| Symbol | Description | Format |
| 1 | 96-wellplatecoatedwithpVTGAntibody
12breakapartstripsof8wells(12x8intotal),inaframe.Readytouse. | 1plate |
| S | StandardStock 10x
0.8µg/ml | 1x0.2ml |
| C1 | ControlC1,10x
ConcentrationseeDataSheet. | 1x0.2ml |
| C2 | ControlC2,10x
ConcentrationseeDataSheet. | 1x0.2ml |
| 2 | WashBuffer50x
Dilute1:50withdeionizedwater. | 1x30ml |
| 3 | DilutionBuffer
Readytouse. | 1x55ml |
| 4 | 4: MatrixSolution
Readytouse. | 1x7ml |
| 5 | 5: BiotinylatedAntibody(Biotin-AB).
Readytouse. | 1x12ml |
| 6 | StreptavidinPeroxidaseConjugate(SA-HRPConjugate). Readytouse. | 1x12ml |
| 7 | TMBSubstrate | 1x12ml |
| 8 | StopSolution–1MHCI.
1Mhydrochloricacid,readytouse. | 1x12ml |
| I | Kitinstruction | 1x |
Materialsrequiredandnotsupplied
- Pipettes10μl–1000μl
- Multichannelpipettesfor50μl–100μl
- Graduatedcylindersforreconstitutingordilutingreagents
- ManualAspirationSystemorautomaticwasherforELISAplates
- Aquadest
- Vortexmixer
- ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(reference:590-650nm)
- ELISAplateshaker(500rpm)(orbitalshaker)
- Softwarepackagefordatagenerationandanalysis
AssayPrinciple&Procedure:
AssayPrinciple
TheTECO®PerchVitellogeninEIAKitisa96wellimmuno-captureELISAproduct.Mucusorserumsamplesareincubatedwiththevitellogeninspecificantibodycoatedmicrotiterplate.Afterunboundmaterialiswashedout,apolyclonalbiotinylatedantibodybindstothevitellogenin.Inthefollowingincubationstep,astreptavidin-peroxidaseconjugatebindstothebiotinylatedantibody.Inthefinalsubstratereaction,thecolordevelopmentisdirectlyproportionaltotheamountofvitellogenininthesample.
AssayProcedure
Alldeterminations(standards,controlsandsamples)shouldbeassayedinduplicate.Whenperformingthe assay,thestandards,controlsandsamplesshouldbepipettedasfastaspossIBLe(<15minutes).
Toavoiddistortionsduetodifferencesinincubationtimes,HRPconjugate,substratesolutionandstop solutionshouldbeaddedtotheplateinthesameorderandwiththesametimeintervalasthesamples. Amultichannelpipetteisessential.
Allowallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.Duringallincubationsteps, platesshouldbesealedwiththeadhesivefoiloraplasticcover.Forlightprotection,incubateinadarkchamber orcoverplatewithaluminumfoil.
- AllocatethewellsoftheMicrotiterplate 1 forstandards,controlsandsamples.
- Pipette50μlmatrixsolution 4 (multichannelpipette)intoallwells.
- Add50μlofeachpreparedstandard( A – F ),pre-dilutedcontrols( C1 and C2 )andsamplesintothe correspondingwells.
- Coverthewellsandincubatetheplatefor120±5minatroomtemperature(18–30°C)onashaker (500rpm).
- Afterincubation,aspiratethecontentsofthewellsandwash3timeswith350μldilutedwashbuffer 2 . Theuseofanautomaticplatewasherisrecommended.
- Followingthelastwashingstep,pipette100μlofthebiotinylatedAB 5 ineachwell (multichannelpipette).
- Coverthewellsandincubatetheplatefor60±5minatroomtemperature(18–30°C)onashaker (500rpm).
- Afterincubation,washthewells3timeswithwashbufferasdescribedinstep5.
- Followingthelastwashingstep,pipette100μloftheSA-HRPconjugate 6 ineachwell (multichannelpipette).
- 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(80ng/ml)exceeds3.0,themeasurementmayberepeatedat405nm.
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
- NondestructiveandtherebyallowingseveralsubsequentsamplingsinordertorecordakineticofVTGinductionwithamaximumknowntoappearafter7daysofexposure.ThereforMucosatestarefullycompatiblewithacuteaswellaschronicalOECDtestmethods.
- Epithelialorganizedepidermisisdirectlyexposedtoexogenousestrogensandtherebyallowingadirectcomparisonwithinvitrotestusingestrogensensitivevitellogeninproducingfishcelllines
- LowerdegreeofinterferencewithendogenousVTGproduction(infemales)andbioconcentrationorenterohepaticcirculationoftheeffectiveestrogen(xenoestrogen)andtherebyshowingacleardoseresponserelationship
- StABIlityofstandardsandsamplesifprescribedstorageconditionsareobserved.
品牌介绍
Diapharma使命宣言位于俄亥俄州西切斯特的Difarma Group，Inc.在诊断和研究领域销售止血、血栓形成、血小板功能测试、仪器和凋亡产品，并提供强大的技术能力和经验，以确保满足或超过客户的期望。地黄止血显色凝块酶联免疫吸附试验试剂盒历史1997年1月1日，由俄亥俄州富兰克林市的Pharmacia Hepar，Inc.成立，最初是Chromogenix基质和分析的独家美国和加拿大经销商。四分之一个多世纪前，Chromogenix开发了第一个显色底物技术，其前身是Kabi Diagnostica。卡比后来与法玛西亚合并。希帕玛目前的一些员工在法玛西亚肝素制造厂的显色部门工作。1998年，夏帕玛搬到了俄亥俄州的西切斯特，至今仍在那里。多年来，迪法玛扩大了其产品线，包括各种止血、细胞死亡、血小板功能、生态毒理学、化验、试剂、抗体和高级制造商的仪器。2017年，夏帕玛庆祝了20年的成功
