Description:TheTECO®HyaluronicAcidPLUSELISAkitisasensitivesandwichenzymelinkedimmunosorbentassayforthequantitativedeterminationofhyaluronicacidinplasma,serumandotherBIOLOGicalfluids.KitComposition:ReagentsandMaterialsSuppliedSymbolDescriptionFormat 1 96-wellplatecoatedwithHABP12breakapartstripsof8wells(12×8intotal),inaframewithcoverplate.Readytouse.1plate A StandardA0ng/ml1x1.5ml B StandardB15ng/ml1x0.5ml C StandardC50ng/ml1x0.5ml D StandardD150ng/ml1x0.5ml E StandardE450ng/ml1x0.5ml F StandardF1000ng/ml1x0.5ml C1 ControlC11x0.5ml C2 ControlC21x0.5ml 2 WashBuffer50x1x30ml 3 SampleDiluentReadytouse.1x50ml 6 HABP-HRPConj.Readytouse.1x12ml 7 TMBSubstrateReadytouse.1x12ml 8 StopSolution–1MHCl1Mhydrochloricacid.Readytouse.1x12ml I Kitinstruction1xMaterialsRequiredandnotSuppliedPipettes10µl–1000µlMultichannelpipettesfor50µl–100µlGraduatedcylindersforreconstitutingordilutingreagentsManualAspirationSystemorAutomaticwasherforELISAplatesAquadestVortexmixerELISAplatereadersuitablefor96wellformatsandcapableofmeasuringat450nm(Reference:590-650nm)ELISAplateshaker(500rpm)(orbitalshaker)SoftwarepackagefordatagenerationandanalysisMeasurementPrinciple:TheTECO®assaykitforhyaluronicacidisasensitivesandwichassayusingamicrotiterplate coatedwithHAbindingprotein(HABP)andHRPconjugatedHABPfordetection.ThisHRPconjugated HABPbindstosampleHAandisfollowedbyasubstratereaction.Thecolordevelopmentiscatalyzed quantitativelydependentonHAlevelsofthesamples.AssayPrinciple&Procedure:AssayPrincipleTheTECO®assaykitforhyaluronicacidisasensitivesandwichassayusingamicrotiterplatecoatedwithHAbindingprotein(HABP)andHRPconjugatedHABPfordetection.ThisHRPconjugatedHABPbindstosampleHAandisfollowedbyasubstratereaction.ThecolordevelopmentiscatalyzedquantitativelydependentonHAlevelsofthesamples.AssayProcedureAlldeterminations(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.AllocatethewellsoftheMicrotiterplate 1 forstandards,controlsandsamples.Dilutestandards(A till F),controls(C1 and C2)andsamples1:50withSampleDiluent 3.Pipette100µlofeachdilutedstandards(A till F),controls(C1 and C2)andsamples intothecorrespondingwells.Coverthewellswithaplasticcoverandincubatetheplatefor2h±5minatroomtemperature (20–25°C)onashaker(500rpm).Afterincubation,aspiratethewellsbyusingaplatewasherormanuallydecantbyinvertingtheplate. Washthewells3timeswith350µldilutedwashbufferperwell.Afterthelastwashcycletapthe invertedwellsonadryabsorbentsurfacetoremoveexcesswashsolution.Theuseofanautomatic platewasherisrecommended.Followingthelastwashingstep,pipette100µloftheHABP-HRPconjugate 6 ineachwell (multichannelpipette).Coverthewellswithaplasticcoverandincubatetheplatefor30±5minatroomtemperature (20–25°C)onashaker(500rpm).Afterincubationwashthewells5timeswithwashbufferasdescribedinstep5.Pipette100µloftheTMBsubstratesolution 7 ineachwell(multichannelpipette).Incubatetheplatefor30min,inthedark,atroomtemperature(20–25°C)onashaker(500rpm).Stopthereactionbyadding100µlofstopsolution 8 (multichannelpipette).Measurethecolorreactionwithin10minutesat450nm(referencefilterbetween590–650nm). Iftheextinctionofthestd F exceeds3.0,themeasurementshouldberepeatedat405nm.ProtocolsforthedifferentautomaticELISAsystemsareavailable.Background:ResearchUseHyaluronicacid(HA),alsoknownashyaluronanorhyaluronateisalargelinearnon-sulfatedglycosaminogly-can(GAG)withamolecularweightbetween106and107Da.Itisamajorcomponentofconnectivetissuesandthusdistributedubiquitouslyintheorganism.Aboutone-halfofthebody’sentirehyaluronanisfoundintheskinandaboutonefourthintheskeletonanditssupportingstructureslikeligamentsandjoints.Hyaluronanissynthesizedbyfibroblastsandotherspecializedconnectivetissuecells.Hyaluronanisespeciallyimportantforthestructureandorganizationofextracellularmatrices.Thehyaluro-nannetworkactsasanosmoticbufferandisreponsibleforwaterhomeostasisaswellasitregulatesproteindistributionviatheformationofflowanddiffusionbarriers.Additionally,hyaluronaninteractswithproteinsandcellsurfacesandthushasastronginfluenceoncellproliferation,differentiationandtissuerepair.TurnoverandcatabolismThetissuehalf-lifeofhyaluronandiffersbetweenspeciesandvariesfromaboutonetoseveraldays.Acertainamountofhyaluronanisdegradedlocally,butthemuchlargerpartisremovedanddegradedbythelympha-ticsystem.Theremainderentersthebloodcirculationwhereitisremovedprimarilybyliverendothelialcells.Aminorportionismetabolizedbythekidneysandthespleen.Adultcartilageisavascularanddependsuponthesynovialfluidtoprovidenutrition;aswellas,thedisposalofmetabolicwastes.Thus,hyaluronanresultingfromcartilagedegradationisfirstreleasedintothesynovialfluidwhereitentersthebloodandlymphstream,respectively,throughthehighlyvascularizedsynovialmembrane.TheSEROlogicalhalf-lifeofhyaluronanisabout2–5minutes.Thenormaladulthumanserologicallevelofhya-luronanvariesbetween10and100μg/landthetotalhyaluronanturnoverinserumisestimatedtobeintherangeof10–100mg/24h.Serumhyaluronanisinfluencedbyvariousfactorsincludingage,sexandethnicityaswellasfoodintakeandthelevelofphysicalactivity.