EnzymeimmunoassayforthequalitativedeterminationofIgGclassantibodiesagainstBordetellapertussisinhumanserumorplasma(citrate).Bordetellaspeciesarenon-spore-formingencapsulatedbipolar,coccoid(pale-staining)Gram-negativebacilli(about0.3-0.5μmthickand1μmlong).ThegenusconsistsofthehumanparasitesB.pertussisandB.parapertussis,andB.bronchisepticaandB.aviumwhichcauseenzooticinfectionsinvariouswildanddomesticanimalspecies.B.pertussisistheclassicalexciterofpertussisandexistsonlyinillpeople;B.parapertussiscauses5-20%ofamilderandoftenclinicalunapparentformofpertussis.B.bronchisepticahasseldomly(egclosecontactwithanimals)humanpathogenicsignificanceasopportuNISTicsecondaryexciterinmixedinfections(bronchitis,pneumonia,woundinfection).Pertussisorwhoopingcoughisabacterialinfectionoftherespiratorysystem.Itisahighlycontagiouschildhooddiseasewhichappearsseldomlyunderadults.Itistransmittedbyrespiratorycontact.B.pertussishastheABIlitytosticktotheciliaoftheepidermalcellsoftherespiratorysystem.Fragmentsofthebacterialcellwall(ExoToxin=trachealCytotoxine,TCT)inhibitthemovementoftheciliainthetrachealmucosa.Afteranincubationtimeofonetothreeweeksthediseaserunsthroughthreestages(s.tablebelow).Thelethalityis0.6%andconcernsbabiesinthefirstsixmonthswithmorethan70%.Fornewbornandprematureinfantsitishigher(1-2%).InAfricabesidemeaslevirusB.pertussisisthemainreasonforhighinfantmortility.Thedistributionofthediseaseisworldwide.Clinicalpertussisisfollowedbynaturalacquiredimmunitywhichislong-lastingbutnotpermanent.Inmostcountriesanactivevaccinationisrecommendedwhichleadsto90%protectionforthreetotwelveyears.Usuallytheimmunizationpreparationiscombinedwithdiphtheriaandtetanustoxoids.ThequalitativeimmunoenzymaticdeterminationofIgG-classantibodiesagainstBordetellapertussisisbasedontheELISA(Enzyme-linkedImmunosorbentAssay)technique.MicrotiterstripwellsareprecoatedwithBordetellapertussis/toxinantigenstobindcorrespondingantibodiesofthespecimen.AfterwashingthewellstoremoveallunboundsamplematerialhorserADIshperoxidase(HRP)labelledanti-humanIgGconjugateisadded.ThisconjugatebindstothecapturedBordetellapertussis/toxinspecificantibodies.Theimmunecomplexformedbytheboundconjugateisvisualizedbyaddingtetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisproportionaltotheamountofBordetellapertussis/toxinspecificIgGantibodiesinthespecimen.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.