Description:

TheTECO®REACHMedakaVitellogeninELISA kitisasensitiveenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserum,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: 2.6-210ng/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-apartwellstripscoatedwithIgGdirectedagainstMedaka VTG)
• Non-SpecificBinding(NSB) Wells: 1break-apartstripof8wells coatedwithoutspecificbindingantibodies
• StandardStockSolution:105 ng,2vials
• ControlC1: lowcontrol,2vials
• ControlC2: highcontrol,2vials
• Inter-AssayReferenceStandard:15.0ng,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).
•ELISAplateshaker(500rpm)

Formucussamples:TheMucusCollectionSet(TE1034),whichincludesExtractionBufferandvalidatedSamplingSwabsis alsorequired.

AssayPrinciple:

AssayPrinciple

TheTECO®REACHMedakaVitellogeninELISAkitisa96-well immuno-captureELISAproduct.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibody-coatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopment
isdirectlyproportionaltotheamountofvitellogenininthesample.

ResultAnalysis

Thestandardrangeoftheassayisbetween0and210ng/ml.Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationsinsamples canthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationcan beobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.Adilutioncorrectionformucusisnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbetestedagain,pre-dilutedwithDilutionBuffer, andthenthis additionaldilutionshould betakeninaccountfortheconcentrationcalculation.

TECO®REACHMedakaVTG ELISAStandardCurveExample

Species:

• Medaka,Japanesericefish(Oryziaslatipes)

Background:

Inoviparousanimals,vitellogenin(VTG)isanestrogen-inducedyolkprecursorproteinmainlysynthesizedinthelivertobedepositedinthematuringoocytes,whereitissplitintheyolkproteinslipovitellin1,lipovitellin2andphosvitin.Theseyolkproteinsserveasnourishmentstorageforthedevelopingembryos.Non-physiologicalinductionofvitellogenininmalesorinjuvenilefishisthoughttoindicateanestrogenmediatedendocrinedisruption.Therefore,VTGdeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidelinesforthetestingofchemicalsforestrogenicactivity.Normally,vitellogeninismeasuredinbloodsamplesorwholebodyhomogenate(WBH)–bothsampletypesrequireinvasiveanddestructivetreatmentofthefish.Bloodcanbe difficulttocollect,inparticularwhereverysmallfishareconcernedand wheretheanimalsmustsurvivesampling.

Recently,severalcelltypeshavebeenshowntoproduceVTGafterestrogenstimulation,includingthoseoftheepidermalmucosa.EventhoughtheVTGconcentrationintheskinmucusisanorderofmagnitudelowerthaninblood,serumorbodyhomogenates(containinglivertissue),theskinmucosaisverywellsuitedasamatrixfor determiningexogenousVTGinductioncausedbyenvironmentalchemicalswithaffinitytoestrogenreceptors.ByusingasensitiveELISAincombinationwithauniquesamplingandextractionsystemthedeterminationofmucosa-bornVTGdeterminationhasthefollowingadvantages:

• Simpleandhighlystandardizedsamplingtechniqueandsamplepreparation.
• Strictlydefinedmatrixwithoutproteasecontaminationcausedbynon-targettissuesorlymphaticfluid.
• Non-destructive,therebyallowingseveralsubsequentsamplingsinordertorecordakineticofVTGinduction.Amaximumisknowntoappearafter7daysofexposure.Therefore,MucosatestsarefullycompatIBLewithacuteaswellaschronicalOECDtestmethods.
• Epithelialorganizedepidermisisdirectlyexposedtoexogenousestrogensandtherebyallowingadirectcomparisonwithinvitrotestusingestrogensensitivevitellogeninproducingfishcelllines.
• LowerdegreeofinterferencewithendogenousVTGproduction(infemales)andbioconcentrationorenterohepaticcirculationoftheeffectiveestrogen(xenoestrogen)andtherebyshowingacleardoseresponserelationship.
• StABIlityofstandardsandsamplesifprescribedstorageconditionsareobserved.

TheTECO®REACHMedakaVitellogeninELISA kitallowsfortestingsamples ofblood,homogenateand/ormucus.Thesesampletypesprovideresearcherswithgoodsampling optionsespeciallywhen conductingtime-coursestudiesorfieldresearchwhereitiscriticaltopreservethelifeofthefish.