Description:
TheTECO®REACHPerch(Perciformes)VitellogeninELISAKitisasensitivesandwichenzymelinkedimmunosorbentassayforthedeterminationofvitellogenininserum,wholebodyhomgenate(WBH) andmucusofperciformesaccordingtoECregulationNr.440/2008(REACH)fromJuly10th2015/DocumentD039048/03.
VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:
- OECD(2009),TestNo.229
- OECD(2009),TestNo.230
- OECD(2011),TestNo.234
REACHkitsmeetstrictqualityassuranceregulationsbyincludingadditionalcomponentsinthekit:
- 1 break-apartstripof8wellsforNon-SpecificBinding(NSB).
- Anadditionalinter-assayreferenceStandardStockofadifferentvitellogeninELISAproductionlot.
- Apackageinsertthatincludes thespecificrequirementsforvitellogenintestingwithinREACHregulations.
Vitellogenindeterminationisusedinecotoxicologicalstudiestodeterminetheeffectsofestrogeniccompoundsinwater.
Range:1-80ng/ml
Sensitivity: <1.0ng/ml
Incubationtime: 4.0hours
Samplevolume: 50µl
Samplepreparation:
- Serum:Storefreshserumsamplesimmediatelyaftercollectionat -20°Corloweruntilassayed.
- WBH:StorefreshWBHsamplesimmediatelyaftercollectionat -20°Corloweruntilassayed.
- Mucus: CollectasdescribedintheTECO®MucusCollectionSet(TE1034).Mucuscontainingswabscanbestoredseveralmonthsat<-20°C.
Referencevalues:
- Serumlevelsareintherangeofµg/mluptomg/ml
- WBHlevelsareintherangeofmg/ml
- Mucuslevelareintherangeofng/ml
StimulationStudiesusingestrADIol(E2),Ethinyl-Estradiol(EE2)andBisphenolAshowclearincreasesinVitellogeninlevelsinserum,WBHandmucus.
KitComposition:
KitContents:
- AntibodyCoatedMicroassayPlate: 96-wellplate (12×8break-apartwellstripscoatedwithIgGdirectedagainstPerciformesVTG)
- Non-SpecificBinding(NSB) Wells: 1break-apartstripof8wells coatedwithoutspecificbindingantibodies
- StandardStockSolution:160ng,1vial
- ControlC1: lowcontrol,1vial
- ControlC2: high control,1vial
- Inter-AssayReferenceStandard:30 ng,1vial
- 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).
•ELISAplateshaker(500rpm)
Formucussamples:TheMucusCollectionSet(TE1034)whichincludesExtractionBufferandvalidatedSamplingSwabsis alsorequired.
AssayPrinciple:
AssayPrinciple
TheTECO®REACHPerch VitellogeninELISAkitisa96wellimmuno-captureELISAproductusinghomologueantibodiesandhomologueVTGstandardmaterial.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.
ResultAnalysis
Thestandardrangeoftheassayisbetween0and80ng/ml.Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationsinthesample canthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationcanbeobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.A dilutioncorrectionformucus isnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbetestedagain,pre-dilutedwithDilutionBuffer, andthentheadditionaldilutionshould betakeninaccountfortheconcentrationcalculation.
TECO®REACHCyprinidVTG ELISAStandardCurveExample
Species:
- Europeanperch/perch/redfinperch/Englishperch(Percafluviatilis)
- Tilapia(Oreochromisniloticus)
- Bluegill(Lepomismacrochirus)
- Ruffe(Gymnocephaluscernua)
- Three-spinedstickleback(Gasterosteusaculeatus)
Background:
Inoviparousanimals,vitellogenin(VTG)isanestrogen-inducedyolkprecursorproteinmainlysynthesizedinthelivertobedepositedinthematuringoocytes,whereitissplitintheyolkproteinslipovitellin1,lipovitellin2andphosvitin.Theseyolkproteinsserveasnourishmentstorageforthedevelopingembryos.Non-physiologicalinductionofvitellogenininmalesorinjuvenilefishisthoughttoindicateanestrogenmediatedendocrinedisruption.ThereforeVTGdeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidelinesforthetestingofchemicalsforestrogenicactivity.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®REACHPerch VitellogeninELISAkitallowsforsamplingofblood,homogenateand/ormucuswhichprovides researcherswithsampling optionswhen conductingtime-coursestudiesorfieldresearchwhereitiscriticaltopreservethelifeofthefish.
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elisa试剂盒回收率是反应待测物在样品分析过程中的损失的程度,损失越少,回收率越高,如果作标液1PPM,就是1毫克/升,而作出标准数据为0.99毫克/升,就是说你的回收率是99%,这个与真实成分有密切的关系,说明方法的准确度。比如水中总无机氯含量测定,样品水中含有无机氯20mg/L,取100mL被测水样品,加入0.1mL浓度为10mg/mL的含无机氯标准样品,测定时忽略体积变化,如果测定出样品中无机氯为2.98mg/L,则认为回收率为99%。向左转|向右转
2、规范包被操作,吸附是否均匀
3、重复性、可靠性
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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
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