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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今天文章就简单整理了一下最近遇到的问题，有些问题对你来说可能很简单，但是还是会给一些科研菜鸟带去小麻烦，特整理了这么个专场来涨涨姿势。
这里就简单整理了5个问题，具体内容如下：
小编自己在各大论坛看到最多的三个答案：
1）48T可做42个样本加6个标准曲线；96T可做90个样本加6个标准曲线
2）以96T试剂盒来算，定量的试剂盒一般可以做90个左右的样品，定性的试剂盒可以做94个左右的样品
3）96T的ELISA试剂盒，去除标准品复孔检测，最多还能检测80个标本；48T的最多30个样本。
2、elisa试剂盒96T的能做检测多少血清样品？板能重复利用吗？
96T，一般做复孔的话，做标准曲线需要16个孔，剩下80个孔就可以做40个样本。这个板不能重复利用。而且配套的试剂也只是够一块板子用的。
3、想请教一下，我想用elisa法测血清里的一种抗原，查了资料，发现没有现成的试剂盒用，现在就有2种备选方案，一种是买抗体对自己包被（查了R&D等大公司都没有可用于elisa的单抗）。哪种比较好呢？自己包被的会不会比国产的试剂盒可靠呢？
如果你觉得技术不错有队其他公司不放心，可以自己购买抗体对自己包被，这个就是麻烦点，自己包被用着放心，舒心；
另外国内还是有一些资质不错的公司，如北京达科为，上海巧伊，依科赛，深圳欣博盛等等
4、western和ELISA处理样本有何差别？
WesternBlot样品制备要加蛋白裂解液（含有蛋白酶抑制剂），然后冰上研磨，收集于EP管，冰上裂解30min，4度12000rpm离心15min，取上清分装，即得到蛋白样品。建议直接电泳，多余的-80保存。建议分装的蛋白每支略多于一次上样量，一次性用完一支，避免反复冻融。
ELISA样品，直接脑组织冰上匀浆，收集于EP管，同上一样离心，大约220-250ul每支分装（因为ELISA上样量基本都是每孔100ul，做复孔，每个样品2孔，就需要200ul，为了避免不够2孔，分装时要大于200ul每支），直接实验。多余的-80保存，一样避免反复冻融。
不过ELISA最好少量样品进行预实验，或者查阅文献，看看大概组织里有多高浓度，选择合适的试剂盒，测量范围要与组织浓度相符。避免浓度过高测量不准，或者浓度过低，小于试剂盒最低可测浓度。
5、Elisa试剂盒该买哪家公司的呢？
提出这个问题的朋友其实该打，目前国际上各个品牌的试剂盒生厂商，都会有其偏重点，像R&D在细胞因子方面的试剂盒就非常牛逼，而其他牌子也一样的。
Bender：粘附分子方面的试剂盒；
Cayman：花生四烯酸类产品的试剂盒；
Merck(Linco)：内分泌研究方面的试剂盒；
Tips：
1、买Elisa试剂盒时，尽量先查文献，通过文献找品牌，再找供应商，找供应商就找正规代理商，千万不要去干“赔夫人又折兵”的蠢事。
2、ELISA中，复孔就是同一个样品加到至少两个孔中（一般3个或4个），最后取其平均值作为样品的读数，这样测得的结果才有说服力！
做复孔是为了较好的较少组内差异，如果血清不足不做复孔也可以。
第二步、明确编码该蛋白的基因与其它哪些物种同源性最好
第三步、寻找检测这些物种蛋白的试剂盒
第四步、咨询商家ELISA是试剂盒使用的抗体类型:多抗还是单抗? 单抗是针对什么抗原决定簇的?
第五步、 做出自己的判断该买哪个试剂盒，或者实在没有可以找公司进行订制实验
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
