Description:

TheTECO®UltraSensitiveCyprinidVitellogeninELISA kitisaverysensitiveenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenin(VTG)incyprinidfishmucusorcellculture*.

VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:

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

Vitellogenindeterminationisusedinecotoxicologicalstudiestodeterminetheeffectsofestrogeniccompoundsinthewater.

AssayMeasuringRange: 0-2.0ng/ml

AssaySensitivity: <0.03ng/ml

Incubationtime:Overnight16-24hours/4h±10min

SampleVolume: 50µl

SamplePreparation:

• Mucus: CollectasdescribedintheTECO®MucusCollectionSet(TE1034).Mucuscontainingswabscanbestoredseveralmonthsat<-20°C.
• Cellculture:Storefreshsamplesimmediatelyaftercollectionat<-20°Cuntilassayed.

ReferenceValues:

• Mucuslevelsareintherangeofng/ml.

*Additionalassayvalidationmayberequiredwiththeuseofcellculturesamples.

KitComposition:

KitContents:

• AntibodyCoatedMicroassayPlate: 96-wellplate (12×8break-apartwellstripscoatedwithIgGdirectedagainstCyprinidVTG)
• StandardStockSolution:35ng,2vials
• ControlC1:  lowcontrol,2vials
• ControlC2:high control,2vials
• 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)
•Softwarepackagefordatagenerationandanalysis

Formucussamples:TheMucusCollectionSet(TE1034)whichincludesExtractionBufferandvalidatedSamplingSwabsis alsorequired.

AssayPrinciple:

AssayPrinciple

TheTECO®UltraSensitiveCyprinidVitellogeninELISA kitisa96wellimmuno-captureELISAproduct.Samplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.

ResultAnalysis

Thestandardrangeoftheassayisbetween0.025and2.0ng/ml.Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationscanthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationwillbeobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.Notethatthedilutioncorrectionformucusisnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbere-testedafterdilutingwithDilutionBufferandthenthis additionaldilutionshould betakeninaccountfortheconcentrationcalculation.

TECO®UltraSensitiveCyprinidVTGELISAStandardCurveExample

Species:

• Carp(Cyprinuscarpio)
• Goldfish(Carassiusgibelioauratus)
• Zebrafish(Daniorerio)
• Fatheadminnow(Pimephalespromelas)
• Commonbream/freshwaterbream/bronzebream/carpbream(Abramisbrama)
• Roach(Rutilusrutilus)
• Commonrudd (Scardiniuserythrophthalmus)
• Chub(Squaliuscephalus)
• Commonnase (Chondrostomanasus)
• Bleak(Alburnusalburnus)
• NeonTetra(Paracheirodoninnesi)
• Gudgeon(Gobiogobio)
• Commondace(Leuciscusleuciscus)
• Stoneloach(Barbatulabarbatula)
• Commonminnow(Phoxinusphoxinus)

Background:

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

Recently,severalcelltypeshavebeenshowntoproduceVTGafterestrogenstimulation,includingthoseoftheepidermalmucosa.EventhoughtheVTGconcentrationintheskinmucusisanorderofmagnitudelowerthaninbloodserumorinbodyhomogenates(containinglivertissue),theskinmucosaisverywellsuitedasamatrixfor determiningexogenousVTGinductioncausedbyenvironmentalchemicalswithaffinitytoestrogenreceptors.ByusingahighlysensitiveELISAincombinationwithauniquesamplingandextractionsystemthedeterminationofmucosa-bornVTGdeterminationhasthefollowingadvantages:

• Simpleandhighlystandardizedsamplingtechniqueandsamplepreparation.
• Strictlydefinedmatrixwithoutproteasecontaminationcausedbynon-targettissuesorlymphaticfluid.
• Non-destructiveandtherebyallowingseveralsubsequentsamplingsinordertorecordakineticofVTGinductionwithamaximumknowntoappearafter7daysofexposure.ThereforeMucosatestarefullycompatIBLewithacuteaswellaschronicalOECDtestmethods.
• Epithelialorganizedepidermisisdirectlyexposedtoexogenousestrogensandtherebyallowingadirectcomparisonwithinvitrotestusingestrogensensitivevitellogeninproducingfishcelllines.
• LowerdegreeofinterferencewithendogenousVTGproduction(infemales)andbioconcentrationorenterohepaticcirculationoftheeffectiveestrogen(xenoestrogen)andtherebyshowingacleardoseresponserelationship.
• StABIlityofstandardsandsamplesifprescribedstorageconditionsareobserved.

The TECO®UltraSensitiveCyprinidVitellogeninELISA kitprovidesahigh-sensitivityELISAoptionforresearchersdeterminingVTGproteinconcentrationinfishmucusorcellculturesamples.