Description
Numeroustechniqueshavebeendevelopedtoprepareimmunoliposomesbasedonthenucleophilicreactivityoffreeaminegroupsofproteinsorpeptides.OneofthemostpopularandcommonlyusedmethodsistocovalentlycouplefreecarboxylicgroupstoprimaryaminesthroughactivationofthecarboxylgroupswithEDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide).EDC,whichisaso-calledzero-lengthcrosslinkingagent,reactswiththecarboxyltoformanaminereactiveintermediate(O-acylisourea).TheproducedO-acylisoureacanbeeasilydisplacedbynucleophilicattackfromprimaryaminogroupsinthereactionmixture.However,thisintermediateisunstableandhydrolyzedinaqueoussolutions.Inordertopreventtheintermediatehydrolysis,sulfo-NHS(N-hydroxysulfosuccinimide)isaddedtoEDCtoproduceasignificantlymorestableandmoresolubleactiveintermediate(NHSester).
Consequently,theimmunoliposomesarepreparedbyatwo-stepcouplingprocedure:first,activatingthefreecarboxylgroupontheantibody,peptideorproteinwithEDCandsulfo-NHS,andthencovalentlyconjugatingantibodytothelipidsthroughdisplacementofsulfo-NHSgroupsbyaminegroupsoftheliposomes,asdepictedbelow.EDC/sulfo-NHScouplingreactionsarehighlyselectiveandhighlyefficient,andtheBIOLOGicalactivityoftheproteinorpeptideispreserved.
Immunodox®-AmineisaPEGylatedproduct.Forotheraminereactive(PEGylatedandnon-PEGyalatedproducts)andalsoImmunodox®productssuitableforothertypesconjugationmethodsseehere.
FormulationInformation
Immunodox®-Amine(PEGylated)
LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
---|---|---|---|
Total | 15.89mg/ml | 21.58mM | 100 |
HydrogenatedSoyPC | 9.58 | 12.22 | 57 |
Cholesterol | 3.19 | 8.25 | 38 |
DSPE-PEG(2000) | 2.5 | 0.89 | 4 |
DSPE-PEG(2000)-Amine | 0.62 | 0.22 | 1 |
Buffers,LiposomeSizeandEncapsulatedDrugConcentration | Specification |
---|---|
InsideBuffer | AmmoniumSulfate |
OutsideBuffer | PhosphateBufferedSaline |
pH | 7.4 |
LiposomeSize | 100nm |
EncapsulatedDoxorubicin | 2mg/ml(3.45mM) |
ConjugationProtocol
MaterialsandEquipment
Inordertoconjugateyourantibody,protein,peptideorligandcontainingcarboxylicacidtoImmunodox®-Amineliposomesyouwillneed:
- EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride).Thesolutionshouldbemadefreshmomentsbeforeuse.
- Sulfo-NHS(N-hydroxysulfosuccinimide).Thesolutionshouldbemadefreshmomentsbeforeuse.
- MESbuffer.ThereasonforusingMESbufferisbecauseMESisanon-amine,non-carboxylatebuffer,andactivationreactionwithEDCandSulfo-NHSismostefficientatpH4.5-7.2.Alternatively,youcanusePBSbuffer.ButyouhavetomakesurethatthepHofthebufferisadjustedto6.00.
- Sephadex®spincolumn.SephadexsizeexclusionspincolumncanbeusedforseparationofliposomesformfreeEDC(MW:191.70).SinceEDCisbeingseparatedfromlargeliposomeparticlesthenanysizesofSephadex®spincolumnsuchasG-10,G-15,G-25,G50canbeused.However,keepinmindthatyouwilllosealargepercentageofyourliposomesonthespincolumn.Alternatively,insteadofremovingtheEDCbyspincolumnyoucanquenchitbyusing2-mercaptoethanol.
- Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein/peptide/ligand.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnordialysiscassette.IfyoudonotusethecorrectMWCO,youcanloseyourentireprep.Forthisprotocol,werecommendMWCOof300,000dalton.
PreparationMethod
Atwo-stepprotocolfortheactivationofproteins,peptidesorantibodieswithEDC/sulfo-NHSandsubsequentconjugationwithamine-containingmoleculesisgivenbelow.
- Dissolvetheprotein,antibody,peptideorligandtobeactivatedin0.1MMES,0.5MNaCl,pH6.0(reactionbuffer)ataconcentrationbetween1-10mg/ml.Formostproteinsandantibodies(dependingontheMW)thisisequaltoaconcentrationof10-5to10-4 molarsolution.Pleasecalculatethemolarityofyourprotein,peptide,Aborligand.
- Addtothesolutioninstep1aquantityof10-foldmolarexcessofEDCand25-foldmolarexcessofsulfo-NHS.Toaidinaliquotingthecorrectamountofthesereagents,theymaybequicklydissolvedintheMESbufferatahigherconcentration,andthenavolumeimmediatelyPipettedintotheproteinsolutiontoobtainthepropermolarquantities. Mixandreactfor15minutesatroomtemperature.
- Beforeaddingtheliposomescontainingamine(Immunodox®-Amine)toactivatedproteinorligand,youneedtomakesurethatEDCwillberemovedfromthesolution.ThiscaneitherbedonebyusingapropersizespincolumnorbyquenchingEDCby2-mercaptoethanol.
- Add2-mercaptoethanoltothereactionsolutiontoobtainafinalconcentrationof20mM.Mixandincubatefor10minatroomtemperature.NOTE:Iftheproteinbeingactivatedissensitivetothislevelof2-mercaptoethanol,insteadofquenchingthereactionchemically,theactivationmaybeterminatedbydesalting(step5).
- Ifthereactionwasquenchedbytheadditionof2-mercaptoethanol,theactivatedproteinmaybeaddeddirectlytotheamine-containingliposomes(Immunodox®-Amine)forconjugation.Alternatively,orifno2-mercaptoethanolwasadded,theactivatedproteinmaybepurifiedfromreactionby-productsbygelfiltrationusingadesaltingspincolumnsuchasSephadex®spincolumn.ThedesaltingoperationshouldbeperformedrapidlytominimizehydrolysisandrecoverasmuchasactiveesterfunctionalityaspossIBLe.
- Afterpurification,addtheactivatedproteintotheImmunodox®-Amine.ThemolarratioofreactiveAminelipidtoprotein,peptideorantibodyispreferredtobearound10:1.Thetotallipidconcentrationinourliposomesis21.58mM.1%molofthelipidinliposomescontainsPEG-NH2groupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.For2mlvolumeliposome,thisisequalto2.20×10-7mol,andfor5mlvolumeliposome,thisisequalto5.50×10-7molofPEG-NH2.Youneedtocalculatethetotalmolofyourpeptide,proteinorligandinyoursolutionandadd1:10molarratioofligandtolipid.Allowtoreactforatleast2hatroomtemperature.
- Removenon-conjugatedantibody,protein,peptideorligandbydialysis.Wepreferdialysistosizeexclusioncolumns.Dialysisisamuchslowerprocessbuttherewillbeminimumlossofimmunoliposomesaftertheprepiscleanedfromnon-conjugatedprotein/peptide/ligand.Spincolumnsarefasterbutyoucaneasilyloseover50%oftheliposomesonthespincolumn.WerecommendusingFloat-A-LyzerdialysiscassettefromSpectrumLabs.YouneedtochooseacassettewithproperMWCOdependingontheMWofyourprotein,ligand,antibodyorantibodyfragment.NOTE:IfyoudecidetouseadialysiscassetteyouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000daltontheporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesandspincolumnsblindly.Theycomeinvarioussizesandyouneedtochoosethecorrectsizewisely.Dialyzetheimmunoliposomesolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletisdialyzeforanother8hours.Afterthisstep,yourcleanedupimmunoliposomeisreadytobeused.
LiposomeParticleCalculator
Immunodox®liposomesareunilamellarandsizedto100nm.Themolarconcentrationofliposomeis21.58 mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.
TechnicalNotes
- Doxorubicinisafluorescentmoleculewithλex470nmandλem585nm.Ifyouareusingafluorescenttagonyourantibodyorligand,youneedtomakesurethattheywillnotinterferewitheachother.
- EDCandsulfo-NHSshouldbepreparedimmediatelyandkeptatroomtemperaturebeforeuse.
- TheactivationreactionwithEDCandSulfo-NHSismostefficientatpH4.5-7.2,andEDCreactionsareoftenperformedinatpH4.7-6.0.Forthisreason,wehaveformulatedtheliposomesinPBSbufferandadjustedthepHto6.
- ReactionofSulfo-NHS-activatedmoleculeswithprimaryaminesismostefficientatpH7-8,andSulfo-NHS-esterreactionsareusuallyperformedinphosphate-bufferedsaline(PBS)atpH7.2-7.5.
- Trisbuffershouldneverbeusedinanystepoftheprocesssinceitcontainsamine.
- IfyouareusingaligandorpeptidethatishydrophobicthenitisrecommendedtosolubilizeitinDMSOorDMFandthenaddthebuffertoit.Itisrecommendednottousemorethan5%volumeofDMSOorDMFinthesolution.DMFandDMSOarebothcompatiblewithliposomesandtheyarealsomiscibleinwater.Otherorganicsolventsuchasethanolandchloroformarenotcompatiblewithliposomesandwillcausetheliposomestolyse.IfyouendupusingDMSOorDMFthenaftertheconjugationreactionisdone,youneedtoremoveDMSOandDMFfromtheliposomes.InordertodothatyouneedtouseadialysiscassettethatismadefromREGENERATEDCELLULOSEMEMBRANE.NOTE:NotallmembranesarecompatiblewithDMFandDMSO.WerecommendusingaSlide-A-Lyzer™MINIDialysisDevicewithMWCOof2KmadefromregeneratedcellulosemembranemanufacturedbyThermofisher.AfterDMSOorDMFisremoved,youcanuseFloat-A-Lyzer®dialysisdeviceforthefinalstepofcleaninguptheprep.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Database
Directlinktothedatabasepageforeasynavigation:ImmunoliposomesConjugationDatabase
Appearance
Immunodox®-Amineisaredtranslucentliquidmadeofnanosizeunilamellarliposomes.Usuallyduetothesmallsizeofliposomesnosettlingwilloccurinthebottomofthevial.Theliposomesarepackagedinanambervial.
EducationalVideo
Ordering/ShippingInformation
- Allliposomebasedformulationsareshippedonblueiceat4°Cininsulatedpackagesusingovernightshippingorinternationalexpressshipping.
- LiposomesshouldNEVERbefrozen.Icecrystalsthatforminthelipidmembranecanrupturethemembrane,changethesizeoftheliposomesandcausetheencapsulateddrugtoleakout.Liposomesinliquidformshouldalwaysbekeptintherefrigerator.
- ClientswhoorderfromoutsideoftheUnitedStatesofAmericaareresponsiblefortheirgovernmentimporttaxesandcustomspaperwork.EncapsulaNanoSciencesisNOTresponsibleforimportationfeestocountriesoutsideoftheUnitedStatesofAmerica.
- WestronglyencouragetheclientsinJapan,Korea,TaiwanandChinatoorderviaadistributor.Toughcustomsclearanceregulationsinthesecountrieswillcausedelayincustomclearanceoftheseperishableformulationsifordereddirectlythroughus.Distributorscaneasilyclearthepackagesfromcustoms.Toseethelistofthedistributorsclickhere.
- ClientsorderingfromuniversitiesandresearchinstitutesinAustraliashouldkeepinmindthattheliposomeformulationsaremadefromsyntheticmaterialandtheformulationsdonotrequirea“permittoimportquarantinematerial”.LiposomesareNOTbiologicalproducts.
- Ifyouwouldlikeyourinstitute’sFedExorDHLaccounttobechargedforshipping,thenpleaseprovidetheaccountnumberatthetimeofordering.
- EncapsulaNanoScienceshasnocontroloverdelaysduetoinclementweatherorcustomsclearancedelays.YouwillreceiveaFedExorDHLtrackingnumberonceyourorderisconfirmed.ContactFedExorDHLinadvanceandmakesurethatthepaperworkforcustomsisdoneontime. AllsubsequentshippinginquiriesshouldbedirectedtoFederalExpressorDHL.
StorageandShelfLife
Storage
Immunodox®productsshouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.IfthesUSPensionisfrozen,theencapsulateddrugcanbereleasedfromtheliposomesthuslimitingitseffectiveness.Inaddition,thesizeoftheliposomeswillalsochangeuponfreezingandthawing.
ShelfLife
Immunodox®-Amineismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin4monthsofthemanufacturingdate.
ReferencesandbackgroundreADIng
1.HermansonGT.Bioconjugatetechniques.Academicpress;2013Jul25.
2.TorchilinV,WeissigV,editors.Liposomes:apracticalapproach.OxfordUniversityPress;2003Jun5.
3.GrabarekZ,GergelyJ.Zero-lengthcrosslinkingprocedurewiththeuseofactiveesters.Analyticalbiochemistry.1990Feb15;185(1):131-5.
4.YanL,CraytonSH,ThawaniJP,AmirshaghaghiA,TsourkasA,ChengZ.ApH‐ResponsiveDrug‐DeliveryPlatformBasedonGlycolChitosan–CoatedLiposomes.Small.2015Oct1;11(37):4870-4.
5. Silva-LópezEI,EdensLE,BardenAO,KellerDJ,BrozikJA.ConditionsforliposomeadsorptionandbilayerformationonBSApassivatedsolidsupports.Chemistryandphysicsoflipids.2014Oct31;183:91-9.
6.HazraM,SinghSK,andRayS.SurfaceModificationofLiposomalVaccinesbyPeptideConjugation.JournalofPharmaSciTech,2011;1(1):41-47.
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过表达慢病毒
>10^8 PFU/ml
干扰慢病毒
>10^8 PFU/ml
主要有: 1.磁珠法核酸提取试剂盒2.磁珠法提取DNA试剂盒3.DNA提取试剂盒(离心柱法)4.磁珠法RNA提取试剂盒5.RNA提取试剂盒(离心柱法)6.磁珠法植物基因组DNA提取试剂盒7.反转录试剂盒8.胶回收试剂盒9.PCR纯化试剂盒10.病毒核酸提取试剂盒
病毒液通过pre-filterdisc的时候是不是要制造一个真空环境产生压力差使液体通过?这个时候是用自备的针管还是试剂盒配的5毫升的针管啊?
对虾白斑综合症病毒(WSSV)核酸检测试剂盒(PCR-荧光探针法)
对虾白斑综合症病毒(WSSV)核酸检测试剂盒(恒温荧光法—一管式)
对虾桃拉综合症病毒(TSV)核酸检测试剂盒(恒温荧光法)
对虾桃拉综合症病毒(TSV)核酸检测试剂盒(PCR-荧光探针法)
对虾传染性皮下及造血组织坏死症病毒(IHHNV)核酸检测试剂盒(恒温荧光法)
对虾传染性皮下及造血组织坏死症病毒(IHHNV)核酸检测试剂盒(PCR-荧光探针法)
对虾肝胰腺细小病毒(HPV)核酸检测试剂盒(恒温荧光法)
对虾黄头病毒(YHV)核酸检测试剂盒(恒温荧光法)
对虾黄头病毒(YHV)核酸检测试剂盒(PCR-荧光探针法)
对虾杆状病毒(BP)核酸检测试剂盒(恒温荧光法)
对虾杆状病毒(BP)核酸检测试剂盒(PCR-荧光探针法)
对虾传染性肌肉坏死病毒(IMNV)核酸检测试剂盒(恒温荧光法)
对虾传染性肌肉坏死病毒(IMNV)核酸检测试剂盒(PCR-荧光探针法)
罗氏沼虾诺达病毒(MrNV)核酸检测试剂盒(恒温荧光法)
罗氏沼虾诺达病毒(MrNV)核酸检测试剂盒(PCR-荧光探针法)
急性肝胰腺坏死病(AHPND/EMS)病原核酸检测试剂盒(恒温荧光法)
急性肝胰腺坏死病(AHPND/EMS)病原核酸检测试剂盒(PCR-荧光探针法)
对虾偷死野田村病毒(CMNV)核酸检测试剂盒(恒温扩增法)
对虾偷死野田村病毒(CMNV)核酸检测试剂盒(PCR-荧光探针法)
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