Description:

TheTECO®REACHPerch(Perciformes)VitellogeninELISAKitisasensitivesandwichenzymelinkedimmunosorbentassayforthedeterminationofvitellogenininserum,wholebodyhomgenate(WBH) andmucusofperciformesaccordingtoECregulationNr.440/2008(REACH)fromJuly10th2015/DocumentD039048/03.

VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:

•  OECD(2009),TestNo.229
•  OECD(2009),TestNo.230
•  OECD(2011),TestNo.234

REACHkitsmeetstrictqualityassuranceregulationsbyincludingadditionalcomponentsinthekit:

• 1 break-apartstripof8wellsforNon-SpecificBinding(NSB).
• Anadditionalinter-assayreferenceStandardStockofadifferentvitellogeninELISAproductionlot.
• Apackageinsertthatincludes thespecificrequirementsforvitellogenintestingwithinREACHregulations.

Vitellogenindeterminationisusedinecotoxicologicalstudiestodeterminetheeffectsofestrogeniccompoundsinwater.

Range:1-80ng/ml

Sensitivity: <1.0ng/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

StimulationStudiesusingestrADIol(E2),Ethinyl-Estradiol(EE2)andBisphenolAshowclearincreasesinVitellogeninlevelsinserum,WBHandmucus.

KitComposition:

KitContents:

• AntibodyCoatedMicroassayPlate: 96-wellplate (12×8break-apartwellstripscoatedwithIgGdirectedagainstPerciformesVTG)
• Non-SpecificBinding(NSB) Wells: 1break-apartstripof8wells coatedwithoutspecificbindingantibodies
• StandardStockSolution:160ng,1vial
• ControlC1:  lowcontrol,1vial
• ControlC2: high control,1vial
• Inter-AssayReferenceStandard:30 ng,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®REACHPerch VitellogeninELISAkitisa96wellimmuno-captureELISAproductusinghomologueantibodiesandhomologueVTGstandardmaterial.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.

ResultAnalysis

Thestandardrangeoftheassayisbetween0and80ng/ml.Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationsinthesample canthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationcanbeobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.A dilutioncorrectionformucus isnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbetestedagain,pre-dilutedwithDilutionBuffer, andthentheadditionaldilutionshould betakeninaccountfortheconcentrationcalculation.

TECO®REACHCyprinidVTG ELISAStandardCurveExample

Species:

• Europeanperch/perch/redfinperch/Englishperch(Percafluviatilis)
• Tilapia(Oreochromisniloticus)
• Bluegill(Lepomismacrochirus)
• Ruffe(Gymnocephaluscernua)
• Three-spinedstickleback(Gasterosteusaculeatus)

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®REACHPerch VitellogeninELISAkitallowsforsamplingofblood,homogenateand/ormucuswhichprovides researcherswithsampling optionswhen conductingtime-coursestudiesorfieldresearchwhereitiscriticaltopreservethelifeofthefish.

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