Description:

ThePerch(Perciformes)VitellogeninELISAKitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofvitellogenininserumandmucusofperciformes.

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

 

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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.

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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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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