Description
Numeroustechniqueshavebeendevelopedtoprepareimmunoliposomesbasedonthenucleophilicreactivityoffreeaminegroupsofproteinsorpeptides.However,themostcommon,versatileandstraightforwardactivationchemistryforcreatingreactiveacylatingreagentsandlabelingpeptides/proteinsistoformNHSesterwithprimaryamines.AsinglestepnucleophilicsubstitutionreactionbetweenNHSesterderivativeandalphaaminesattheNterminalorthebetaaminesoflysinesidechainsleadstotheformationofastableamidebond.
N-Hydroxysuccinimide(NHS)estersofDSPE-PEG-NHSliposomesreactwiththeprimaryaminegroupsonthepeptides,proteins,antibodiesorotherfunctionalligandsfortargeteddrugdeliveryapplications.ThenucleophilicattackoftheaminegroupsontheNHS-activatedcarbonylgroupoftheDSPE-PEG-NHSresultsintheeliminationoftheNHSgroupandformationofamidelinkage.
ImmunoFluor™-NHSisaPEGylatedproduct.Forotheraminereactive(PEGylatedandnon-PEGyalated)productsandalsoImmunoFluor™productssuitableforothertypesofconjugationmethodsseehere.
FORMULATIONINFORMATION
ImmunoFluor™-NHS(PEGylated)(Post-insertion)
| PostInsertionKit(3Vials) | Specification |
|---|---|
| Vial1 | PreformedliposomescomposedofHSPC:Cholesterol:FluorescentLipid(59.5:40:0.5molarratio) |
| Vial2 | DSPE-PEG(2000)-NHSlipid(reactivePEGylatedlipid)inpowderform |
| Vial3 | DSPE-PEG(2000)lipid(non-reactivePEGylatedlipid)inpowderform |
| LipidCompositionforVial1* | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| HydrogenatedSoyPC | ~11.5 | ~14.66 | ~60 |
| Cholesterol | 3.83 | 9.9 | 40 |
| FluorescentLipid(seebelow) | Variesbasedonthedye | Variesbasedonthedye | Variesbasedonthedye |
| Total | ~15.33mg/ml | ~24.56mM | 100 |
| *Forthe5-mlkit,thevolumeofvial1is4ml.1mlofmicellesolutionthatareformedusingvials2and3willbeaddedtothisvialtomakethefinalvolumeof5mlinthefinalproduct.Forthe2-mlkit,thevolumeofvial1is1.6ml.0.4mlofmicellesolutionthatisformedusingvials2and3willbeaddedtothisvialtomakethefinalvolumeof2mlinthefinalproduct. | |||
| FluorescentDye | Excitation/Emission(nm) | MolecularStructure |
|---|---|---|
| 1,1"-Dioctadecyl-3,3,3",3"-tetramethylindocarbocyanineperchlorate(DiI) | 549/565 |  |
| 3,3"-Dilinoleyloxacarbocyanineperchlorate(DiO) | 484/501 |  |
| 1,1"-Dioctadecyl-3,3,3",3"-tetramethylindodicarbocyanine,4-chlorobenzenesulfonatesalt(DiD) | 644/665 |  |
| 1,1"-Dioctadecyl-3,3,3",3"-tetramethylindotricarbocyanineiodide(DiR) | 750/780 |  |
| 4-(4-(Dihexadecylamino)styryl)-N-methylpyridiniumiodide(DiA) | 456/590 |  |
| 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxADIazol-4-yl)(ammoniumsalt)(NBDonheadgroup) | 460/535 |  |
| 1-Palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphocholine(NBDonfattyacidtail) | 460/534 |  |
| 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissaminerhodamineBsulfonyl)(ammoniumsalt)(Rhodaminelipid) | 560/583 |  |
| 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl)(ammoniumsalt)(Dansyllipid) | 336/513 |  |
| 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(1-pyrenesulfonyl)(ammoniumsalt)(Pyrenelipid) | 351/379 |  |
| ReZolve-L1™ | 350/500 |  |
| IraZolve-L1™ | 405/600 |  |
| BufferandLiposomeSizeforVial1 | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 100nm |
| Vial2* | Specification |
|---|---|
| DSPE-PEG(2000)-NHSLipid | ThisvialcontainsreactiveDSPE-PEG(2000)-NHSlipidinpowderform.Thislipidisconjugatedtoareactiveprotein,peptideorligandcontainingamineandthenmixedwithnon-reactiveDSPE-PEG(2000)lipidinaqueoussolutiontoformmicelles.ThePEGylatedlipidmicellesareincubatedwithpreformedliposomesinvial1andPEGlipidswillpost-insertthemselvesintotheliposomes. |
| *TheamountofthepowderedPEG(2000)-NHSlipidforthe2-mlkitis1.34mgandforthe5-mlkitis3.34mg. | |
| Vial3* | Specification |
|---|---|
| DSPE-PEG(2000)Lipid | Thisvialcontainsnon-reactiveDSPE-PEG(2000)lipidinpowderform.ThislipidinmixedwithDSPE-PEG(2000)-NHSlipidwhichisalreadyconjugatedtoaligand(protein,peptide,etc.)inaqueoussolutiontoformmicelles.ThePEGylatedlipidmicellesareincubatedwithpreformedliposomesinvial1andPEGlipidswillpost-insertthemselvesintotheliposomes. |
| *TheamountofthepowderedPEG(2000)-DSPElipidforthe2-mlkitis5mgandforthe5-mlkitis12.5mg. | |
ConjugationProtocol(Post-insertion)
MaterialsandEquipment
The3-vialpost-insertionkitcontainspreformedliposomes(vial1),non-reactivePEGylatedlipidinpowderform(vial2)andDSPE-PEG(2000)-NHSlipidinpowderform(vial3). Inordertousethepost-insertionkit,youwillneed:
- Laboratoryvortexmixerisrecommendedtohave.
- Laboratorymagneticstirrerisneededfordialysis.
- Twosmall10-mlroundbottomflasksortwosmallglassvials.
- Arotaryevaporator. Weunderstandthatmanylabsmightnothavearotovap.Alternatively,youcanuseanitrogentankconnectedtoathinhoseforcreatingastreamofnitrogenflowtodrythelipidandmakeathinfilm.
- Asmallamountofasolventsuchachloroformormethylenechloride(youwillonlyneedafewmilliliters).
- Phosphatebufferedsaline(PBS).
- ASonicator.Itisbettertohaveabathsonicator.Ifyoudonot,thatisfine.Youstillcanfollowtheprotocol.Youmayalsouseavortexinsteadofthesonicatorforagitationofthesolutionaswell.
- Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein,peptideorantibody.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000daltontheporesizeonthedialysismembranegetscloseto100nmandtherefore,yourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnordialysiscassette.IfyoudonotusethecorrectMWCO,youcanloseyourentireprep.Forthisprotocol,werecommendMWCOof300,000dalton.
PreparationMethod
- Dissolvethecontentofvial3in100µlofchloroformormethylenechloride.Transferthesolutiontoa10mlroundbottomflask.Drythechloroformusingarotaryevaporatororunderastreamofnitrogenandmakeadriedlipidfilm.
- Add100µlofPBSbuffertothedriedlipidfilm.Itispreferredtosonicatethehydratedlipidfilmusingabathsonicatorandsonicatethemicellesolutionfor5minutes.IfyoudonothaveabathsonicatorthenhydratethedriedlipidfilmwithPBSforatleast1hourandconstantlyrotatethesolutionintheroundbottomflaskusingarotavap(notconnectedtovacuum)orbyhandtomakesurethatallthedriedlipidonthewalloftheroundbottomflaskwillgotothesolutionandformmicelles.Alternatively,youcanuseavortextoagitatethesolution.Thegoalistohaveallthedriedlipidonthewalloftheroundbottomglasstogothemicellesolution.CoverthemouthoftheroundbottomflaskwithParafilm.Refrigeratethemicellesolutionofnon-reactivePEGlipidsuntilitisreadytobemixedwithmicellesformedinstep4.
- Thereis 1.34mg(0.22µmol)ofDSPE-PEG-NHSinthe2-mlpost-insertionkit.Dissolvethecontentofvial2in100µlofchloroformormethylenechloride.Transferthesolutiontoa10mlroundbottomflask.Drythechloroformusingarotaryevaporatororunderastreamofnitrogen.
- Addthewater-solubleprotein,peptideorligandat1:2molarratioofligandtodriedfilmofDSPE-PEG-NHS.Add300µlofwater-solublesolutionofproteinofligandinPBS(pH7.4)tothedriedlipidfilm.Itispreferredtosonicatethehydratedlipidfilmusingabathsonicatorandsonicatethemicellesolutionfor5minutes.IfyoudonothaveabathsonicatorthenhydratethedriedlipidfilmwithPBSforatleast1hourandconstantlyrotatethesolutionintheroundbottomflaskusingarotavap(notconnectedtovacuum)orbyhandtomakesurethatallthedriedlipidonthewalloftheroundbottomflaskwillgotothesolutionandformmicelles.Alternatively,youcanuseavortextoagitatethesolution.Thegoalistohaveallthedriedlipidonthewalloftheroundbottomglasstogothemicellesolution.Thesolutionisincubatedatroomtemperaturefor6hoursandinrefrigeratorfor24hours.ThereactionispHsensitive.Readthetechnicalnotebelowformoreinformation.
- Mixthemicellesinstep2tothemicellesinstep4.Thetotalvolumeofmicellesshouldbe400µl.
- Toconductpost-insertion,themicellardispersionisthenco-incubatedwithpreformedplainliposomesat60℃for30min.
- Removethenon-conjugatedprotein,peptideorantibodyfromtheimmunoliposomesbydialysis.Wepreferdialysistosizeexclusioncolumns.Dialysisisamuchslowerprocessbuttherewillbeminimumlossofimmunoliposomesaftertheprepiscleanedfromnon-conjugatedprotein/peptide/ligand.Spincolumnsaremuchfaster;however,youcaneasilyloseover50%oftheliposomesonthespincolumn.WerecommendusingFloat-A-Lyzer® dialysiscassettefromSpectrumLabs.YouwillneedtochooseacassettewithproperMWCOdependingontheMWofyourprotein,peptide,antibodyorantibodyfragment.NOTE:Ifyoudecidetouseadialysiscassette,youwillneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandtherefore,yourliposomescanbedialyzedout.Youcannotusedialysiscassettesandspincolumnsblindly.Theycomeinvarioussizesandyouneedtochoosethecorrectsizewisely.Dialyzetheimmunoliposomesolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletisdialyzeforanother8hours.Afterthisstep,yourcleanedupimmunoliposomeisreadytobeused.
LiposomeParticleCalculator
ImmunoFluor™liposomesareunilamellarandsizedto100nm.Themolarconcentrationofliposomeis24.56mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.
TechnicalNotes
- HydrolysisofDSPE-PEG-NHSinaqueoussolutionscompeteswiththeprimaryaminereaction,resultingintheeliminationoftheNHSgroup,whichcanconsequentlydecreasethecouplingyieldpriortothereactionwiththeprotein/antibody.InordertominimizetheimpactofthehydrolysisofNHSester,useahighconcentrationofprotein/antibodytoincreasetheefficiencyofthecross-linking.
- ThereactionofNHSesterswithaminesisstronglypH-dependent:atlowpH,theaminogroupisprotonated,andnomodificationtakesplace.Athigher-than-optimalpH,hydrolysisofNHSesterisquick,andmodificationyielddiminishes.Thehalf-lifeofNHSestersatpH7and8is4-5hoursand1hour,respectively.WhilstNHSestershaveahalf-lifeofonly10minutesatpH8.6.Therefore,toavoidthehydrolysisofNHSester,DSPE-PEG-NHSlipidshouldbeusedimmediatelyforconjugationtoantibodies,proteinsorpeptidescontainingfreeamines.NHSesterreactionsareconductedincommonbuffersatpH7-8.
- PrimaryaminebufferssuchasTrisshouldNOTbeusedbecausetheycompeteforreaction;however,insomeprocedures,itisusefultoaddTrisorglycinebufferattheendofaconjugationproceduretoquench(stop)thereaction.
- Ifyouareusingaligandorpeptidethatishydrophobic,itisrecommendedtosolubilizeitinDMSOorDMFandthenaddthebuffertoit.Itisrecommendednottousemorethan5%volumeofDMSOorDMFinthesolution.DMFandDMSOarebothcompatIBLewithliposomesandtheyarealsomiscibleinwater.Otherorganicsolventsuchasethanolandchloroformarenotcompatiblewithliposomesandwillcausetheliposomestolyse.IfyouendupusingDMSOorDMFthenaftertheconjugationreactionisdone,youneedtoremoveDMSOandDMFfromtheliposomes.Inordertodothat,youneedtouseadialysiscassettethatismadefromREGENERATEDCELLULOSEMEMBRANE.NOTE:NotallmembranesarecompatiblewithDMFandDMSO.WerecommendusingaSlide-A-Lyzer™MINIDialysisDevicewithMWCOof2KmadefromregeneratedcellulosemembranemanufacturedbyThermofisher.AfterDMSOorDMFisremovedyoucanuseFloat-A-Lyzer®dialysisdeviceforthefinalstepofcleaninguptheprep.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Database
Directlinktothedatabasepageforeasynavigation:ImmunoliposomesConjugationDatabase
Appearance
ImmunoFluor™-NHS(PEGylated)post-insertionkitcomesinthreevials:vial1formulationiscoloredandthecolordependsonthetypeofthefluorescentdyethatisused.Itcontainsnanosizeunilamellarliposomeswhichdoesnotcontainanyreactiveofnon-reactivePEGylatedlipid. Usuallyduetothesmallsizeofliposomesnosettlingwilloccurinthebottomofthevial.Vial2containsreactiveDSPE-PEG(2000)-NHSlipidinwhitepowderform.Vial3containsnon-reactiveDSPE-PEG(2000)lipidinwhitepowderform.
Ordering/ShippingInformation
- Allliposomebasedformulationsareshippedonblueiceat4°Cininsulatedpackagesusingovernightshippingorinternationalexpressshipping.
- LiposomesshouldNEVERbefrozen.Icecrystalsthatisformedinthelipidmembranecanrupturethemembrane,changethesizeoftheliposomesandcausetheencapsulateddrugtoleakout.Liposomesinliquidformshouldalwaysbekeptintherefrigerator.
- ClientswhoorderfromoutsideoftheUnitedStatesofAmericaareresponsiblefortheirgovernmentimporttaxesandcustomspaperwork.EncapsulaNanoSciencesisNOTresponsibleforimportationfeestocountriesoutsideoftheUnitedStatesofAmerica.
- WestronglyencouragetheclientsinJapan,Korea,TaiwanandChinatoorderviaadistributor.Toughcustomsclearanceregulationsinthesecountrieswillcausedelayincustomclearanceoftheseperishableformulationsifordereddirectlythroughus.Distributorscaneasilyclearthepackagesfromcustoms.Toseethelistofthedistributorsclickhere.
- ClientsorderingfromuniversitiesandresearchinstitutesinAustraliashouldkeepinmindthattheliposomeformulationsaremadefromsyntheticmaterialandtheformulationsdonotrequirea“permittoimportquarantinematerial”.LiposomesareNOTBIOLOGicalproducts.
- Ifyouwouldlikeyourinstitute’sFedExorDHLaccounttobechargedforshipping,thenpleaseprovidetheaccountnumberatthetimeofordering.
- EncapsulaNanoScienceshasnocontroloverdelaysduetoinclementweatherorcustomsclearancedelays.YouwillreceiveaFedExorDHLtrackingnumberonceyourorderisconfirmed.ContactFedExorDHLinadvanceandmakesurethatthepaperworkforcustomsisdoneontime. AllsubsequentshippinginquiriesshouldbedirectedtoFederalExpressorDHL.
StorageandShelfLife
Storage
ImmunoFluor™productsshouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.IfthesUSPensionisfrozen,theencapsulateddrugcanbereleasedfromtheliposomesthuslimitingitseffectiveness.Inaddition,thesizeoftheliposomeswillalsochangeuponfreezingandthawing.
ShelfLife
ImmunoFluor™-NHSismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin2monthsofthemanufacturingdate.
Referencesandbackgroundreading
1.NaK,LeeSA,JungSH,HyunJ,ShinBC.Elastin-likepolypeptidemodifiedliposomesforenhancingcellularuptakeintotumorcells.ColloidsandSurfacesB:Biointerfaces.2012Mar1;91:130-6.
2.DaiW,FanY,ZhangH,WangX,ZhangQ,WangX.AcomprehensivestudyofiRGD-modifiedliposomeswithimprovedchemotherapeuticefficacyonB16melanoma.Drugdelivery.2015Jan2;22(1):10-20.
3.FerreiraDdosS,FariaSD,deAraújoLopesSC,TeixeiraCS,MalachiasA,Magalhães-PaniagoR,deSouzaFilhoJD,OliveiraBL,GuimarãesAR,CaravanP,FerreiraLA.Developmentofabone-targetedpH-sensitiveLiposomalformulationcontainingdoxorubicin:physicochemicalcharacterization,cytotoxicity,andbiodistributionevaluationinamousemodelofbonemetastasis.Internationaljournalofnanomedicine.2016;11:3737-51.
4.WangD,FuJ,ShiY,PengD,YuanL,HeB,DaiW,ZhangH,WangX,TianJ,ZhangQ.ThemodulationoftumorvesselpermeABIlitybythalidomideanditsimpactsondifferenttypesoftargeteddrugdeliverysystemsinasarcomamousemodel.JournalofControlledRelease.2016Sep28;238:186-96.
5.FeiW,ZhangY,HanS,TaoJ,ZhengH,WeiY,ZhuJ,LiF,WangX.RGDconjugatedliposome-hollowsilicahybridnanovehiclesfortargetedandcontrolleddeliveryofarsenictrioxideagainsthepaticcarcinoma.Internationaljournalofpharmaceutics.2017Mar15;519(1):250-62.
6.AbuchowskiA,KazoGM,VerhoestJrCR,VanEsT,KafkewitzD,NucciML,ViauAT,DavisFF.Cancertherapywithchemicallymodifiedenzymes.I.Antitumorpropertiesofpolyethyleneglycol-asparaginaseconjugates.Cancerbiochemistrybiophysics.1984Jun;7(2):175-86.
7. SartoreL,CalicetiP,SchiavonO,VeroneseFM.EnzymemodificationbyMPEGwithanaminoacidorpeptideasspacerarms.Appliedbiochemistryandbiotechnology.1991Jan1;27(1):45-54.
8. ShahinM,SoudyR,El-SikhryH,SeubertJM,KaurK,LavasanifarA.Engineeredpeptidesforthedevelopmentofactivelytumortargetedliposomalcarriersofdoxorubicin.Cancerletters.2013Jul1;334(2):284-92.
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DAS-ELISA试剂盒中抗体有包被抗体和酶标抗体，这两种抗体是一种抗体，前者没有酶标，后者用酶进行了标记。
NCM-ELISA一般有一抗和二抗，这两种抗体不一样，前者（一抗）一般是鼠抗体或兔抗体，没有酶标，相对应的二抗一般是羊抗鼠或羊抗兔抗体，用酶进行标记。也有用马大规模备抗体的。
TDS-ELISA是三抗体ELISA，第三级的抗体是将信号级联放大。
以上三种是比较常见的，还有其他一些试剂盒，可参考免疫学的内容。
首先要什么有什么的，你得好好考虑一下。
2、看生产地址
根本没有生产地址，我们知道做实验做产品需要很多的仪器、试剂、耗材，没有人相信一间简单的屋子可以生产各种样的试剂盒。
3、看产品包装
没有任何的生产地址、联系方式等信息，这种产品有问题了连个投诉的地方都没有。
4、看公司网站
有些打着国外原装旗号，整个公司网站为英文页面，实际注册IP地址在中国。如果写着国外的地址，让你国外的朋友实地去看一下！
5、做交叉验证
拿对方提供的几个种类的试剂盒，把里面的关键组份相互替换做做实验，如果交叉严重，只能说明是一种原料生产的试剂盒贴了不同的标签。
6、看价格
价格低得离谱，却打着进口大公司原料分装，核算成本，这种低得离谱的价格是连原料都买不起的。
