| Kitsize | 12x8 |
| Method | ELISA |
| Incubationtime | 18h,1h,15min |
| Standardrange | 3-180pg/mL |
| Specimen/Volumes | 500µLSerum |
| Substrate/isotope | TMB450nm |
| RegulatoryStatus: | EU:CE |
TheLC-MS/MSisourreference
ThequantificationofthephysiologicallyactiveVitaminDmetabolite1,25-dihydroxyVitaminDisanecessarydiagnostictoolforseveralpathologicalconditionssuchas:
- Kidneydiseases
- Kidneytransplantation
- Sarcoidosis
- Rickets
- Osteomalacia
Thequalityofthemeasuringmethod,whichourassayfulfillsahundredpercent,thereforeisindispensable.
Thelevelsof1,25-dihydroxyVitaminDinplasmaorserumis100to1000-foldlessthanthatof25-hydroxyVitaminD.Duetoitslowconcentrationsandthepresenceofmanysimilarmetabolites,theexactmeasurementof1,25-dihydroxyvitaminDstillrequirestodoanextractionandseparationstepwithorganicsolventsofthesampleprioranymeasurementsystem.Thisextractionwithorganicsolventsstilltodayisthegoldstandardextractionmethod.
"Thesuccessiveabandonmentofsampleextraction,chromatography,[…]inimmunoassayshasundoubtedlycontributedtotheinconsistenciesreportedbyFarrelletal,[…]."(GrahamCarter,DEQASorganizer,CarterGD.ClinChem.2012;58(3):486-8.)
Theextractionsuppressesthepotentialinterferencefromsamplematrix,heterophilicantibodiesandabnormalconcentrationsofVitaminDbindingproteins.AndtheadditionalseparationonsolidphasecartridgesfurthersuppressestheinterferencefromotherVitaminDmetabolitessuchas25-hydroxyVitaminD,24,25-dihydroxyVitaminD,3-epi-25-hydroxyVitaminDand25,26-dihydroxyVitaminD.
ThisleadstogoodcorrelationofourELISAwiththegoldstandardtechnologyLC-MS/MS.
FurThermoretheELISAwasalsocorrelatedtothestillwidelyusedrADIoimmunoassayfromDiasorin.AlsoheretheELISAshowedagoodfit.
KeyfeaturesofourELISAare:
- GoodcorrelationwithLC-MS/MS
- VerylowLimitofDetection(1.7pg/mL)
- Limitednumberofincubationsteps
- DEQASsamplesaccuratelymeasured
- 108%cross-reactivityagainst1,25(OH)2VitaminD2
- GoldStandardextractionprotocolensuringnointerference
Generalinformation
1,25(OH)2VitaminDistheactiveformofVitaminDwithregardtotheknownfunctionswhereas25OHVitaminDandVitaminDitselfcanbeexcludedasbeingphysiologicallyfunctional.1,25(OH)2VitaminDstimulatestheintestinalabsorptionofbothcalciumandphosphorus.Italsostimulatesboneresorptionandmineralizationtherebypreventingthedevelopmentofricketsandosteomalacia.1,25(OH)2VitaminDisalsoactiveinothertissuesresponsibleforCalciumtransport(placenta,kidney,mammarygland,...)andendocrineglandssuchasparathyroidglands.1,25(OH)2VitaminDisrapidlymetabolizedanditshalflifeinplasmaisapproximately12h.Itsisfurthermetabolizedtoeithercalcitroicacid,essentiallywithoutanyBIOLOGicalactivity,orvia24-hydroxylationto1,24,25-trihydroxyvitaminD.Thiscompoundhaslessbiologicalactivitythanitsparentandthismetabolicrouteisconsideredasaminorpathway.Thelevelsof1,25(OH)2VitaminDinplasmaorserumis100-to1000-foldlessthanthatof25OHVitaminD.Duetoitslowconcentrationsandthepresenceofmanysimilarmetabolites,themeasurementof1,25(OH)2VitaminDrequiresextractionandseparationbychromatography.
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