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 |
Materialsrequiredandnotsupplied
- Pipettes10μl–1000μl
- Multichannelpipettesfor50μl–100μl
- Graduatedcylindersforreconstitutingordilutingreagents
- ManualAspirationSystemorautomaticwasherforELISAplates
- Aquadest
- Vortexmixer
- ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(Reference:590-650nm)
- ELISAplateshaker(500rpm)(orbitalshaker)
- Softwarepackagefordatagenerationandanalysis
Formucussamples:ExtractionBufferandvalidatedSamplingSwabsarenotpartofthiskit.PleaseorderTECO®MucusCollectionSet(TE1034)separately.
AssayPrinciple&Procedure:
AssayPrinciple
TheTECO®CyprinidVitellogeninEIAKitisa96wellimmuno-captureELISAproduct.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.
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.
- Allocatethewellsofthemicrotiterplate 1 forstandards,controlsandsamples.
- Pipette50μlmatrixsolution 4 (multichannelpipette)intoallwells.
- Add50μlofeachpreparedstandard( A – F ),preparedcontrols( C1 and C2 )and(pre-diluted)samples intothecorrespondingwells.
- Coverthewellsandincubatetheplatefor120±10minatroomtemperature(18–30°C)onashaker (500rpm).
- Afterincubation,aspiratethecontentsofthewellsandwash3timeswith350μldilutedwashbuffer 2 . Theuseofanautomaticplatewasherisrecommended.
- Followingthelastwashingstep,pipette100μlfthebiotinylatedAB 5 ineachwell(multichannel pipette).
- Coverthewellsandincubatetheplatefor60±5minatroomtemperature(18–30°C)onashaker (500rpm).
- Afterincubation,washthewells3timeswithwashbufferasdescribedinstep5.
- Followingthelastwashingstep,pipette100μloftheSA-HRPconjugate 6 ineachwell(multichannel pipette).
- Coverthewellsandincubatetheplatefor30±5minatroomtemperature(18–30°C)onashaker(500rpm).
- Afterincubation,washthewells5timeswithWashBufferasdescribedinstep5.
- Pipette100μloftheTMBsubstratesolution 7 ineachwell(multichannelpipette).
- Incubatetheplatefor15-30min,inthedark,atroomtemperature(18–30°C)onashaker(500rpm).
- Stopthereactionbyadding100μlofstopsolution 8 (multichannelpipette).
- 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.
