GenWay/AFP/GWBF49883/1x96 Assays

Description:

ProductName:AFP

#ofSamples:1x96Assays

IntendedUse:Immunoenzymaticcolorimetricmethod(ELISA)forquantitativedeterminationofAFPinserumorplasma.

Introduction:AlphaFetoprotein(AFP)isa68kDaglycoprotein,whichisnormallyonlyproducedinthefetusduringitsdevelopment.Itisanormallyproducedbytheliverandyolksacofthefetus.
AFPlevelsdecreasesoonafterbirthandprobablyhasnofunctioninnormaladults.Itbindsthehormoneestradioltokeepitfromaffectingthefetalbrain.Itsmeasurementduringpregnancyhasbeenusefultodetectcertainabnormalities-specifically,ifhighlevelsofAFParefoundinamnioticfluid,itcanindicateadevelopmentaldefectinthebaby.
InsomepatientswhoarenotpregnantatumorcanproduceAFP,thusitcanbeusedasatumourMarker.AFPisthemaintumourmarker(alongwithHCG)todiagnosetesticularcanceranditsvaluesovertimecanhavesignificanteffectonthetreatmentplan.
Likealltumourmarkers,thedetectionofAFPbyitselfisnotdiagnosticofanything,althoughifitisdetecteditiscertainlyadvisabletoruleoutthediseasescouldcauselevelstorise.Theprimaryreasontumormarkersareusedaretomeasurethesuccessofatreatment(e.g.chemotherapy),iflevelsofAFParegoingdown,itisanindicationthatadiseaseisimproving.NewresearchexhibitsthatanisoformofAFPwhichbindsLensculinarisagglutinin(AFP-L3)canbeparticularlyusefulinearlyidentificationofaggressivetumorsassociatedwithhepatocellularcarcinoma(HCC).

Principlesoftheassay:TheAFPassayisbasedonsimultaneousbindingofhumanAFPtotwomonoclonalantibodies;oneisimmobilizedonthemicroplate,theotherissolubleandconjugatedwithhorseradishperoxidase(HRP).Microtiterstripwellsareprecoatedwithanti-AFPIgGantibodies.AFPinsamplesandstandardsbindstotheimmobilisedantibodiesonthesurfaceofthemicrotiterwellsandthesecond,solubleanti-AFPantibody-enzymeconjugatebindstotheimmobileantibody-AFP-complexduringthefirstincubation.Afterwardsabound/freeseparationisperformedbysolid-phasewashing.TheimmunecomplexisvisualizedbyaddingTetramethylbenzidine(TMB)substrate,whichgivesabluereactionproduct.TheintensityofthisproductisproportionaltotheamountofAFPinsamplesandstandards.Sulphuricacidisaddedtostopthereaction.Thisproducesayellowendpointcolour.Absorptionat450nmisreadusinganELISAmicrowellplatereader.

StorageandStability:Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°C.Afterfirstusethestandardsolutionsarestillstabileforanother6monthsifstoredat2…8°C.

LimitationsoftheTest:Sample(s),whicharecontaminatedmicrobiologically,shouldnotbeusedintheassay.Highlylipemeicorhaemolysedspecimen(s)shouldsimilarlynotbeused.Itisimportantthatthetimeofreactionineachwellisheldconstantforreproducibleresults.Pipettingofsamplesshouldnotextendbeyondtenminutestoavoidassaydrift.Ifmorethanoneplateisused,itisrecommendedtorepeatthedoseresponsecurve.Additionofthesubstratesolutioninitiatesakineticreaction,whichisterminatedbytheadditionofthestopsolution.Therefore,theadditionofthesubstrateandthestoppingsolutionshouldbeaddedinthesamesequencetoeliminateanytimedeviationduringreaction.Platereadersmeasurevertically.Donottouchthebottomofthewells.Failuretoremoveadheringsolutionadequatelyintheaspirationordecantationwashstep(s)mayresultinpoorreplicationandspuriousresults.

ReferencesWisdom,G.B.(1976)Clin.Chem.22/8,1243-1255Shome,B.andParlow,A.F.(1974)J.Clin.Endocr.Metab.39,199–202Uotila,M.,Ruoslahti,E.,Engvall,E.(1981)J.Immunol.Methds42,11-15Acosta,A.A.M.D.andWright,G.L.(1983)J.ofClinicalImmunoassays6,41JacobsenG.K.(1983)ActaPathMicrob.Immun.Scand.91,183–190KohnJ.,OeeA.H.,McElwainT.J.(1976)Lancet2,433–436WaaldmannT.A.andMcIntireK.R.(1974)Cancer34,1510-1515

AdditionalInformation:

Name	AFP
RelatedProductNames	AFP
MolecularWeight	0.5
Storage	Thereagentsarestableuptotheexpirydatestatedonthelabelwhenstoredat2...8°C.Afterfirstusethestandardsolutionsarestillstabileforanother6monthsifstoredat2 8°C.
IntendedUse	ResearchUseOnly