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.
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2、规范包被操作,吸附是否均匀
3、重复性、可靠性
6、是否提供技术
7、适用于血浆、血清、组织匀浆液、细胞培养上清液、尿液等多种类型的样本
8、可检测动物类型是否丰富
9、可检测指标是否齐全
ELISA检测试剂盒是用于体外定性检测人血清或血浆中的抗人类戊型肝炎(HEV)病毒 IgM抗体.
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