EnzymeimmunoassayforthequantitativedeterminationofIgG-classantibodiesagainstBordetellapertussistoxin(PT)inhumanserumorplasma(citrate).Bordetellaspeciesarenon-spore-formingencapsulatedbipolar,coccoid(pale-staining)Gram-negativebacilli(about0.3-0.5μmthickand1μmlong).ThegenusconsistsofthehumanpathogensB.pertussisandB.parapertussis,andB.bronchisepticawhichcauseenzooticinfectionsinvariouswildanddomesticanimalspecies.B.pertussisisthepathogenicagentofpertussisandexistsonlyinillpeople;B.parapertussiscauses5-20%ofamilderandoftenclinicalunapparentformofpertussis.B.bronchisepticahasseldomly(e.g.closecontactwithanimals)humanpathogenicsignificanceasopportuNISTicsecondaryinfectiousagentinmixedinfections(bronchitis,pneumonia,woundinfection).Pertussisorwhoopingcoughisabacterialinfectionoftherespiratorysystem.Itisahighlycontagiouschildhooddiseasewhichappearsseldomlyinadults.Itistransmittedbyairbornedroplets.B.pertussishastheABIlitytosticktotheciliaoftheepidermalcellsoftherespiratorysystem.Fragmentsofthebacterialcellwall(ExoToxin=trachealCytotoxin,TCT)inhibitthemovementoftheciliainthetrachealmucosa.Afteranincubationtimeofonetothreeweeksthediseaserunsthroughthreestages(s.tablebelow).Thelethalityis0.6%andconcernsbabiesinthefirstsixmonthswithmorethan70%.Fornewbornandprematureinfantsitishigher(1-2%).InAfricabesidemeaslesvirusB.pertussisisthemainreasonforhighinfantmortality.Thedistributionofthediseaseisworldwide.Clinicalpertussisisfollowedbynaturalacquiredimmunitywhichislong-lastingbutnotpermanent.Inmostcountriesanactivevaccinationisrecommendedwhichleadsto90%protectionforthreetotwelveyears.Usuallytheimmunizationpreparationiscombinedwithdiphtheriaandtetanustoxoids.ThequantitativeimmunoenzymaticdeterminationofIgG-classantibodiestoBordetellapertussistoxin(PT)isbasedontheELISA(Enzyme-linkedImmunosorbentAssay)technique.MicrotiterstripwellsarecoatedwithB.pertussistoxintobindcorrespondingantibodiesofthespecimen.AfterwashingthewellstoremoveallunboundsamplematerialhorserADIshperoxidase(HRP)labelledanti-humanIgGconjugateisadded.ThisconjugatebindstothecapturedB.pertussistoxinspecificantibodies.Theimmunecomplexformedbytheboundconjugateisvisualizedbyaddingtetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisproportionaltotheamountofB.pertussistoxinspecificIgGantibodiesinthespecimen.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.