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.
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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。
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
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
