Description:ProductNameBorreliaburgdorferiIgGELISA(recombinant).#ofSamples1x96Assays.IntendedUseTherecombinantBorreliaburgdorferiIgG-ELISAisintendedforthequalitativedeterminationofIgGclassantibodiesagainstBorreliaburgdorferiinhumanserumorplasma(citrate)offeringincreaseddiagnosticspecificityandsensitivitybyemployingimmunodominantantigens.IncombinationwiththerecombinantIgMELISAeachclinicalphaseofLymedisease-fromErythmachronicummigransoverBannwarth-SyndromuptoLymearthritis-maybedetectedeveninborderlandcasesprovidingunerringresultsanddiagnosticaidfortheclinicalstaff.IntroductionSpiRochetesaremotilebacteriawithaperiplasmaticaxialfilament.AllpathogenicspeciesbelongtothefamilyTreponemataceae,whichincludesthethreegenera:Treponema,Borrelia,andLeptospira.TheTreponemaeareextremelylong,flexIBLe,filamentouscellsthatareusuallyheldinacharacteristicspiral,orcoiled-springshape.BorreliaearethelargestTreponemataceaewithverycoarseandirregularspirals.Borreliaburgdorferi,thecausativeagentofLymedisease,istransmittedmainlybyticksbutprobablyalsobyotherblood-suckinginsects.HABItatsarethewooded,humidandtemperateregionsofNorthAmerica,Europe,NorthAfrica,AustraliaandJapan;foresters,farmers,andanybodyenteringinfestedforestsmaybeaffected.Thedegreeofcontaminationofticksamountsto3-60%dependentonseasonalandregionaldifferences;upto30%ofthepopulationmaybeinfected(about1500casesannuallyinUSA,severalhundredinEurope).Becauseofitsmultisystemiccharacter,unequivocaldiagnosisofLymediseaseisdifficult.Onlyearlydetectionpermitsefficientcontrolbyantibiotics,sinceinthechronicphaseborreliaearenearlyinaccessible.Thepresenceofbacteriaresp.infectionmaybeidentifiedbyMicroscopy:GiemsaorWright-stainedbloodsmears.PCRSEROlogy:DetectionofantibodiesbyIF,ELISA,immunoblotting.PrinciplesoftheassayThemajorconstituentofB.burgdorferiflagellaisflagellin(41kDa,p41).WhereasthelipoproteinOspC(22kDa,p22)withintheoutermembraneofthespirocheteinducesanearlyantibodyformationintheECM-phaseofLymedisease,thespecies-specificMarkersp100,p18andVlsEareespeciallyreliablefordetectingtheIgGresponseandtheyareresponsibleforthehighsensitivity.P41iisincludedinthepresentedantigenstoavoidcrossreactivitywithantibodiesofsyphiliticsera.TheBorreliaburgdorferiIgG-ELISAcontainstherecombinantepitopeOspCofthephylumB31(B.sensustricto),20047andT25(B.garinii),p100,andp18ofthephylumPKo(B.afzelii)andp41iofthephylumPBi(B.garinii).MicrotiterstripwellsareprecoatedwithrecombinantBorreliaburgdorferiantigens.DilutedpatientspecimensandreadytousecontrolsareaddedtothesewellsandantibodiesrecognizingtheimmobilizedB.burgdorferiantigenbindduringthefirstincubation.AfterwashingthewellstoremoveallunboundsampleandcontrolmaterialhorserADIshperoxidaselabelledanti-humanIgGconjugateisadded.Duringasecondincubationthisconjugatebindstothecapturedantibodies,andtheexcessunboundconjugateisremovedbyafurtherwashstep.TheimmunecomplexformedbytheboundconjugateisvisualizedwithTMBSubstrateSolutionwhichgivesabluereactionproduct,theintensityofwhichisproportionaltotheamountofB.burgdorferi-specificIgGantibodyinthepatientspecimen.Sulphuricacidisaddedtoeachwelltostopthereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.StorageandStabilityThereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2-8°C.LimitationsoftheTestBacterialcontaminationorrepeatedfreeze-thawcyclesofthespecimenmayaffecttheabsorbancevalues.Diagnosisofaninfectiousdiseaseshouldnotbeestablishedonthebasisofasingletestresult.Aprecisediagnosisshouldtakeintoconsiderationclinicalhistory,symptomatologyaswellasserologicaldata.Inimmunosuppremizedpatientsandnewbornsserologicaldataonlyhaverestrictedvalue.Aneg.result(IgGorIgM)cannotexcludeaninfectionwithB.burgdorferi.Especiallyintheearlyphaseofinfectionthereisthepossibilitythatnoneornodetectionquantitiesoftheantibodiesdoexist.Inthecaseofinfectionoragreyzoneresultwerecommendafurthersampleafter2-3weeks.ApositiveresultofIgGdoesnotalwaysmeanthatanacuteinfectionexistsbecausetheantibodiesofapreviousinfectioncanpersist.Theuseofrecombinantantigensavoidextensivelycrossreactionswiththefollowingantibodies:Treponemapallidum,Leptospira,Borreliarecurrentis.Antibodiesofalues-infectiontop41iwereoccasionallydeterminated.Thereforealues-infectionshouldbeexcluded.ReferencesJohnsonR.C.,SchmidG.P.,HydeF.W.,SteigerwaldtA.G.,BrennerD.J.(1984)Borreliaburgdorferisp.nov.:etiologicagentofLymedisease.IntJSystBacteriol34,496-497AsbrinkE.,HovmarkA.(1988)EarlyandlatecutaneousmanifestationsofIxodes-bornborreliosis(erythemamigransborreliosis,LymeBorreliose).AnnNYAcadSci539,4-15KarlsonM.,Hovind-HouganK.,SvenungsenB.,StiernstedG.(1990)CultivationandcharacterizationofSpirochetesfromcerebrospinalfluidofpatientswithLymeborreliosis.JClinMicrobiol28,473-479Preac-MursicdV.,WilskeB.,SchierzG.(1986)EurpeanBorreliaBurgdorferiisolatedfromhumanandticks:Cultureconditionsandantibioticsusceptibility.ZblBaktHygA263,112-118WilskeB.,Preac-MursicV.,FuchsR.,SchierzG.(1990)DiagnostikderLymeBorreliose.DiagnoseundLabor,Laboratoriumsblätter40,24-36WilskeB.(2003)DiagnosisofLymeBorreliosisinEurope.Vector-BorneandZoonoticDiseases3(4),215–227Jauris-HeipkeS.etal.(1990):Osp17,anovelimmunodominantoutersurfaceproteinofBorreliaafzelli:recombinantexpressioninEscherichiacolianditsuseasadiagnosticantigenforserodiagnosisofLymeborreliosis.MedMicrobiolImmunol187,213–219Schulte-SpechtelU.etal.(2003)SignifikantImprovementoftheRecombinantBorrelia-SpecificImmunoglobulinGImmunoblotTestbyAdditionofVlsEandaDbpAHomologueDerivedfromBorreliagariniiforDiagnosisofEarlyNeuroborreliosis.J.ClinMircrobiol41,1299–1303EickenC.etal.(2002)CrystalStructureofLymeDiseaseVariableSurfaceAntigenVlsEofBorreliaburgdorferi.J.BiolChem277,21691–21696WangD.,BotkinD.J.andNorrisS.J.(2003)CharacterisationofthevlsantigenicvariationlocioftheLymediseasespirochaetesBorreliagariniiIp90andBorreliaafzeliiACAI.MolMicrobiol47(5),1407–1417OhnishiJ.etal.(2003)GeneticVariationatthevlsELocusofBorreliaburgdorferiwithinTicksandMiceovertheCourseofaSingleTransmissionCycle.J.Bacteriol185,4432-4441.AdditionalInformation:NameBorreliaburgdorferiIgGrecombinantantigens+liquorRelatedProductNamesBorreliaburgdorferiIgG-ELISA(recombinant)MolecularWeight0.5StorageThereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2°C-8°C.IntendedUseResearchUseOnly