EnzymeimmunoassayforthequalitativedeterminationofIgM-classantibodiesagainstBrucellainhumanserumorplasma(citrate).BrucellaisasmallGram-negativebacterium(0.4-0.8μmindiameterand0.4-3.0μminlength)whichisnonflagellated,andnon-spore-forming.TheyarenamedafterthemilitarydoctorDavidBruce,whoonMaltain1887isolatedthepathogensfromthespleenofasoldierwhodiedofundulatingfever.Fourspeciesarepathogenictohuman:Brucellaabortus,Brucellamelitensis,BrucellasuisandBrucellacanis.AllfourspeciesareexcitersofBrucellosis,adiseasecharacterizedbyundulatingfever.DependingonexciterthediseaseisalsocalledMorbusBang(B.abortus)orMaltafever(B.melitensis).Thepathogensaretransmittedfromanimals,whicharemainlyaffected.Theinfectioniscausedbycontactwithillanimalsortheirexcrementsaswellasbynon-pasteurizedmilkandmilkproductslikefreshcheesefromsheeporgoat.Mainentrancesareskinwounds,conjunctivesanddigestivetract.Theintactpathogensaretransportedbygranulocytesintolocallymphnodes,fromwheretheyspreadhaematogenous.Allkindoforganscanbeinfected.Symptomsdependupontheinfectedorgan,whereaninflammationtakesplace.Untilnowthepathogenicmechanismisnotcompletelyunderstood.Brucellosisappearsworldwide.Innon-pasteurizedmilkandmilkproductsBrucellaisviablyandinfectiousforweeks.BovinebrucellosiscausedbyBrucellaabortusisstillthemostwidespreadform,althoughreportedincidenceandprevalenceofthediseasevarywidelyfromcountrytocountry(from<0.01 to >200per100,000population).Brucellamelitensisisendemicinareaswherekeepingofsheepsandgoatsisfrequent.Itcausesserioushumaninfections.Workingwiththesebacteriainlaboratoriesnecessitateshighestcarefulnessbecauseofhighcontagiosity.Riskgroupsincludeabattoirworkers,meatinspectors,animalhandlers,veterinarians,andlaboratorians.Brucellosisisanationallynotifiablediseaseandreportabletothelocalhealthauthority.ThequalitativeimmunoenzymaticdeterminationofIgM-classantibodiesagainstBrucellaisbasedontheELISA(Enzyme-linkedImmunosorbentAssay)technique.MicrotiterstripwellsareprecoatedwithBrucellaantigenstobindcorrespondingantibodiesofthespecimen.AfterwashingthewellstoremoveallunboundsamplematerialhorserADIshperoxidase(HRP)labelledantihumanIgMconjugateisadded.ThisconjugatebindstothecapturedBrucella-specificantibodies.TheimmunecomplexformedbytheboundconjugateisvisualizedbyaddingTetramethylbenzidine(TMB)substratewhichgivesabluereactionproduct.TheintensityofthisproductisproportionaltotheamountofBrucellaspecificIgMantibodiesinthespecimen.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorbanceat450nmisreadusinganELISAmicrowellplatereader.ForconcretedatapleaseconsulttheInstructionforUseinthedownloadboxontherightside.