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 |
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.
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.
- 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:
ResearchUse
Hyaluronicacid(HA),alsoknownashyaluronanorhyaluronateisalargelinearnon-sulfatedglycosaminogly-can(GAG)withamolecularweightbetween106and107Da.Itisamajorcomponentofconnectivetissuesandthusdistributedubiquitouslyintheorganism.Aboutone-halfofthebody’sentirehyaluronanisfoundintheskinandaboutonefourthintheskeletonanditssupportingstructureslikeligamentsandjoints.Hyaluronanissynthesizedbyfibroblastsandotherspecializedconnectivetissuecells.
Hyaluronanisespeciallyimportantforthestructureandorganizationofextracellularmatrices.Thehyaluro-nannetworkactsasanosmoticbufferandisreponsibleforwaterhomeostasisaswellasitregulatesproteindistributionviatheformationofflowanddiffusionbarriers.Additionally,hyaluronaninteractswithproteinsandcellsurfacesandthushasastronginfluenceoncellproliferation,differentiationandtissuerepair.
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.
