Description:

Advanatages:

• ForthespecificdeterminationofIgAanti-β₂GP1antibodies
• Excellentclinicalcorrelation
• Colorcodedreagents
• Totalincubationtime:40minutes
• Convenient,costeffective
• Choiceofsingleormulti-pointcalibration

KitComposition:

Reagents

• 12x8stABIlizedβ₂GPl(fromhumanserum)coatedmicrowellswithframe
• 60ml SampleDiluentIV(blue-greensolution)
• 3vials(0.250mL)IgAβ₂GPlCalibratorSerum*(1-high,2-moderate,3-low)(human);seeviallabelforantibodyconcentrationinAunits.Calibrator2shouldbeusedwhenperformingsinglepointcalibration
• 0.250mlIgAβ₂GPlPositiveControlSerum*(human);seeviallabelforexpectedAunitrange
• 0.250mlNormalControlSerum*(human);seeviallabelforexpectedAunitrange
• 15mlanti-humanIgA(goat)HRP-ConjugatedAntibodySolution(orangesolution)
• 15mlOneComponentSubstrateSolution(TMBandH₂O₂);readytouse
• 15mlStoppingSolution(0.36Nsulfuricacid)
• 2bottles(30ml)WashConcentrate(33XPBS/Tween)

Storeat2–8°C.DoNotFreeze.

MaterialsRequiredbutnotincluded

• ReagentgradewatertopreparePBSwashsolutionandtozeroorblanktheplatereaderduringthefinalassaystep
• Graduatedcylinders
• Precisionpipettorscapableofdeliveringbetween5µland1000µl,withappropriatetips
• Miscellaneousglasswareappropriateforhandlingsmallvolumes
• Flasksorbottles,1liter
• Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
• Disposablegloves
• Plate-reADIngspectrophotometercapableofreadingabsorbanceat450nm(650nmreferenceifdualbeam)
• Multichannelpipettorscapableofdeliveringto8wellssimultaneously
• Microdilutiontubesanda96-wellmicrodilutiontubeholderforsampledilutionsandrapiddeliverytomicrowellplate

MeasurementPrinciple:

TheREAADSIgAanti-β₂GPITestKitsareindirectenzymelinkedimmunosorbentassaysforthesemiquantitativedeterminationofanti-β₂GP1antibodiesinhumanserum.AsinglepointormultipointcalibratorisusedtomeasureIgAanti-β₂GP1antibodyconcentrationsinAunits.

ThetestisperformedasanindirectELISA.Dilutedserumorplasmasamples,calibratorsera,andcontrolsareincubatedinmicrowellscoatedwithpurifiedhumanβ₂GPl.Incubationallowstheanti-β₂GPlantibodiespresentinthesamplestoreactwiththeimmobilizedantigen.Aftertheremovalofunboundserumorplasmaproteinsbywashing,antibodiesspecificforhumanIgA,labeledwithhorseradishperoxidase(HRP),areaddedformingcomplexeswiththeβ₂GPlboundantibodies.Followinganotherwashingstep,theboundenzyme-antibodyconjugateisassayedbytheadditionofasinglesolutioncontainingtetramethylbenzidine(TMB)andhydrogenperoxide(H₂O₂)asthechromogenicsubstrate.Colordevelopsinthewellsatanintensityproportionaltotheserumconcentrationofanti-β₂GPlantibodies.

ResultsareobtainedbyreadingtheO.D.(opticaldensityorabsorbance)ofeachwellinaspectrophotometer.Calibratorseraareprovided,withtheIgAanti-β₂GPlantibodyconcentrationsexpressedinAunits.Theuserhastheoptionofrunningeitherasinglepointcalibratororafour-pointcalibrationcurve.Forsinglepointcalibration,dividingtheconcentrationvalueofthecalibratorserabytheO.D.valueofthecalibratorprovidesaconversionfactor.TheO.D.valuesoftheothersamplesaremultipliedbytheconversionfactortoobtainIgAanti-β₂GPlantibodyconcentrationsinAunits.Formultipointcalibration,performalinearregressionanalysiswithcalibratorvaluesagainstcalibratorO.D.s.Controlsandpatientresultsaredeterminedfromthecalibrationcurve.

AssayProcedure:

1. Theassaycanbeperformedwithasinglepointcalibration(Calibrator2)orafour-pointcalibrationcurve(Calibrators1,2,and3plussamplediluent/reagentblankasCalibrator4equalto0Aunits).Areagentblankcontrolshouldalsoberunwiththesinglepointandmultipointcalibrationmethod.SampleDiluentwithoutserumorplasmaisaddedtothewell.Thiswellwillbetreatedthesameasacontrolorpatientsampleinsubsequentassaysteps.
2. Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebagprovided.
3. Preparea1:50dilutionofthecalibrators,controls,andpatientsamplesinsamplediluent(blue-greensolution);e.g.,10µlsampleaddedto490µlSampleDiluentequalsa1:50sampledilution.
4. Add100µlofdilutedcalibrators(includingthereagentblank/Calibrator4),controls,andpatientsamplestotheappropriatemicrowells.
5. Incubate15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptythesamplefluid.Donotallowsamplestocontaminateothermicrowells.
6. Wash4timeswithwashsolution.Eachwellshouldbefilledwithwashsolutionperwash.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnappingmotionofthewristtoshaketheliquidfromthewells.Toretainmicrowellmodulesduringwashing,theframemustbesqueezedatthetopandbottomofthelongestsides.Blotonabsorbentpapertoremoveresidualwashfluid.Donotallowwellstodryoutbetweensteps.
7. Add100µlanti-humanIgAHRP-ConjugatedAntibodySolution(orange)tothewells.
8. Incubatefor15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptytheconjugatesolution.
9. Wash4timeswithwashsolution,asinstep 6.Useasnappingmotiontodraintheliquid,andblotonabsorbenttowelsafterthefinalwash.Donotallowthewellstodryout.
10. Add100µlOneComponentSubstratetoeachwellandincubatefor10minutesatroomtemperature.Addsubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwellswithpositivesamples.
11. Add100µlStoppingSolution(0.36Nsulfuricacid)toeachwelltostoptheenzymereaction.BesuretoaddtheacidtothewellsinthesameorderandatthesamerateastheSubstratewasadded.BlueSubstratewillturnyellowandcolorlesssolutionwillremaincolorless.Blankorzerotheplatereaderagainstanairorawaterblankwell.ReadtheO.D.ofeachwellat450nm(and650nmreferenceifdualbeam).TheO.D.valuesshouldbemeasuredwithin5minutesoftheadditionoftheStoppingSolution.

ClinicalPerformance:

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:

Background:

Anti-phospholipidantibodiesareaheterogeneousgroupofimmunoglobulinsthatbindtoseveralanionicphospholipids,includingcardiolipinandphosphatidylserine.Highserumlevelsofanti-phospholipidantibodiesarefrequentlydetectedinpatientswithautoimmune(e.g.,SLE)andnon-autoimmunediseases,aswellasinapparentlyhealthyindividuals.Theseantibodieshavebeenassociatedwithanincreasedriskforrecurrentarterialandvenousthromboticevents,thrombocytopenia,andfetalloss.Thesemanifestationsarethemainfeaturesoftheanti-phospholipidsyndrome(APS).

Mostautoimmuneanti-phospholipidantibodiesrequireaserumcofactor(β₂GPl)foroptimalbinding.Ithasbeenshownthatmanyanti-phospholipidantibodiesmayreacttoaneoepitopeformedontheβ₂GPlmoleculebytheinteractionbetweenthephospholipidandβ₂GPl. Mostassaysforanti-phospholipidantibodiescontainbovineserumasthesourceofcofactor.Morerecently,ithasbeenshownthatthebindingofβ₂GPltothemicrowellsurfacemayproduceaneoepitopesimilartothatwhencombinedwithaphospholipidandtheresultswiththissystemshowedagoodcorrelationwiththeanti-phospholipidsyndrome.TheSEROlogicdetectionofanti-β₂GPlantibodiesprovidesenhancedclinicalsensitivityforthrombosis.TheREAADSAnti-β₂GPlELISAtestkitusesthewellknownELISAformattodetectanti-β₂GPlantibodiesinhumanserum.

Patientswithpositivereactionstobothanti-phospholipidandanti-β₂GPlassaysweremorelikelytohaveclinicalcomplicationsthanthosepositiveforonlyone.HigherprevalenceandmeanserumlevelsofIgAanti-β₂GPlantibodieshavebeenreportedinautoimmunepatients.Inaddition,anti-β₂GPlantibodiesinSLEpatientscorrelatedwithclinicalmanifestationsofanti-phospholipidsyndrome.