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Diapharma/TECO® Perch (Perciformes) Vitellogenin ELISA/TE1035/Kit/96 tests

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货号:TE1035
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品牌:Diapharma
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商品描述

Description:

ThePerch(Perciformes)VitellogeninELISAKitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserumandmucusofperciformes.

Cyprinid Vitellogenin ELISA, Perch (Perciformes) Vitellogenin ELISA

VitellogenindeterminationisoneofthecoreendpointsinscreeningandtestingforendocrinedisruptingchemicalsstandardizedintheOECDGuidlinesforthetestingofchemicalsforestrogenicactivity:

  •  OECD(2009),TestNo.229
  •  OECD(2009),TestNo.230
  •  OECD(2011),TestNo.234

Vitellogenindeterminationisusedinecotoxicologicalstudiestodetermineestrogeniccompoundsinthewater.

Vitellogeninlevelscanbeusedtodeterminethesexandthestatusofsexualmaturationoffish.

Studies

StimulationStudiesusingestrADIol(E2),Ethinyl-Estradiol(EE2,)BisphenolAshowclearincreasesinVitellogeninlevelsinserumandmucus.

Range: 0-80ng/ml

Sensitivity: <0.5ng/ml

Incubationtime: 4.0hours

Samplevolume: 50µl

Samplepreparation:

  • Serum:Storefreshserumsamplesimmediatelyaftercollectionat
    -20°Corloweruntilassayed.
  • Mucus:CollectmucusasdescribedintheTECO®MucusCollectionSet(TE1034).Mucus-containingswabscanbestoredseveralmonthsat<-20°C

Referencevalues:

  • Serumlevelsareintherangeofµg/mluptomg/ml
  • Mucuslevelareintherangeofng/ml

Species:

  • FishPerciformes
  • Bluegill(Lepomismacrochirus)
  • EuropeanPerch(Percafluviatilis)

KitCompostion:

Reagents

 


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.

  1. AllocatethewellsoftheMicrotiterplate 1 forstandards,controlsandsamples.
  2. Pipette50μlmatrixsolution 4  (multichannelpipette)intoallwells.
  3. Add50μlofeachpreparedstandard( A  – F ),pre-dilutedcontrols( C1 and C2 )andsamplesintothe correspondingwells.
  4. Coverthewellsandincubatetheplatefor120±5minatroomtemperature(18–30°C)onashaker (500rpm).
  5. Afterincubation,aspiratethecontentsofthewellsandwash3timeswith350μldilutedwashbuffer 2 . Theuseofanautomaticplatewasherisrecommended.
  6. Followingthelastwashingstep,pipette100μlofthebiotinylatedAB 5  ineachwell (multichannelpipette).
  7. Coverthewellsandincubatetheplatefor60±5minatroomtemperature(18–30°C)onashaker (500rpm).
  8. Afterincubation,washthewells3timeswithwashbufferasdescribedinstep5.
  9. Followingthelastwashingstep,pipette100μloftheSA-HRPconjugate 6  ineachwell (multichannelpipette).
  10. Coverthewellsandincubatetheplatefor30±5minatroomtemperature(18–30°C)onashaker (500rpm).
  11. Afterincubation,washthewells5timeswithwashbufferasdescribedinstep5.
  12. Pipette100μloftheTMBsubstratesolution 7  ineachwell(multichannelpipette).
  13. Incubatetheplatefor15-30min,inthedark,atroomtemperature(18–30°C)onashaker(500rpm).
  14. Stopthereactionbyadding100μlofstopsolution 8  (multichannelpipette).
  15. 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.
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/**/
SymbolDescriptionFormat
 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  Kitinstruction1x