Description:
TheTECO®REACHMedakaVitellogeninELISA kitisasensitiveenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserum,wholebodyhomgenate(WBH) andmucusofcyprinidsaccordingtoECregulationNr.440/2008(REACH)fromJuly10th2015/DocumentD039048/03.
VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:
- OECD(2009),TestNo.229
- OECD(2009),TestNo.230
- OECD(2011),TestNo.234
REACHkitsmeetstrictqualityassuranceregulationsbyincluding,inadditiontothestandardELISAmaterials,thefollowing componentsinthekit:
- 1 break-apartstripof8wellsforNon-SpecificBinding(NSB)assessment.
- Anextra inter-assayreferenceStandardStockfrom adifferentvitellogeninELISAproductionlot.
- Apackageinsertthatincludes thespecificrequirementsforvitellogenintestingwithinREACHregulations.
Vitellogenindeterminationisusefulinecotoxicologicalstudiestodeterminethepresenceoreffectofestrogeniccompoundsinthewater.
AssayMeasuringRange: 2.6-210ng/ml
AssaySensitivity: <0.1ng/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
KitComposition:
KitContents:
- AntibodyCoatedMicroassayPlate: 96-wellplate (12×8break-apartwellstripscoatedwithIgGdirectedagainstMedaka VTG)
- Non-SpecificBinding(NSB) Wells: 1break-apartstripof8wells coatedwithoutspecificbindingantibodies
- StandardStockSolution:105 ng,2vials
- ControlC1: lowcontrol,2vials
- ControlC2: highcontrol,2vials
- Inter-AssayReferenceStandard:15.0ng,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®REACHMedakaVitellogeninELISAkitisa96-well immuno-captureELISAproduct.Serum,WBHandmucussamplesareincubatedwiththevitellogeninspecificantibody-coatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopment
isdirectlyproportionaltotheamountofvitellogenininthesample.
ResultAnalysis
Thestandardrangeoftheassayisbetween0and210ng/ml.Acalibrationcurvecanbeestablishedbyplottingstandardconcentrationonthex-axis(linearscale)againsttheabsorbanceofthestandardsonthey-axis(linearscale).Thevitellogeninconcentrationsinsamples canthenbereadoffthecalibrationcurve. A4-parametercurvefitshouldbeusedforautomaticdatareduction.Ifsampleswerepre-diluted,theconcentrationcan beobtainedbymultiplyingthevaluereadoffthecalibrationcurvebythedilutionfactor.Adilutioncorrectionformucusisnotnecessaryifonlythe0.5mlExtractionBufferwasaddedtotheswab.SampleswithhigherabsorbancevaluesthanStandardAshouldbetestedagain,pre-dilutedwithDilutionBuffer, andthenthis additionaldilutionshould betakeninaccountfortheconcentrationcalculation.
TECO®REACHMedakaVTG ELISAStandardCurveExample
Species:
- Medaka,Japanesericefish(Oryziaslatipes)
Background:
Inoviparousanimals,vitellogenin(VTG)isanestrogen-inducedyolkprecursorproteinmainlysynthesizedinthelivertobedepositedinthematuringoocytes,whereitissplitintheyolkproteinslipovitellin1,lipovitellin2andphosvitin.Theseyolkproteinsserveasnourishmentstorageforthedevelopingembryos.Non-physiologicalinductionofvitellogenininmalesorinjuvenilefishisthoughttoindicateanestrogenmediatedendocrinedisruption.Therefore,VTGdeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidelinesforthetestingofchemicalsforestrogenicactivity.Normally,vitellogeninismeasuredinbloodsamplesorwholebodyhomogenate(WBH)–bothsampletypesrequireinvasiveanddestructivetreatmentofthefish.Bloodcanbe difficulttocollect,inparticularwhereverysmallfishareconcernedand wheretheanimalsmustsurvivesampling.
Recently,severalcelltypeshavebeenshowntoproduceVTGafterestrogenstimulation,includingthoseoftheepidermalmucosa.EventhoughtheVTGconcentrationintheskinmucusisanorderofmagnitudelowerthaninblood,serumorbodyhomogenates(containinglivertissue),theskinmucosaisverywellsuitedasamatrixfor determiningexogenousVTGinductioncausedbyenvironmentalchemicalswithaffinitytoestrogenreceptors.ByusingasensitiveELISAincombinationwithauniquesamplingandextractionsystemthedeterminationofmucosa-bornVTGdeterminationhasthefollowingadvantages:
- Simpleandhighlystandardizedsamplingtechniqueandsamplepreparation.
- Strictlydefinedmatrixwithoutproteasecontaminationcausedbynon-targettissuesorlymphaticfluid.
- Non-destructive,therebyallowingseveralsubsequentsamplingsinordertorecordakineticofVTGinduction.Amaximumisknowntoappearafter7daysofexposure.Therefore,MucosatestsarefullycompatIBLewithacuteaswellaschronicalOECDtestmethods.
- Epithelialorganizedepidermisisdirectlyexposedtoexogenousestrogensandtherebyallowingadirectcomparisonwithinvitrotestusingestrogensensitivevitellogeninproducingfishcelllines.
- LowerdegreeofinterferencewithendogenousVTGproduction(infemales)andbioconcentrationorenterohepaticcirculationoftheeffectiveestrogen(xenoestrogen)andtherebyshowingacleardoseresponserelationship.
- StABIlityofstandardsandsamplesifprescribedstorageconditionsareobserved.
TheTECO®REACHMedakaVitellogeninELISA kitallowsfortestingsamples ofblood,homogenateand/ormucus.Thesesampletypesprovideresearcherswithgoodsampling optionsespeciallywhen conductingtime-coursestudiesorfieldresearchwhereitiscriticaltopreservethelifeofthefish.
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
的试剂:免疫吸附剂、结合物和酶的底物。
完整的ELISA试剂盒包含以下各组分:
(1)已包被抗原或抗体的固相载体(免疫吸附剂);
(2)酶标记的抗原或抗体(结合物);
(3)酶的底物;
(4)阴性对照品和阳性对照品(定性测定中),参考标准品和控制血清(定量测定
中);
(5)结合物及标本的稀释液;
(6)洗涤液;
(7)酶反应终止液。
Elisa生物试验是一种敏感性高,特异性强,重复性好的实验诊断方法。
但是在选择的时候,也需要注意其他的一些问题。
1、采用何种原料和抗体,是否高效、灵敏、特异
2、规范包被操作,吸附是否均匀
3、重复性、可靠性
6、是否提供技术服务
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试剂盒 就查下博欧特生物
使用方法:
1、 血清:操作过程中避免任何细胞刺激。使用不含热原和内毒素的试管。收集血液后,1000×g离心10分钟将血红细胞迅速小心地分离。
2、 血浆:EDTA、柠檬酸盐、肝素血浆可用于检测。1000×g离心30分钟去除颗粒。
3、 细胞上清液:1000×g离心10分钟去除颗粒和聚合物。
4、 组织匀浆:将组织加入适量生理盐水捣碎。1000×g离心10分钟,取上清液。
5、 保存:如果样品不立即使用,应将其分成小部分-70℃保存,避免反复冷冻。尽可能的不要使用溶血或高血脂血。如果血清中大量颗粒,检测前先离心或过滤。不要在37℃或更高的温度加热解冻。应在室温下解冻并确保样品均匀地充分解冻。
1、是不是获得SCI文献引用;
2、试剂盒是不是具备高灵敏,质量好的;
3、售后服务完全,提供代测服务;
4、后期实验提供全程技术支持!
暂无品牌问答