Description:

TheTECO®Salmonid VitellogeninELISAkitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserum,WBHandmucusofsalmonids.

VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:

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

Vitellogenindeterminationisfrequentlyusedasanendpointinecotoxicologicalstudiestodeterminethepresenceoreffectofestrogeniccompoundsinthewater.

Range: 0.4-35ng/ml

Sensitivity: <0.1ng/ml

Incubationtime: 4.0hours

SampleVolume: 50µl

SamplePreparation:

• Serum:Storefreshserumsamplesimmediatelyaftercollectionat -20°Corloweruntilassayed.
• WBH:StorefreshWBHsamplesimmediatelyaftercollectionat -20°Corloweruntilassayed.
• CollectmucusasdescribedintheTECO®MucusCollectionSet(TE1034).Mucuscontainingswabscanbestoredseveralmonthsat<-20°C.

ReferenceValues:

• Serumlevelsareintherangeofµg/mluptomg/ml
• WBHlevelsareintherangeofmg/ml
• Mucuslevelareintherangeofng/ml

KitComposition:

KitContents:

• AntibodyCoatedMicroassayPlate: 96-wellplate (12×8break-apartwellstripscoatedwithIgGdirectedagainstSalmonid VTG)
• StandardStockSolution:17.5 ng,2vials
• ControlC1:  lowcontrol,2vials
• ControlC2: highcontrol,2vials
• WashBuffer: 1x30ml,50X (Dilute1:50withdeionizedwater.)
• DilutionBuffer:1x55ml.Readytouse.
• MatrixSolution:1x7ml.Readytouse.
• HRP-AbConjugate:1x0.2ml.
• HRP- ConjugateBuffer:1x13ml. 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®Salmonid VitellogeninELISAkitisa96-wellimmuno-captureELISAproduct.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibody-coatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalHRP-conjugated antibodybindstothevitellogenin.Inthefinalsubstratereaction,colordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.

ResultAnalysis

Thestandardrangeoftheassayisbetween0and35ng/ml.Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationsinsamples canthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationcan beobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.Adilutioncorrectionformucusisnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbetestedagain, pre-dilutedwithDilutionBuffer, andthenthis additionaldilutionshould betakeninaccountfortheconcentrationcalculation.

TECO®Salmonid VTG ELISAStandardCurveExample

Species:

• Atlanticsalmon(Salmosalar)
• Browntrout(Salmotrutta)
• Chumsalmon(Oncorhynchusketa)
• Pinksalmon/humpbacksalmon(Oncorhynchusgorbuscha)
• Rainbowtrout(Oncorhynchusmykiss)
• Brooktrout(Salvelinusfontinalis)
• “Commonwhitefish,Europeanwhitefish”(Coregonuslavaretus)
• Maraenawhitefish(Coregonusmaraena)

Background:

Inoviparousanimals,vitellogenin(VTG)isanestrogen-inducedyolkprecursorproteinmainlysynthesizedinthelivertobedepositedinthematuringoocytes,whereitissplitintheyolkproteinslipovitellin1,lipovitellin2andphosvitin.Theseyolkproteinsserveasnourishmentstorageforthedevelopingembryos.Non-physiologicalinductionofvitellogenininmalesorinjuvenilefishisthoughttoindicateanestrogenmediatedendocrinedisruption.Therefore,VTGdeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidelinesforthetestingofchemicalsforestrogenicactivity.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®Salmonid VitellogeninELISAkitallowsfortestingsamples ofblood,homogenateand/ormucus.Theseprovideresearcherswithgoodsampling optionswhen conductingtime-coursestudiesorfieldresearchwhereitiscriticaltopreservethelifeofthefish.