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ProductName:BorreliaburgdorferiIgM–ELISA(recombinant)

#ofSamples:1x96Assays

IntendedUse:TherecombinantBorreliaburgdorferiIgM-ELISAisintendedforthequalitativedeterminationofIgMclassantibodiesagainstBorreliaburgdorferiinhumanserumorplasma(citrate)offeringincreaseddiagnosticspecificityandsensitivitybyemployingimmunodominantantigens.IncombinationwiththerecombinantIgGELISAeachclinicalphaseofLymedisease-fromErythmachronicummigransoverBannwarth-SyndromuptoLymearthritis-maybedetectedeveninborderlandcasesprovidingunerringresultsanddiagnosticaidfortheclinicalstaff.

Introduction:SpiRochetesaremotilebacteriawithaperiplasmaticaxialfilament.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.infectionmaybeidentifiedby:
Microscopy:GiemsaorWright-stainedbloodsmears
PCR
SEROlogy:DetectionofantibodiesbyIF,ELISA,immunoblotting

Principlesoftheassay:ThemajorconstituentofB.burgdorferiflagellaisflagellin(41kDa,p41).WhereasthelipoproteinOspC(22kDa,p22)withintheoutermembraneofthespirocheteinducesanearlyantibodyformationintheECM-phaseofLymedisease.Recombinantp41iisincludedinthepresentedantigenstoavoidcrossreactivitywithantibodiesofsyphiliticsera.
TheBorreliaburgdorferiIgM-ELISAcontainstherecombinantepitopeOspCofthephylumPKo(B.afzelii)and20047(B.garinii)andp41iofthephylumPBi(B.garinii).
Microtiterstripwellsarepre-coatedwithrecombinantBorreliaburgdorferiantigens.DilutedpatientspecimensandreadytousecontrolsareaddedtothesewellsandantibodiesrecognizingtheimmobilizedB.burgdorferiantigenbindduringthefirstincubation.AfterwashingthewellstoremoveallunboundsampleandcontrolmaterialhorserADIshperoxidaselabelledanti-humanIgMconjugateisadded.Duringasecondincubationthisconjugatebindstothecapturedantibodies,andtheexcessunboundconjugateisremovedbyafurtherwashstep.TheimmunecomplexformedbytheboundconjugateisvisualizedwithTMBSubstrateSolutionwhichgivesabluereactionproduct,theintensityofwhichisproportionaltotheamountofB.burgdorferi-specificIgMantibodyinthepatientspecimen.Sulphuricacidisaddedtoeachwelltostopthereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.

StorageandStability:Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°C.

LimitationsoftheTest:Bacterialcontaminationorrepeatedfreeze-thawcyclesofthespecimenmayaffecttheabsorbancevalues.Diagnosisofaninfectiousdiseaseshouldnotbeestablishedonthebasisofasingletestresult.Aprecisediagnosisshouldtakeintoconsiderationclinicalhistory,symptomatologyaswellasserologicaldata.Inimmunsuppremizedpatientsandnewbornsserologicaldataonlyhaverestrictedvalue.Aneg.result(IgMorIgG)cannotexcludeaninfectionwithB.burgdorferi.Especiallyintheearlyphaseofinfectionthereisthepossibilitythatnoneornodetectionquantitiesoftheantibodiesdoexist.Inthecaseofinfectionoragreyzoneresultwerecommendafurthersampleafter2-3weeks.ApositiveresultofIgMdoesnotalwaysmeanthatanacuteinfectionexistsbecausetheantibodiesofapreviousinfectioncanpersist.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,Laboratoriumsblätter40,24-36

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Name	BorreliaburgdorferiIgMrecombinantantigens+liquor
RelatedProductNames	BorreliaburgdorferiIgM–ELISA(recombinant)
MolecularWeight	0.5
Storage	Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°C.
IntendedUse	ResearchUseOnly