Description:

TheTECO®HyaluronicAcidPLUSELISAkitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofhyaluronicacidinplasma,serumandotherBIOLOGicalfluids.

KitComposition:

ReagentsandMaterialsSupplied

Symbol	Description	Format
1	96-wellplatecoatedwithHABP 12breakapartstripsof8wells(12×8intotal),inaframewithcoverplate.Readytouse.	1plate
A	StandardA 0ng/ml	1x1.5ml
B	StandardB 15ng/ml	1x0.5ml
C	StandardC 50ng/ml	1x0.5ml
D	StandardD 150ng/ml	1x0.5ml
E	StandardE 450ng/ml	1x0.5ml
F	StandardF 1000ng/ml	1x0.5ml
C1	ControlC1	1x0.5ml
C2	ControlC2	1x0.5ml
2	WashBuffer50x	1x30ml
3	SampleDiluent Readytouse.	1x50ml
6	HABP-HRPConj. Readytouse.	1x12ml
7	TMBSubstrate Readytouse.	1x12ml
8	StopSolution–1MHCl 1Mhydrochloricacid.Readytouse.	1x12ml
I	Kitinstruction	1x

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MaterialsRequiredandnotSupplied

• Pipettes10µl–1000µl
• Multichannelpipettesfor50µl–100µl
• Graduatedcylindersforreconstitutingordilutingreagents
• ManualAspirationSystemorAutomaticwasherforELISAplates
• Aquadest
• Vortexmixer
• ELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(Reference:590-650nm)
• ELISAplateshaker(500rpm)(orbitalshaker)
• Softwarepackagefordatagenerationandanalysis

MeasurementPrinciple:

TheTECO®assaykitforhyaluronicacidisasensitivesandwichassayusingamicrotiterplate coatedwithHAbindingprotein(HABP)andHRPconjugatedHABPfordetection.ThisHRPconjugated HABPbindstosampleHAandisfollowedbyasubstratereaction.Thecolordevelopmentiscatalyzed quantitativelydependentonHAlevelsofthesamples.

AssayPrinciple&Procedure:

AssayPrinciple

TheTECO®assaykitforhyaluronicacidisasensitivesandwichassayusingamicrotiterplatecoatedwithHAbindingprotein(HABP)andHRPconjugatedHABPfordetection.ThisHRPconjugatedHABPbindstosampleHAandisfollowedbyasubstratereaction.ThecolordevelopmentiscatalyzedquantitativelydependentonHAlevelsofthesamples.

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AssayProcedure

Alldeterminations(standards,controlsandsamples)shouldbeassayedinduplicate.Whenperforming theassay,thestandards,controlsandsamplesshouldbepipettedasfastaspossIBLe(<15minutes). Toavoiddistortionsduetodifferencesinincubationtimes,HABP-HRPconjugate,substratesolutionand stopsolutionshouldbeaddedtotheplateinthesameorderandwiththesametimeintervalasthesamples. Amultichannelpipetteisessential.

Allowallreagentstostandatroomtemperature(20–25°C)foratleast30minutes.Duringallincubation steps,platesshouldbesealedwiththeadhesivefoiloraplasticcover.Forlightprotection,incubatein adarkchamberorcoverplatewithaluminiumfoil.

1. AllocatethewellsoftheMicrotiterplate 1 forstandards,controlsandsamples.
2. Dilutestandards(A till F),controls(C1 and C2)andsamples1:50withSampleDiluent 3.
3. Pipette100µlofeachdilutedstandards(A till F),controls(C1 and C2)andsamples intothecorrespondingwells.
4. Coverthewellswithaplasticcoverandincubatetheplatefor2h±5minatroomtemperature (20–25°C)onashaker(500rpm).
5. Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinvertingtheplate. Washthewells3timeswith350µldilutedwashbufferperwell.Afterthelastwashcycletapthe invertedwellsonadryabsorbentsurfacetoremoveexcesswashsolution.Theuseofanautomatic platewasherisrecommended.
6. Followingthelastwashingstep,pipette100µloftheHABP-HRPconjugate 6 ineachwell (multichannelpipette).
7. Coverthewellswithaplasticcoverandincubatetheplatefor30±5minatroomtemperature (20–25°C)onashaker(500rpm).
8. Afterincubationwashthewells5timeswithwashbufferasdescribedinstep5.
9. Pipette100µloftheTMBsubstratesolution 7 ineachwell(multichannelpipette).
10. Incubatetheplatefor30min,inthedark,atroomtemperature(20–25°C)onashaker(500rpm).
11. Stopthereactionbyadding100µlofstopsolution 8 (multichannelpipette).
    Measurethecolorreactionwithin10minutesat450nm(referencefilterbetween590–650nm). Iftheextinctionofthestd F exceeds3.0,themeasurementshouldberepeatedat405nm.

ProtocolsforthedifferentautomaticELISAsystemsareavailable.

Background:

ResearchUse

Hyaluronicacid(HA),alsoknownashyaluronanorhyaluronateisalargelinearnon-sulfatedglycosaminogly-can(GAG)withamolecularweightbetween106and107Da.Itisamajorcomponentofconnectivetissuesandthusdistributedubiquitouslyintheorganism.Aboutone-halfofthebody’sentirehyaluronanisfoundintheskinandaboutonefourthintheskeletonanditssupportingstructureslikeligamentsandjoints.Hyaluronanissynthesizedbyfibroblastsandotherspecializedconnectivetissuecells.

Hyaluronanisespeciallyimportantforthestructureandorganizationofextracellularmatrices.Thehyaluro-nannetworkactsasanosmoticbufferandisreponsibleforwaterhomeostasisaswellasitregulatesproteindistributionviatheformationofflowanddiffusionbarriers.Additionally,hyaluronaninteractswithproteinsandcellsurfacesandthushasastronginfluenceoncellproliferation,differentiationandtissuerepair.

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Turnoverandcatabolism

Thetissuehalf-lifeofhyaluronandiffersbetweenspeciesandvariesfromaboutonetoseveraldays.Acertainamountofhyaluronanisdegradedlocally,butthemuchlargerpartisremovedanddegradedbythelympha-ticsystem.Theremainderentersthebloodcirculationwhereitisremovedprimarilybyliverendothelialcells.Aminorportionismetabolizedbythekidneysandthespleen.

Adultcartilageisavascularanddependsuponthesynovialfluidtoprovidenutrition;aswellas,thedisposalofmetabolicwastes.Thus,hyaluronanresultingfromcartilagedegradationisfirstreleasedintothesynovialfluidwhereitentersthebloodandlymphstream,respectively,throughthehighlyvascularizedsynovialmembrane.

TheSEROlogicalhalf-lifeofhyaluronanisabout2–5minutes.Thenormaladulthumanserologicallevelofhya-luronanvariesbetween10and100μg/landthetotalhyaluronanturnoverinserumisestimatedtobeintherangeof10–100mg/24h.Serumhyaluronanisinfluencedbyvariousfactorsincludingage,sexandethnicityaswellasfoodintakeandthelevelofphysicalactivity.

 

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