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.
