoantigens, when data are compared with the gold standard IP. Advantages of this new method include easy application without technically demanding RNA analysis by urea-PAGE and without the use of toxic chemicals (for example, acrylamide, silver staining) or 32P radioisotopes. The results obtained by our IP-qPCR assay are consistent and reproducible, and are expressed as semi-quantitative data, while the silver staining gel only detects the presence or absence of specific bands. The high sensitivity of our new method is demonstrated by requiring cell extract from only one million cells and 1 µl of serum to obtain a signal that is at least 128- to 512-fold more sensitive than standard IP-silver staining method. Another important advantage, when considering its use in a large laboratory setting, is the high number of samples that the new assay can handle at a time. In fact, a 96-well plate can be used for qPCR, while only 10 to 16 samples can be tested in a standard gel for RNA analysis by urea-PAGE.
A minor limitation of the IP-qPCR assay is the lack of information on other RNAs immunoprecipitated by the same serum sample. Examples are co-IP of 7-2 and 8-2 RNA by La antigen 18B22 22, and U3 RNA by rare autoimmune sera that recognize the TMG cap structure of U1-U5RNA 17. In practice, it will be reasonable to apply this qPCR method for anti-Th/To and U3RNP antibodies only to SSc patients who are negative for anti-topo I, RNAPIII, and ACA, since the coexistence of multiple SSc autoantibodies is uncommon 1. Also, nucleolar immunofluorescence staining in antinuclear antibody (ANA) screening will be helpful to decide which patients should be tested by this IP-qPCR method, since the majority of anti-Th/To- and U3RNP-positive patients show clear nucleolar staining 13B23 23, in contrast to anti-RNAPI/III sera which often do not show a nucleolar pattern 79. It should be noted that the specificity of the IP-qPCR assay is nearly 100% in SSc samples and that non-SSc sera that are expected to be, or potentially are, false positives (such as anti-La, anti-TMG) were included in the study to clarify the potential limitations of the assay. The SSc sera we tested were not randomly selected, however, the proportion of each specificity is not far from a randomly selected SSc cohort, and it should give us a reasonable idea of the specificity of the assay in general SSc patients. Thus, the positive results obtained by the new IP-qPCR assay in the presence of rare antibodies in SSc, such as anti-La and anti-TMG, do not represent a clinically significant problem.
The new method can be readily applied to detect other autoantibodies, such as autoantibodies to aminoacyl-tRNA synthetases and signal recognition particle (SRP) in polymyositis/dermatomyositis patients, just by using appropriate specific primers for the RNA species of interest. With the IP-qPCR method, each tRNA specificity can be readily defined without the complicated aminoacylation assay B24 24. Although clinical manifestations of many anti-synthetase antibodies appear similar and they are known as the 'anti-synthetase syndrome', it is possible that fine specificity still has clinical significance as there are some suggestions of different clinical manifestations in patients with different anti-synthetase antibodies 15B25 25B26 26.
Conclusions
We have established a new method to detect autoantibodies to Th/To and U3RNP based on qPCR detection of the RNA components of the autoantigens. High specificity and sensitivity compared with the gold standard IP were confirmed. This new assay will be useful for detection of autoantibodies to any RNA-protein complex autoantigen and may allow clinical utilization of several autoantibodies that have been mainly detected by IP and not utilized widely in the past.
Abbreviations
ACA: anti-centromere antibodies; ANA: anti-nuclear antibodies; cDNA: complementary DNA; Ct: cycle threshold; dcSSc: diffuse cutaneous SSc; ELISA: enzyme-linked immunosorbent assay; IP: immunoprecipitation; lcSSc: limited cutaneous SSc; NHS: normal human serum; qPCR: quantitative polymerase chain reaction; RNAPIII: RNA polymerase III; RT reverse transcription; SD: standard deviation; SRP: signal recognition particle; SSc: scleroderma: systemic sclerosis; TMG: trimethylguanosine; topo I: topoisomerase I; U3 RNP: U3 ribonucleoprotein; urea-PAGE: urea-polyacrylamide gel electrophoresis.
Competing interests
Patent application: 'Autoantibody to RNA-protein complex detected by quantitative PCR'. U.S. PCT International Application No. PCT/US11/60789. Filing Date: November 15, 2011. Inventors: Edward K. L. Chan, Minoru Satoh, and Angela Ceribelli.
Authors' contributions
AC and MS carried out the immunoassays. MS and EKLC designed the study. MS performed the statistical analysis. AC, MS and EKLC drafted the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgements
We thank Dr. W.H. Reeves, Dr. E.S. Sobel, Dr. M.R. Bubb from the Division of Rheumatology and Clinical Immunology, Department of Medicine, University of Florida (Gainesville, Fl, USA) and Dr. F. Franceschini, Dr. I. Cavazzana and Prof. A. Tincani from the Rheumatology Unit, Spedali Civili (Brescia, Italy) for providing some of the samples. We also thank Mr. Paul R. Dominguez-Gutierrez for helping with primer design and initial technical assistance with RT and qPCR. Supported in part by a grant from the Lupus Research Institute and the National Institutes of Health grant AI47859. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.
refgrp Autoantibodies in systemic sclerosisSteenVDSemin Arthritis Rheum20053535lpage 4210.1016/j.semarthrit.2005.03.005link fulltext 16084222Autoantibodies in the diagnosis of systemic rheumatic diseasesvon MuhlenCATanEMSemin Arthritis Rheum19952432335810.1016/S0049-0172(95)80004-27604300Autoantibody profiles in systemic sclerosis: predictive value for clinical evaluation and prognosisHamaguchiYJ Dermatol201037425320175839Predictive value of antinuclear autoantibodies: The lessons of the systemic sclerosis autoantibodiesKoenigMDieudeMSenecalJLAutoimmun Rev2008758859310.1016/j.autrev.2008.06.01018617021Autoantibodies in systemic sclerosis (scleroderma): clues for clinical evaluation, prognosis and pathogenesisGrasseggerAPohla-GuboGFrauscherMHintnerHWien Med Wochenschr2008158192810.1007/s10354-007-0451-518286246Enzyme-linked immunosorbent assay for detection of anti-RNA polymerase III antibody: analytical accuracy and clinical associations in systemic sclerosisKuwanaMOkanoYPandeyJPSilverRMFertigNMedsgerTAsuf JrArthritis Rheum2005522425243210.1002/art.2123216052583Clinical interpretation of antinuclear antibody tests in systemic rheumatic diseasesSatohMVazquez-Del MercadoMChanEKLMod Rheumatol20091921922810.1007/s10165-009-0155-3pmcid 287609519277826Clinical usefulness of anti-RNA polymerase III antibody measurement by enzyme-linked immunosorbent assaySatohTIshikawaOIhnHEndoHKawaguchiYSasakiTGotoDTakahashiKTakahashiHMisakiYMimoriTMuroYYazawaNSatoSTakeharaKKuwanaMRheumatology (Oxford)2009481570157410.1093/rheumatology/kep290Clinical implication of autoantibodies in patients with systemic rheumatic diseasesSatohMChanEKSobelESKimpelDLYamasakiYNarainSMansoorRReevesWHExpert Rev Clin Immunol2007372173810.1586/1744666X.3.5.72120477023Evidence-based guidelines for the use of immunologic tests: anticentromere, Scl-70, and nucleolar antibodiesReveilleJDSolomonDHArthritis Rheum20034939941210.1002/art.1111312794797A comparison between anti-Th/To- and anticentromere antibody-positive systemic sclerosis patients with limited cutaneous involvementMitriGMLucasMFertigNSteenVDMedsgerTAJrArthritis Rheum20034820320910.1002/art.1076012528120Clinical and serological associations with anti-RNA polymerase antibodies in systemic sclerosisHarveyGRButtsSRandsALPatelYMcHughNJClin Exp Immunol199911739540210.1046/j.1365-2249.1999.00964.x190533810444276Anti-Th/To are common antinucleolar autoantibodies in Italian patients with sclerodermaCeribelliACavazzanaIFranceschiniFAiroPTincaniACattaneoRPauleyBAChanEKSatohMJ Rheumatol2010372071207510.3899/jrheum.10031620682663Antibodies to fibrillarin, PM-Scl and RNA polymerase III detected by ELISA assays in patients with systemic sclerosisVillaltaDMorozziGTampoiaMAlpiniCBruscaISalgaroloVPapischWBizzaroNClin Chim Acta201041171071310.1016/j.cca.2010.01.03720138166Unusually high frequency of autoantibodies to PL-7 associated with milder muscle disease in Japanese patients with polymyositis/dermatomyositisYamasakiYYamadaHNozakiTAkaogiJNicholsCLyonsRLoyACChanEKReevesWHSatohMArthritis Rheum2006542004200910.1002/art.2188316732549Restricted heterogeneity and changing spectrotypes in autoantibodies to La/SS-BSatohMAkizukiMYamagataHNakayamaSHommaMAutoimmunity19962422923610.3109/089169396089947159147581Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNAOkanoYMedsgerTAJrJ Immunol1992149109310981634764Autoantibody to Th ribonucleoprotein (nucleolar 7-2 RNA protein particle) in patients with systemic sclerosisOkanoYMedsgerTAJrArthritis Rheum1990331822182810.1002/art.17803312101701994Precursors to the U3 small nucleolar RNA lack small nucleolar RNP proteins but are stabilized by La bindingKufelJAllmangCChanfreauGPetfalskiELafontaineDLTollerveyDMol Cell Biol2000205415542410.1128/MCB.20.15.5415-5424.20008599310891482Autoantibody reactive with RNA polymerase III in systemic sclerosisOkanoYSteenVDMedsgerTAJrAnn Intern Med1993119100510138214977Anti-U3 RNP autoantibodies in systemic sclerosisAggarwalRLucasMFertigNOddisCVMedsgerTAJrArthritis Rheum2009601112111810.1002/art.2440919333934Sequential association of nucleolar 7-2 RNA with two different autoantigensHashimotoCSteitzJAJ Biol Chem1983258137913826185483Gender and ethnicity differences in the prevalence of scleroderma-related autoantibodiesKrzyszczakMELiYRossSJCeribelliAChanEKBubbMRSobelESReevesWHSatohMClin Rheumatol2011301333133910.1007/s10067-011-1751-021523365Antibody to threonyl-transfer RNA synthetase in myositis seraTargoffINArnettFCReichlinMArthritis Rheum19883151552410.1002/art.17803104083128989Clinical profile of anti-PL-12 autoantibody. Cohort study and review of the literatureKalluriMSahnSAOddisCVGharibSLChristopher-StineLDanoffSKCasciola-RosenLHongGDellaripaPFHighlandKBChest20091351550155610.1378/chest.08-223319225060Clinical and immunogenetic features of patients with autoantibodies to asparaginyl-transfer RNA synthetaseHirakataMSuwaATakadaTSatoSNagaiSGenthESongYWMimoriTTargoffINArthritis Rheum2007561295130310.1002/art.2250617393393