Description:

TheREAADSAnti-Beta2GlycoproteinI(Aβ₂GPI)IgMtestkitisan indirectenzyme-linkedimmunosorbentassay(ELISA)forthesemi-quantitativedeterminationofIgManti-β₂GP1antibodiesinhumanserumorcitratedplasma(3.2%sodiumcitrate).AsinglepointormultipointcalibratorisusedtomeasureIgManti-β₂GP1antibodyconcentrationsinMunits. ForthedetectionofIgManti-β₂GPlantibodiesinindividualswithsystemiclupuserythematosus(SLE)andlupus-likedisorders(anti-phospholipidsyndrome).

Advantages:

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

KitComposition:

Reagents

• 12x8stABIlizedβ₂GPl(fromhumanserum)coatedmicrowellswithframe
• 60mlSampleDiluentIV(blue-greensolution)
• 3vials(0.250ml)IgMβ₂GPlCalibratorSerum*(1-high,2-moderate,3-low)(human);seeviallabelforantibodyconcentrationinMunits.Calibrator2shouldbeusedwhenperformingsinglepointcalibration
• 0.250mlIgMβ₂GPlPositiveControlSerum*(human);seeviallabelforexpectedMunitrange
• 0.250mlNormalControlSerum*(human);seeviallabelforexpectedMunitrange
• 15mlanti-humanIgM(goat)HRP-ConjugatedAntibodySolution(redsolution)
• 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:

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

ResultsareobtainedbyreadingtheO.D.(opticaldensityorabsorbance)ofeachwellinaspectrophotometer.Calibratorseraareprovided,withtheIgManti-β₂GPlantibodyconcentrationsexpressedinMunits.Theuserhastheoptionofrunningeitherasinglepointcalibratororafour-pointcalibrationcurve.Forsinglepointcalibration,dividingtheconcentrationvalueofthecalibratorserabytheO.D.valueofthecalibratorprovidesaconversionfactor.TheO.D.valuesoftheothersamplesaremultipliedbytheconversionfactortoobtainIgManti-β₂GPlantibodyconcentrationsinMunits.Formultipointcalibration,performalinearregressionanalysiswithcalibratorvaluesagainstcalibratorO.D.s.Controlsandpatientresultsaredeterminedfromthecalibrationcurve.

AssayProcedure:

1. Theassaycanbeperformedwithasinglepointcalibration(Calibrator2)orafour-pointcalibrationcurve(Calibrators1,2,and3plussamplediluent/reagentblankasCalibrator4equalto0Munits).Areagentblankcontrolshouldalsoberunwiththesinglepointandmultipointcalibrationmethod.SampleDiluentwithoutserumorplasmaisaddedtothewell.Thiswellwillbetreatedthesameasacontrolorpatientsampleinsubsequentassaysteps.
2. Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebagprovided.
3. Preparea1:50dilutionofthecalibrators,controls,andpatientsamplesinsamplediluent(blue-greensolution);e.g.,10µlsampleaddedto490µl SampleDiluentequalsa1:50sampledilution.
4. Add100µlofdilutedcalibrators(includingthereagentblank/Calibrator4),controls,andpatientsamplestotheappropriatemicrowells.
5. Incubate15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptythesamplefluid.Donotallowsamplestocontaminateothermicrowells.
6. Wash4timeswithwashsolution.Eachwellshouldbefilledwithwashsolutionperwash.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnappingmotionofthewristtoshaketheliquidfromthewells.Toretainmicrowellmodulesduringwashing,theframemustbesqueezedatthetopandbottomofthelongestsides.Blotonabsorbentpapertoremoveresidualwashfluid.Donotallowwellstodryoutbetweensteps.
7. Add100µlanti-humanIgMHRP-ConjugatedAntibodySolution(red)tothewells.
8. Incubatefor15minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthemicrowellsandemptytheconjugatesolution.
9. Wash4timeswithwashsolution,asinstep6.Useasnappingmotiontodraintheliquidandblotonabsorbenttowelsafterthefinalwash.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:

Group1:SLE+Thrombosisand/orthrombocytopenia	IgG	IgM	IgA
AverageValue	69Gunits	24Munits	106Aunits
%positive	58%	42%	67%
Group2:SLEcontrol
AverageValue	9Gunits	9Munits	22Aunits
%positive	20%	11%	11%

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.HigherprevalenceandmeanserumlevelsofIgManti-β₂GPlantibodieshavebeenreportedinautoimmunepatients.Inaddition,anti-β₂GPlantibodiesinSLEpatientscorrelatedwithclinicalmanifestationsofanti-phospholipidsyndrome.

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