EnzymeimmunoassayforthequantitativedeterminationofIgG-classantibodiesagainstCoryNEBacteriumdiphtheriaetoxininhumanserumorplasma(citrate).Corynebacteriaareaerobicnonspore-forminggram-positiverodsofirregularshape(0.5–1μmthickand2-6μmlong).Theycompriseskincommensals,opportuNISTpathogensandseveralmajorpathogens,includingCorynebacteriumdiphtheriae.Ingeneral,theyareisolatedfromthroatswabsonselectivemediacontainingtellurite.ThebacterialinfectioncausedbyC.diphtheriae,Diphtheria,hastwoforms.Respiratorydiphtheriaistypicallycausedbytoxin-producing(toxigenic)strains;cutaneousdiseasecanbecausedbyeithertoxigenicornontoxigenicstrains.Intherespiratoryformofthedisease,amembraneisformed;thismembraneisusuallyvisIBLeonthethroatortonsils.Personsmaydiefromasphyxiationwhenthemembraneobstructsbreathing.Othercomplicationsarecausedbyremoteeffectsofthediphtheriatoxin(myocarditis,nerveparalysis)Cutaneousdiphtheriaisusuallymild,typicallyconsistingofnon-distinctivesoresorshallowulcersandonlyrarelyinvolvingtoxiccomplications(1-2%ofinfectionswithtoxigenicstrains).Diphtheriawasoneofthemostcommoncausesofdeathamongchildrenduringtheprevaccineera.Sincetheintroductionandwidespreaduseofdiphtheriatoxoidvaccine(formalin-inactivateddiphtheriatoxin)inmostindustrializedcountriesthediseaseisnowcharacterizedbysporADIccasesandintermittentoutbreaksoflowintensity.ButrecentlargeepidemicsofdiphtheriainseveraleasternEuropeancountrieshaveagaindrawnattentiontothis„forgotten“disease–and,themajorityofthesecaseshaveoccurredamongadolescentsandadultsinsteadofchildren.ThequantitativeimmunoenzymaticdeterminationofIgG-classantibodiesagainstC.diphtheriaetoxinisbasedontheELISA(Enzyme-linkedImmunosorbentAssay)technique.MicrotiterstripwellsareprecoatedwithinactivatedspecificCorynebacteriumdiphtheriaetoxin(toxoid)antigenstobindcorrespondingantibodiesofthespecimen.Afterwashingthewellstoremoveallunboundsamplematerialhorseradishperoxidase(HRP)labelledanti-humanIgGconjugateisadded.ThisconjugatebindstothecapturedC.diphtheriaetoxin-specificantibodies.TheimmunecomplexformedbytheboundconjugateisvisualizedbyaddingTetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisproportionaltotheamountofC.diphtheriaetoxin-specificIgGantibodiesinthespecimen.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.