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。
ELISA的基础是抗原或抗体的固相化及抗原或抗体的酶标记。结合在固相载体表面的抗原或抗体仍保持其免疫学活性，酶标记的抗原或抗体既保留其免疫学活性，又保留酶的活性。在测定时，受检标本（测定其中的抗体或抗原）与固相载体表面的抗原或抗体起反应。用洗涤的方法使固相载体上形成的抗原抗体复合物与液体中的其他物质分开。再加入酶标记的抗原或抗体，也通过反应而结合在固相载体上。此时固相上的酶量与标本中受检物质的量呈一定的比例。加入酶反应的底物后，底物被酶催化成为有色产物，产物的量与标本中受检物质的量直接相关，故可根据呈色的深浅进行定性或定量分析。由于酶的催化效率很高，间接地放大了免疫反应的结果，使测定方法达到很高的敏感度。
1．取出试剂盒室温平衡30min，取出血样放至室温。
2．配标准品：取150uL标准品加入150uL标准品稀释液稀释，依次稀释5次。
3．加样：分别于各反应孔中加入标准品50uL，样品40UL，标准品做复孔，样品做3孔。
4．分别于样品孔中加入10UL抗体。
5．标准品和样品孔中分别加入50uL链酶亲和素-HRP，盖上封板膜,轻轻震荡混匀，37℃温育60min。
6．配洗涤液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用。
7．洗涤：小心揭开封板膜，弃去液体，甩干，每孔加200uL洗涤液，静置30s后弃去，如此重复5次，拍干。
8．显色：每孔先加入显色剂A 50uL，再加显色剂B 50uL，轻轻震荡混匀，37℃避光显色6min。
9． 终止：每孔加入终止液50uL，终止反应（此时蓝色立即转为黄色）。
10．测定：以空白孔调零，450nm波长依序测量各孔的吸光度。测定应在加终止液10min之内进行。
11．保存结果，收拾桌面。
12．分析处理数据
注意事项
1. 取出板条前恢复到室温后再打开外包装袋，实验中不用的板条立即放回包装中，密闭封口，其余不用试剂应盖好。
2. 实验操作中请使用一次性的吸头，避免交叉污染。
3．实验板孔加入试剂的顺序应一致，以保证所有反应孔的孵育时间一致。
4．洗涤过程中反应孔中残留的洗涤液应在滤纸上充分拍干，勿将滤纸直接放入反应孔中吸水。
5．试剂盒内试剂请在保质期内使用，不同批号试剂不要混用。
6．1000pg/ml以上的结果为非线性的，根据此标准曲线无法得到精确的结果。大于1000pg/ml 的样品应以标准稀释缓冲液稀释后重做。在结果分析时，结合考虑相应的稀释度。
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。
