Description:

REAADSAnti-Cardiolipin(aCL)IgAis an ELISAforthedeterminationofIgAanti-cardiolipin antibodiesinhumanserumorplasma inindividualswithsystemiclupus erythematosus(SLE)andlupus-likedisorders(anti-phospholipidsyndrome).

Advantages:

• SpecificdeterminationofIgAaCLantibodies
• ConvenientELISAprocedure
• Objective,accurateandreproducIBLe
• Reagentcompletekit
• Shortincubationsatroomtemperature

KitComposition:

Reagents,StorageandStABIlity

• Stabilizedbeefheartcardiolipin(diphosphatidylglycerol)coatedmicrowells:96(12stripsof8breakawaywells), withframe.
• SampleDiluent: 1bottle, 60ml(greensolution),containsbovinecalfserumand sodiumazide.
• aCLIgACalibratorSerum: 3vials, 0.250ml(1-high,2-moderate,3-low)(human),seeviallabelforantibody concentrationinAPLunits.Calibrator2shouldbeusedwhenperformingsinglepointcalibration.Containssodiumazide.
• aCLIgAPositiveControlSerum: 1vial,0.250ml(human),seeviallabelforexpectedAPLrange.Containssodiumazide.
• aCLNormalControlSera: 1vial, 0.250ml,(human),seeviallabelforexpectedAPLrange.Containssodiumazide.
• anti-humanIgA(goat)HRP-conjugatedantibodysolution:1bottle,15ml(orangesolution).
• OneComponentSubstrate: 1bottle, 15ml(TMBandH2O2),readytouse.
• StoppingSolution:1bottle, 15ml(0.36Nsulfuricacid).
• WashConcentrate2bottles, 30ml (33XPBS).

Storeat2–8°C.DoNotFreeze.

PrincipleandProcedure:

Principle

ThetestisperformedasanindirectELISA.Dilutedserumsamples,calibratorsera,andcontrolsareincubatedin cardiolipincoatedmicrowells,allowingaCLantibodiespresentinthesamplestoreactwiththeimmobilizedantigen. Aftertheremovalofunboundserumproteinsbywashing,antibodiesspecificforhumanIgAlabeledwith horserADIshperoxidase(HRP)areaddedformingcomplexeswiththecardiolipinboundantibodies.Following anotherwashingstep,theboundenzyme-antibodyconjugateisassayedbytheadditionoftetramethylbenzidine (TMB)andhydrogenperoxide(H2O2)asthechromogenicsubstrate.Colordevelopsinthewellsatanintensity proportionaltotheserumconcentrationofIgAaCLantibodies. ResultsareobtainedbyreadingtheO.D.(opticaldensityorabsorbance)ofeachwellwithaspectrophotometer. Calibratorseraareprovided,withtheIgAaCLconcentrationexpressedinAPLunits.Theuserhastheoptionof runningeitherasinglepointcalibratororafour-pointcalibrationcurve.Forsinglepointcalibration,dividingthe concentrationvalueofthecalibratorbytheO.D.valueofthecalibratorprovidesaconversionfactor.TheO.D. valuesofthecontrolsandpatientsamplesaremultipliedbytheconversionfactortoobtainIgAaCLvalues, expressedinAPLunits.Formultipointcalibration,performalinearregressionanalysiswithcalibratorvalues againstcalibratorO.D.s.Controlandpatientresultsaredeterminedfromthecalibrationcurve.

Procedure

Dilutedpatientserum(preferred)orplasmaisincubatedincoatedmicrowells.Cardiolipinantibodiespresentinthesamplewillbindtothecoatedwells.Afterwashingtoremoveunboundplasmaproteins,enzymeconjugatedanti-humanimmunoglobulinspecificforIgAisadded.Thewellsarewashedagain,andachromogenicsubstrateisadded,resultinginacoloredproduct.Thereactionisstoppedbytheadditionofaweakacidsolution,andthecoloredproductismeasuredinaspectro-photometerat450nm.Testresultsareavailableinlessthanonehour.ValuesforaCLantibodiesarereportedinGPL,MPL,andAPLunits,withassaycutoffsestablishedat23GPL,11MPL,and22APL.

ClinicalPerformance*:

ClinicalSpecificity:Assaycutoffswerechallengedwithahealthyblooddonorpopulation.Usingthestatedcutoffs,theassayswere97%specificforIgG,96%specificforIgM,and95%specificforIgA.

UnselectedSLEpopulationsweretestedtodetermineassaysensitivity;21%ofthesampleswerepositiveforIgG,10%forIgMand26%forIgAantibodieswiththeREAADSaCLassays.TheclinicalsensitivityforthrombosisandthrombocytopeniawasdeterminedbycomparingaCLtestresultsfromtwogroupsofselectedSLEpatients:Group1?withaclinicalhistoryofthrombosisand/orthrombocytopenia;andGroup2?withnohistoryofthrombosisorthrombocytopenia(control).Theresultsareshowninthechartsbelow:

Group1:SLE+Thrombosisand/orthrombocytopenia	IgGaCL	IgMaCL	IgAaCL
Averagevalue	30GPL	9 MPL	32APL
%positive	45%	25%	79%
Group2:SLEControl	IgGaCL	IgMaCL	IgAaCL
Averagevalue	8 GPL	3 MPL	15 APL
%positive	0%	0%	0%

KitSizes:

CATALOG#	TYPE	WELLS	FORMAT
K023-001	IgG/IgM	96	rapidformat(15-15-10)
K023-002*	IgG/IgM	288	rapidformat(15-15-10)
K026-001	IgA	96	rapidformat(15-15-10)
K026-006*	IgA	288	rapidformat(15-15-10)
K11139*	IgG	96	extendedincubations(30-30-30)
K12747*	IgG	288	extendedincubations(30-30-30)
K11140*	IgM	96	extendedincubations(30-30-30)
K12748*	IgM	288	extendedincubations(30-30-30)
K11141*	IgA	96	extendedincubations(30-30-30)
K12770*	IgA	288	extendedincubations(30-30-30)

*Thisproductmayrequireaspecialorder.Pleaseinquireatinfo@Diapharma.comor1-800-526-5224.

Background:

Anti-cardiolipin(aCL)antibodiesareassociatedwiththepresenceofbothvenousandarterialthrombosis,thrombocytopenia,andrecurrentfetalloss.Theseautoantibodiesarefrequentlyfoundinpatientswithsystemiclupuserythematosus(SLE)andotherautoimmunediseases,aswellasinsomeindividualswithnoapparentpreviousunderlyingdisease.

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