Description:

TheTECO® CyprinidVitellogeninELISAkitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserum,WBHandmucusofcyprinids.

VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:

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

Vitellogenindeterminationisusedinecotoxicologicalstudiestodetermineestrogeniccompoundsinthewater.

Vitellogeninlevelscanbeusedtodeterminethesexandthesexualmaturationoffish.

Range: 0-35ng/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

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

KitComposition:

Reagents

Symbol	Description	Format
1	96-wellplatecoatedwithcypVTGAntibody 12breakapartstripsof8wells(12x8intotal),inaframe.Readytouse.	1plate
S	StandardStock lyophilized 17.5ng	2x
C1	ControlC1 lyophilized. Concentration: seeDataSheet.	2x
C2	ControlC2 lyophilized Concentration: seeDataSheet.	2x
2	WashBuffer50x Dilute1:50withdeionizedwater.	1x 30ml
3	DilutionBuffer Readytouse.	1x55ml
4	MatrixSolution Readytouse.	1x7ml
5	BiotinylatedAntibody(Biotin-AB) Readytouse.	1x12ml
6	StreptavidinPeroxidaseConjugate(SA-HRPConj) Readytouse.	1x12ml
7	TMBSubstrate Readytouse.	1x12ml
8	StopSolution–1MHCI 1Mhydrochloricacid.Readytouse.	1x12ml
I	KitInstructions	1x

 

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Materialsrequiredandnotsupplied

• Pipettes10μl–1000μl
• Multichannelpipettesfor50μl–100μl
• Graduatedcylindersforreconstitutingordilutingreagents
• ManualAspirationSystemorautomaticwasherforELISAplates
• Aquadest
• Vortexmixer
• ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(Reference:590-650nm)
• ELISAplateshaker(500rpm)(orbitalshaker)
• Softwarepackagefordatagenerationandanalysis

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Formucussamples:ExtractionBufferandvalidatedSamplingSwabsarenotpartofthiskit.PleaseorderTECO®MucusCollectionSet(TE1034)separately.

AssayPrinciple&Procedure:

AssayPrinciple

TheTECO®CyprinidVitellogeninEIAKitisa96wellimmuno-captureELISAproduct.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.

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AssayProcedure

Alldeterminations(standards,controlsandsamples)shouldbeassayedinduplicate.Whenperformingthe assay,thestandards,controlsandsamplesshouldbepipettedasfastaspossIBLe(<15minutes).

Toavoiddistortionsduetodifferencesinincubationtimes,HRPconjugate,substratesolutionandstopsolutionshouldbeaddedtotheplateinthesameorderandwiththesametimeintervalasthesamples.A multichannelpipetteisessential.

Allowallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.Duringallincubationsteps, platesshouldbesealedwiththeadhesivefoiloraplasticcover.Forlightprotection,incubateinadarkchamber orcoverplatewithaluminumfoil.

1. Allocatethewellsofthemicrotiterplate 1 forstandards,controlsandsamples.
2. Pipette50μlmatrixsolution 4  (multichannelpipette)intoallwells.
3. Add50μlofeachpreparedstandard( A  – F ),preparedcontrols( C1 and C2 )and(pre-diluted)samples intothecorrespondingwells.
4. Coverthewellsandincubatetheplatefor120±10minatroomtemperature(18–30°C)onashaker (500rpm).
5. Afterincubation,aspiratethecontentsofthewellsandwash3timeswith350μldilutedwashbuffer 2 . Theuseofanautomaticplatewasherisrecommended.
6. Followingthelastwashingstep,pipette100μlfthebiotinylatedAB 5  ineachwell(multichannel pipette).
7. Coverthewellsandincubatetheplatefor60±5minatroomtemperature(18–30°C)onashaker (500rpm).
8. Afterincubation,washthewells3timeswithwashbufferasdescribedinstep5.
9. Followingthelastwashingstep,pipette100μloftheSA-HRPconjugate 6  ineachwell(multichannel pipette).
10. Coverthewellsandincubatetheplatefor30±5minatroomtemperature(18–30°C)onashaker(500rpm).
11. Afterincubation,washthewells5timeswithWashBufferasdescribedinstep5.
12. Pipette100μloftheTMBsubstratesolution 7  ineachwell(multichannelpipette).
13. Incubatetheplatefor15-30min,inthedark,atroomtemperature(18–30°C)onashaker(500rpm).
14. Stopthereactionbyadding100μlofstopsolution 8  (multichannelpipette).
15. Measurethecolorreactionwithin10minutesat450nm(referencefilterbetween590–650nm). IftheextinctionofthestandardA(35ng/ml)exceeds3.0,themeasurementmayberepeatedat405nm.

Species:

• Fish
• Cyprinid
• Carp(Caprinuscarpio)
• Goldfish(Carassiusauratus)
• Zebrafish(Daniorerio)
• Medaka,Japanesericefish(Oryziaslatipes)
• FatheadMinnow(Pimephalespromelas)

Background:

Inoviparousanimals,vitellogenin(VTG)isanestrogeninducedyolkprecursorproteinmainlysynthesizedinthelivertobedepositedinthematuringoocytes,whereitissplitintheyolkproteinslipovitellin1,lipovitellin2andphosvitin.Theseyolkproteinsserveasnourishmentstorageforthedevelopingembryos.Non-physiologicalinductionofvitellogenininmalesorinjuvenilefishisthoughttoindicateanestrogenmediatedendocrinedisruption.ThereforeVTGdeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidelinesforthetestingofchemicalsforestrogenicactivity.Normallyvitellogeninismeasuredinbloodsamplesorwholebodyhomogenate(WBH)–bothsampletypesrequireinvasiveanddestructivetreatmentofthefish.Bloodisdifficulttocollect,inparticularwhereverysmallfishareconcerned,orinapproacheswheretheanimalsmustsurvivesampling.Thisisparticularlyimportantinfieldmonitoringinordertoavoidimpactonthepopulationunderinvestigation.RecentlyseveralcelltypeshavebeenshowntoproduceVTGafterestrogenstimulation,includingthoseoftheepidermalmucosa.EventhoughtheVTGconcentrationintheskinmucusisanorderofmagnitudelowerthaninbloodserumorinbodyhomogenates(containinglivertissue),theskinmucosaisverywellsuitedasamatrixtodetermineexogenousVTGinductioncausedbyenvironmentalchemicalswithaffinitytoestrogenreceptors.ByusingahighlysensitiveELISAincombinationwithanuniquesamplingandextractionsystemthedeterminationofmucosabornVTGdeterminationhasthefollowingadvantages:

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

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