EnzymeimmunoassayforthequalitativedeterminationofIgGclassantibodiesagainstBorreliaburgdorferiinhumanserumorplasma(citrate)andCSF(cerebrospinalfluid).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).ThemajorconstituentofB.burgdorferiflagellaisflagellin(41kDa,p41).WhereasthelipoproteinOspC(22kDa,p22)withintheoutermembraneofthespirocheteinducesanearlyantibodyformationintheECMphaseofLymedisease,thespecies-specificMarkersp100,p18andVlsEareespeciallyreliablefordetectingtheIgGresponseandtheyareresponsibleforthehighsensitivity.P41iisincludedinthepresentedantigenstoavoidcrossreactivitywithantibodiesofsyphiliticsera.TheBorreliaburgdorferiIgG-ELISAcontainsthefollowingrecombinantepitopes:OspCofthephylumB31(B.sensustricto),20047andT25(B.garinii),p100,andp18ofthephylumPKo(B.
afzelii)andp41iofthephylumPBi(B.garinii).MicrotiterstripwellsareprecoatedwithrecombinantBorreliaburgdorferiantigens.DilutedpatientspecimensandreadytousecontrolsareaddedtothesewellsandantibodiesrecognizingtheimmobilizedB.burgdorferiantigenbindduringthefirstincubation.AfterwashingthewellstoremoveallunboundsampleandcontrolmaterialhorserADIshperoxidaselabelledanti-humanIgGconjugateisadded.Duringasecondincubationthisconjugatebindstothecapturedantibodies,andtheexcessunboundconjugateisremovedbyafurtherwashstep.TheimmunecomplexformedbytheboundconjugateisvisualizedwithTMB
SubstrateSolutionwhichgivesabluereactionproduct,theintensityofwhichisproportionaltotheamountofB.burgdorferi-specificIgGantibodyinthepatientspecimen.Sulphuricacidisaddedtoeachwelltostop
thereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.