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Mission StatementDiapharma Group, Inc. in West Chester, Ohio sells hemostasis, thrombosis, platelet function testing, instrumentation, and apoptosis products in the diagnostic and research fields and provides strong technical competence and experienc...
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Diapharma/TECO® Perch (Perciformes) Vitellogenin ELISA/TE1035/Kit/96 tests

Diapharma/TECO® Perch (Perciformes) Vitellogenin ELISA/TE1035/Kit/96 tests
  • Diapharma/TECO® Perch (Perciformes) Vitellogenin ELISA/TE1035/Kit/96 tests
商品介绍

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.
品牌介绍

Mission Statement

Diapharma Group, Inc. in West Chester, Ohio sells hemostasis, thrombosis, platelet function testing, instrumentation, and apoptosis products in the diagnostic and research fields and provides strong technical competence and experience to ensure customer expectations will be met or exceeded.

diapharma hemostasis chromogenic clotting elisa assay test kit

 

History

The Diapharma Group, Inc. formed on January 1, 1997 from Pharmacia Hepar, Inc. in Franklin, Ohio, and began as the exclusive US and Canadian distributor of the Chromogenix substrates and assays.

Over a quarter-century ago, Chromogenix developed the first chromogenic substrate technology under the former name, Kabi Diagnostica. Kabi later merged with Pharmacia. Some of Diapharma’s current staff worked inside the Chromogenix department within Pharmacia’s heparin manufacturing plant.

In 1998, Diapharma moved to West Chester, Ohio, where it remains today. Over the years, Diapharma expanded its product line to include a variety of hemostasis, cell death, platelet function, ecotoxicology, assays, reagents, antibodies and instruments from superior manufacturers.

In 2017, Diapharma celebrated twenty years of success


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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