Description:
TheREAADSAnti-Beta2GlycoproteinI(Aβ2GPI)IgG testkitisan indirectenzymelinkedimmunosorbentassay(ELISA)forthesemi-quantitativedeterminationofIgGanti-β2GP1antibodiesinhumanserumorcitratedplasma(3.2%sodiumcitrate).AsinglepointormultipointcalibratorisusedtomeasureIgGanti-β2GP1antibodyconcentrationsinGunits. ForthedetectionofIgGanti-β2GPlantibodiesinindividualswithsystemiclupuserythematosus(SLE)andlupus-likedisorders(anti-phospholipidsyndrome).
Advantages:
- ForthespecificdeterminationofIgGanti-B2GP1antibodies
- Excellentclinicalcorrelation
- Colorcodedreagents
- Totalincubationtime:40minutes
- Convenient,costeffective
- Choiceofsingleormulti-pointcalibration
KitComposition:
Reagents
EachREAADSIgGAnti-β2GPl96-MicrowellTestKitcontainsthefollowingreagents(volumesmayvarydependingonthekitsizeandconfiguration):
- 12x8stABIlizedβ2GPl(fromhumanserum)coatedmicrowellswithframe
- 60mlSampleDiluentIV(blue-greensolution)
- 3vials(0.250ml)IgGβ2GPlCalibratorSerum*(1-high,2-moderate,3-low)(human);seeviallabelforantibodyconcentrationinGunits.Calibrator2shouldbeusedwhenperformingsinglepointcalibration
- 0.250mlIgGβ2GPlPositiveControlSerum*(human);seeviallabelforexpectedGunitrange
- 0.250mlNormalControlSerum*(human);seeviallabelforexpectedGunitrange
- 15mlanti-humanIgG(goat)HRP-ConjugatedAntibodySolution(bluesolution)
- 15mlOneComponentSubstrateSolution(TMBandH2O2);readytouse
- 15mlStoppingSolution(0.36Nsulfuricacid)
- 2bottles(30ml)WashConcentrate(33XPBS/Tween)
Storeat2–8°C.DoNotFreeze.
MaterialsRequiredbutnotSupplied
- ReagentgradewatertopreparePBSwashsolutionandtozeroorblanktheplatereaderduringthefinalassaystep
- Graduatedcylinders
- Precisionpipettorscapableofdeliveringbetween5μland1000μl,withappropriatetips
- Miscellaneousglasswareappropriateforhandlingsmallvolumes
- Flasksorbottles,1liter
- Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
- Disposablegloves
- Plate-reADIngspectrophotometercapableofreadingabsorbanceat450nm(650nmreferenceifdualbeam)
- Multichannelpipettorscapableofdeliveringto8wellssimultaneously
- Microdilutiontubesanda96-wellmicrodilutiontubeholderforsampledilutionsandrapiddeliverytomicrowellplate
MeasurementPrinciple:
TheREAADSIgG,IgMandIgAanti-B2GPItestkitsareindirectenzymelinkedimmunosorbentassaysforthesemiquantitativedeterminationofanti-B2GP1antibodiesinhumanserum.AsinglepointormultipointcalibratorisusedtomeasureIgG,IgM,orIgAanti-B2GP1antibodyconcentrationsinG,MorAunits.
TheREAADSIgGtestisperformedasanindirectELISA.Dilutedserumorplasmasamples,calibratorsera,andcontrolsareincubatedinmicrowellscoatedwithpurifiedhumanβ2GPl.Incubationallowstheanti-β2GPlantibodiespresentinthesamplestoreactwiththeimmobilizedantigen.Aftertheremovalofunboundserumorplasmaproteinsbywashing,antibodiesspecificforhumanIgG,labeledwithhorseradishperoxidase(HRP),areaddedformingcomplexeswiththeβ2GPlboundantibodies.Followinganotherwashingstep,theboundenzyme-antibodyconjugateisassayedbytheadditionofasinglesolutioncontainingtetramethylbenzidine(TMB)andhydrogenperoxide(H2O2)asthechromogenicsubstrate.Colordevelopsinthewellsatanintensityproportionaltotheserumconcentrationofanti-β2GPlantibodies.
ResultsareobtainedbyreadingtheO.D.(opticaldensityorabsorbance)ofeachwellinaspectrophotometer.Calibratorseraareprovided,withtheIgGanti-β2GPlantibodyconcentrationsexpressedinGunits.Theuserhastheoptionofrunningeitherasinglepointcalibratororafour-pointcalibrationcurve.Forsinglepointcalibration,dividingtheconcentrationvalueofthecalibratorserabytheO.D.valueofthecalibratorprovidesaconversionfactor.TheO.D.valuesoftheothersamplesaremultipliedbytheconversionfactortoobtainIgGanti-β2GPlantibodyconcentrationsinGunits.Formultipointcalibration,performalinearregressionanalysiswithcalibratorvaluesagainstcalibratorO.D.s.Controlsandpatientresultsaredeterminedfromthecalibrationcurve.Theseunitsaretraceabletoavailablereferencepreparations.
AssayProcedure:
- Theassaycanbeperformedwithasinglepointcalibration(Calibrator2)orafour-pointcalibrationcurve(Calibrators1,2,and3plussamplediluent/reagentblankasCalibrator4equalto0Gunits).Areagentblankcontrolshouldalsoberunwithboththesinglepointandmultipointcalibrationmethods.SampleDiluentwithoutserumorplasmaisaddedtothewell.Thiswellwillbetreatedthesameasacontrolorpatientsampleinsubsequentassaysteps.
- Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebagprovided.
- Preparea1:50dilutionofthecalibrators,controls,andpatientsamplesinsamplediluent(blue-greensolution);e.g.,10μlsampleaddedto490μlSampleDiluentequalsa1:50sampledilution.
- Add100μlofdilutedcalibrators(includingthereagentblank/Calibrator4),controls,andpatientsamplestotheappropriatemicrowells.
- Incubate15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptythesamplefluid.Donotallowsamplestocontaminateothermicrowells.
- Wash4timeswithwashsolution.Eachwellshouldbefilledwithwashsolutionperwash.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnappingmotionofthewristtoshaketheliquidfromthewells.Toretainmicrowellmodulesduringwashing,theframemustbesqueezedatthetopandbottomofthelongersides.Blotonabsorbentpapertoremoveresidualwashfluid.Donotallowwellstodryoutbetweensteps.
- Add100μlanti-humanIgGHRP-ConjugatedAntibodySolution(blue)tothewells.
- Incubatefor15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptytheconjugatesolution.
- Wash4timeswithwashsolution,asinstep6.Useasnappingmotiontodraintheliquidandblotonabsorbenttowelsafterthefinalwash.Donotallowthewellstodryout.
- Add100μlOne-ComponentSubstratetoeachwellandincubatefor10minutesatroomtemperature.Addsubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwellswithpositivesamples.
- 11.Add100μlStoppingSolution(0.36Nsulfuricacid)toeachwelltostoptheenzymereaction.BesuretoaddtheacidtothewellsinthesameorderandatthesamerateastheSubstratewasadded.BlueSubstratewillturnyellowandcolorlesssolutionwillremaincolorless.Blankorzerotheplatereaderagainstanairorawaterblankwell.ReadtheO.D.ofeachwellat450nm(and650nmreferenceifdualbeam).TheO.D.valuesshouldbemeasuredwithin5minutesoftheadditionoftheStoppingSolution.
ClinicalProcedure:
ClinicalSpecificity:serumsamplesfrommultiplehealthyblooddonorpopulationswereassayedforthepresenceofIgG,IgM,IgAanti-B2GPIantibodies.Specificitywasshowntobe100%,93%,and96%,respectivelyforthethreeisotypes.
ClinicalSensitivity:anunselectedSLEpopulationwastestedtodeterminetheclinicalsensitivityoftheanti-B2GPIassays.
Sensitivitywas23%forIgG,20%forIgMand25%forIgA.Theclinicalsensitivityoftheassayforthrombosiswasdeterminedbycomparinganti-B2GPItestresultsfromtwogroupsofselectedSLEpatients:Group1,withaclinicalhistoryofthrombosisand/orthrombocytopenia;andGroup2,withnohistoryofthrombosis(control).Theresultsareshownbelow:
| Group1:SLE+Thrombosisand/orthrombocytopenia | |||
| IgG | IgM | IgA | |
| AverageValue | 69Gunits | 24Munits | 106Aunits |
| %positive | 58% | 42% | 67% |
| Group2:SLEcontrol | |||
| IgG | IgM | IgA | |
| AverageValue | 9Gunits | 9Munits | 22Aunits |
| %positive | 20% | 11% | 11% |
Background:
Anti-phospholipidantibodiesareaheterogeneousgroupofimmunoglobulinsthatbindtoseveralanionicphospholipids,includingcardiolipinandphosphatidylserine.1,2Highserumlevelsofanti-phospholipidantibodiesarefrequentlydetectedinpatientswithautoimmune(e.g.,SLE)andnon-autoimmunediseases,aswellasinapparentlyhealthyindividuals.Theseantibodieshavebeenassociatedwithanincreasedriskforrecurrentarterialandvenousthromboticevents,thrombocytopenia,andfetalloss.Thesemanifestationsarethemainfeaturesoftheanti-phospholipidsyndrome(APS).
Mostautoimmuneanti-phospholipidantibodiesrequireaserumcofactor(β2GPl)foroptimalbinding.Ithasbeenshownthatmanyanti-phospholipidantibodiesmayreacttoaneoepitopeformedontheβ2GPlmoleculebytheinteractionbetweenthephospholipidandβ2GPl.Mostassaysforantiphospholipidantibodiescontainbovineserumasthesourceofcofactor.Morerecently,ithasbeenshownthatthebindingofβ2GPltothemicrowellsurfacemayproduceaneoepitopesimilartothatwhencombinedwithaphospholipidandtheresultswiththissystemshowedagoodcorrelationwiththeanti-phospholipidsyndrome.TheSEROlogicdetectionofanti-β2GPlantibodiesprovidesenhancedclinicalsensitivityforthrombosis.TheREAADSAnti-β2GPlELISATestKitusesthewellknownELISAformattodetectanti-β2GPlantibodiesinhumanserum.
Patientswithpositivereactionstobothanti-phospholipidandanti-β2GPlassaysweremorelikelytohaveclinicalcomplicationsthanthosepositiveforonlyone.HigherprevalenceandmeanserumlevelsofIgGanti-β2GPlantibodieshavebeenreportedinautoimmunepatients.Inaddition,anti-β2GPlantibodiesinSLEpatientscorrelatedwithclinicalmanifestationsofanti-phospholipidsyndrome.
