Description:
TheTECO®REACHCyprinidVitellogeninELISA kitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserum,wholebodyhomgenate(WBH) andmucusofcyprinidsaccordingtoECregulationNr.440/2008(REACH)fromJuly10th2015/DocumentD039048/03.
VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:
- OECD(2009),TestNo.229
- OECD(2009),TestNo.230
- OECD(2011),TestNo.234
REACHkitsmeetstrictqualityassuranceregulationsbyincluding,inadditiontothestandardELISAmaterials,thefollowing componentsinthekit:
- 1 break-apartstripof8wellsforNon-SpecificBinding(NSB)assessment.
- Anextra inter-assayreferenceStandardStockfrom adifferentvitellogeninELISAproductionlot.
- Apackageinsertthatincludes thespecificrequirementsforvitellogenintestingwithinREACHregulations.
Vitellogenindeterminationisusefulinecotoxicologicalstudiestodeterminethepresenceoreffectofestrogeniccompoundsinthewater.
AssayMeasuringRange: 0-70ng/ml
AssaySensitivity: <0.1ng/ml
Incubationtime: 4.0hours
SampleVolume: 50µl
SamplePreparation:
- Serum:Storefreshserumsamplesimmediatelyaftercollectionat -20°Corloweruntilassayed.
- WBH:StorefreshWBHsamplesimmediatelyaftercollectionat -20°Corloweruntilassayed.
- Mucus: CollectasdescribedintheTECO®MucusCollectionSet(TE1034).Mucuscontainingswabscanbestoredseveralmonthsat<-20°C.
ReferenceValues:
- Serumlevelsareintherangeofµg/mluptomg/ml
- WBHlevelsareintherangeofmg/ml
- Mucuslevelareintherangeofng/ml
KitComposition:
KitContents:
- AntibodyCoatedMicroassayPlate: 96-wellplate (12×8break-apartwellstripscoatedwithIgGdirectedagainstCyprinidVTG)
- Non-SpecificBinding(NSB) Wells: 1break-apartstripof8wells coatedwithoutspecificbindingantibodies
- StandardStockSolution:35ng,2vials
- ControlC1: lowcontrol,2vials
- ControlC2: middlecontrol,2vials
- ControlC3:highcontrol,2vials
- Inter-AssayReferenceStandard: 5.0ng,1vial
- WashBuffer: 1x30ml,50X (Dilute1:50withdeionizedwater.)
- DilutionBuffer:1x55ml.Readytouse.
- MatrixSolution:1x7ml.Readytouse.
- BiotinylatedAntibody(Biotin-AB):1x12ml.Readytouse.
- StreptavidinPeroxidaseConjugate(SA-HRPConj.):1x12ml. Readytouse.
- TMBSubstrate:1x12ml. Readytouse.
- StopSolution: 1x12ml 1MHCl.Readytouse.
Materialsrequiredbut notsupplied:
•Pipettes,10μl–1000μl
•Multichannelpipettes,50μl–100μl
•Graduatedcylindersforreconstitutingand dilutingreagents
•ManualAspirationSystemorautomaticwasherforELISAplates
•Distilledwater
•Vortexmixer
•ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(Reference:590-650nm).
Forextendedstandardrange:ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat 405nmand450nm(Reference:590-650nm)
•ELISAplateshaker(500rpm)
Formucussamples:TheMucusCollectionSet(TE1034)whichincludesExtractionBufferandvalidatedSamplingSwabsis alsorequired.
AssayPrinciple:
AssayPrinciple
TheTECO®REACHCyprinidVitellogeninELISAkitisa96wellimmuno-captureELISAproductusinghomologueantibodiesandhomologueVTGstandardmaterial.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.
ResultAnalysis
Thestandardrangeoftheassayisbetween0and35ng/ml.However, toavoidadditionalsampledilution,thiskitprovidesanoptionalstandardrangeextensionupto70ng/ml. Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationsinsamples canthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationcan beobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.Adilutioncorrectionformucusisnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbetestedagain, pre-dilutedwithDilutionBuffer, andthenthis additionaldilutionshould betakeninaccountfortheconcentrationcalculation.
TECO®REACHCyprinidVTG ELISAStandardCurveExample
Species:
- Carp(Caprinuscarpio)
- Goldfish(Carassiusauratus)
- Zebrafish(Daniorerio)
- FatheadMinnow(Pimephalespromelas)
Background:
Inoviparousanimals,vitellogenin(VTG)isanestrogen-inducedyolkprecursorproteinmainlysynthesizedinthelivertobedepositedinthematuringoocytes,whereitissplitintheyolkproteinslipovitellin1,lipovitellin2andphosvitin.Theseyolkproteinsserveasnourishmentstorageforthedevelopingembryos.Non-physiologicalinductionofvitellogenininmalesorinjuvenilefishisthoughttoindicateanestrogenmediatedendocrinedisruption.ThereforeVTGdeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidelinesforthetestingofchemicalsforestrogenicactivity.Normally,vitellogeninismeasuredinbloodsamplesorwholebodyhomogenate(WBH)–bothsampletypesrequireinvasiveanddestructivetreatmentofthefish.Bloodcanbe difficulttocollect,inparticularwhereverysmallfishareconcernedand wheretheanimalsmustsurvivesampling.
Recently,severalcelltypeshavebeenshowntoproduceVTGafterestrogenstimulation,includingthoseoftheepidermalmucosa.EventhoughtheVTGconcentrationintheskinmucusisanorderofmagnitudelowerthaninbloodserumorinbodyhomogenates(containinglivertissue),theskinmucosaisverywellsuitedasamatrixfor determiningexogenousVTGinductioncausedbyenvironmentalchemicalswithaffinitytoestrogenreceptors.ByusingahighlysensitiveELISAincombinationwithauniquesamplingandextractionsystemthedeterminationofmucosa-bornVTGdeterminationhasthefollowingadvantages:
- Simpleandhighlystandardizedsamplingtechniqueandsamplepreparation.
- Strictlydefinedmatrixwithoutproteasecontaminationcausedbynon-targettissuesorlymphaticfluid.
- Non-destructiveandtherebyallowingseveralsubsequentsamplingsinordertorecordakineticofVTGinductionwithamaximumknowntoappearafter7daysofexposure.ThereforMucosatestarefullycompatIBLewithacuteaswellaschronicalOECDtestmethods.
- Epithelialorganizedepidermisisdirectlyexposedtoexogenousestrogensandtherebyallowingadirectcomparisonwithinvitrotestusingestrogensensitivevitellogeninproducingfishcelllines.
- LowerdegreeofinterferencewithendogenousVTGproduction(infemales)andbioconcentrationorenterohepaticcirculationoftheeffectiveestrogen(xenoestrogen)andtherebyshowingacleardoseresponserelationship.
- StABIlityofstandardsandsamplesifprescribedstorageconditionsareobserved.
TheTECO®REACHCyprinidVitellogeninELISAkitallowsforsamplingofblood,homogenateand/ormucuswhichprovides researcherswithsampling optionswhen conductingtime-coursestudiesorfieldresearchwhereitiscriticaltopreservethelifeofthefish.
