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.
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（1）优级纯试剂 亦称保证试剂，为一级品，纯度高，杂质极少，主要用于精密分析和科学研究，常以GR表示。
（2）分析纯试剂 亦称分析试剂，为二级品，纯度略低于优级纯，杂质含量略高于优级纯，适用于重要分析和一般性研究工作，常以AR表示。
（3）化学纯试剂 为三级品，纯度较分析纯差，但高于实验试剂，适用于工厂、学校一般性的分析工作，常以CP表示。
（4）实验试剂 为四级品，纯度比化学纯差，但比工业品纯度高，主要用于一般化学实验，不能用于分析工作，常以 LR表示。
以上按试剂纯度的分类法已在我国通用。根据化学工业部颁布的“化学试剂包装及标志”的规定，化学试剂的不同等级分别用各种不同的颜色来标志，见表1。
表1 我国化学试剂的等级及标志
bhclna2so4nano3na2co3
chclnaohna2co3nacl
dba(oh)2nahco3alcl3nahso4
刚入这行，谢谢大家
A.H2SO4Na2SO4AgNO3BaCl2.
B.NaOHNa2CO3NaHSO4MgCl2
C.CaCl2NaNO3MgSO4BaCl2
D.HNO3KOHKClK2CO3
