Description
InordertodeveloparapidandstraightforwardcouplingprocedureatthePEGterminus,amethodofdirectcouplingantibodiestothePEGterminusofliposomeswasintroducedbyBendasetal.Inthismethodology,antibodiesaresimplyattachedtothePEGterminusofliposomes,whichhadbeenendgroup-functionalizedwithcyanuricchloride,inmildbasicconditions(pH8.8)withoutpriorantibodyderivatizations.Ithasbeenshownthatinordertoobtainastableattachmentofproteinsonliposome,theDSPE-PEG-cyanurwasaddedintotheliposomestochemicallyconjugatewithproteinstoformastablecomplexandminimizethedenaturationofproteins.
Proteinscanbecovalentlycoupledtotheliposomesviaamine-reactivecyanur-groups,eitherdirectlytothevesiclesurfaceusingcyanuricchloride-activatedDSPE(cyanur-DSPE)ortothedistalendsofPEG-spacersusingactivatedcyanur-PEG-PE(ammoniumsalt).CyanuricchlorideatthePEGterminusfunctionstolinkpeptides,antibodiesandotheramine-containingbiomoleculesornanoparticlesviaanucleophilicsubstitutionreactionunderbasicconditions.Antibodiesorotherproteinscanbeconjugatedwithoutanypreviousderivatization.
Immunodox®-CyanurisaPEGylatedproduct.Forotheraminereactive(PEGylatedandnon-PEGyalatedproducts)andalsoImmunodox®productssuitableforothertypesconjugationmethodsseehere.
FormulationInformation
Immunodox®-Cyanur(PEGylated)
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 15.92mg/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)-Cyanur | 0.65 | 0.22 | 1 |
| Buffer,LiposomeSizeandEncapsulatedDrugConcentration | Specification |
|---|---|
| Buffer | BorateBuffer |
| pH | 8.8 |
| LiposomeSize | 100nm |
| EncapsulatedDoxorubicin | 2mg/ml(3.45mM) |
ConjugationProtocol
MaterialsandEquipment
Inordertoconjugateyourantibody,protein,peptideorligandtoImmunodox®-Cyanur(PEGylated)youwillneed:
- Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein/peptide/ligand.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnsordialysiscassettes.IfyoudonotusethecorrectMWCO,youcanloseyourentireprep.InthiscasewerecommendusingadialysiscassettewithMWCOof300,000dalton.
- Boratebufferyoucaneithermaketheboratebufferorpurchaseitfromachemicalvendor.Inanycase,youneedtomakesurethatthepHisadjustedto8.8.
PreparationMethod
- ThetotallipidconcentrationinImmunodox®-Cyanuris21.58mM.1%molofthelipidinliposomescontainsPEG-Cyanurgroupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.Fora2mlvolumeliposomethisisequalto2.2×10-7molandfor5mlvolumeliposomesthisisequalof5.50×10-7molofPEG-Cyanur.
- Add1:1000molarratioofantibody,protein,peptideorligandtototallipid.Thiswillbeequalto1:5molarratioofantibody,protein,peptideorligandtoPEG-Cyanurlipid.Forexampleina2mlkit,for2.2×10-7molofPEG-Cyanurlipid,4.4×10-8molofantibody,protein,peptideorligandisneeded.
- TheconjugationhastobedoneundermildbasicconditionsuchaboratebufferpH8.8.Dissolveyourantibody,protein,peptideorligandinboratebufferpH8.8.
- IncubateImmunodox®-Cynaurwithantibody,protein,peptideorligandfor16hoursatroomtemperature.
- Removenon-conjugatedantibody,protein,peptideorligandbydialysis.Wepreferdialysistosizeexclusioncolumns.Dialysisisamuchslowerprocessbuttherewillbeminimumlossofimmunoliposomesaftertheprepiscleanedfromnon-conjugatedprotein/peptide/ligand.Spincolumnsaremuchfaster,butyoucaneasilyloseover50%oftheliposomesonthespincolumn.WerecommendusingFloat-A-Lyzer®dialysiscassettefromSpectrumLabs.YouneedtochooseacassettewithproperMWCOdependingontheMWofyourprotein,ligand,antibodyorantibodyfragment.InthiscasewerecommendusingadialysiscassettewithMWCOof300,000dalton.NOTE:IfyoudecidetouseadialysiscassetteyouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesandspincolumnsblindly.Theycomeinvarioussizesandyouneedtochoosethecorrectsizewisely.Dialyzetheimmunoliposomesolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletisdialyzeforanother8hours.Afterthisstep,yourcleanedupimmunoliposomeisreadytobeused.
LiposomeParticleCalculator
Immunodox®liposomesareunilamellarandsizedto100nm.Themolarconcentrationofliposomeis21.58mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.
TechnicalNotes
- Doxorubicinisafluorescentmoleculewithλex470nmandλem585nm.Ifyouareusingafluorescenttagonyourantibodyorligand,thenyouneedtomakesurethattheywillnotinterferewitheachother.
- Trisbuffershouldneverbeusedinanystepoftheprocesssinceitcontainsamine.
- Cyanuricchlorideisconsideredasasensoryrespiratoryirritant.However,despitethenameofthecyanur-modifiedliposomes,theyhavenotshownanysignofacute,chronicorgenotoxicity.
- Cyanurgroupsareamine-reactive,however,somerandomattachmentsoftheantibodiescanbeexpectedsincecyanuricchloridecanreactwithawiderangeofnucleophilicfunctionalities,suchasalcoholsandthiols.Thismayinterferewiththebindingoftheantibodytotheliposome,andtherefore,thebindingaffinitywouldchange.
- SizeexclusionspincolumnssuchasSepharose®CL-4BcanbeusedinsteadofFloat-A-Lyzer®dialysiscassette.However,averylargeamountofliposomeswillsticktothecolumnduringthecleanupprocessandthereforewestronglysuggestusingdialysisthansizeexclusivebeads.
- 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®-Cyanurisaredtranslucentliquidmadeofnanosizeunilamellarliposomes.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®-Cyanurismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin4monthsofthemanufacturingdate.
ReferencesandbackgroundreADIng
1.BendasG,KrauseA,BakowskyU,VogelJ,RotheU.TargetABIlityofnovelimmunoliposomespreparedbyanewantibodyconjugationtechnique.Internationaljournalofpharmaceutics.1999Apr20;181(1):79-93.
2.LeeHY,MohammedKA,KayeF,SharmaP,MoudgilBM,ClappWL,NasreenN.Targeteddeliveryoflet-7amicroRNAencapsulatedephrin-A1conjugatedLiposomalnanoparticlesinhibittumorgrowthinlungcancer.Internationaljournalofnanomedicine.2013;8:4481.
3. NyanhongoGS,SteinerW,GübitzGM,editors.BiofunctionalizationofPolymersandtheirApplications.SpringerScience&BusinessMedia;2011Aug6.
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的试剂：免疫吸附剂、结合物和酶的底物。
完整的ELISA试剂盒包含以下各组分：
（1）已包被抗原或抗体的固相载体（免疫吸附剂）；
（2）酶标记的抗原或抗体（结合物）；
（3）酶的底物；
（4）阴性对照品和阳性对照品（定性测定中），参考标准品和控制血清（定量测定
中）；
（5）结合物及标本的稀释液；
（6）洗涤液；
（7）酶反应终止液。
elisa试剂盒回收率是反应待测物在样品分析过程中的损失的程度，损失越少，回收率越高，如果作标液1PPM，就是1毫克/升，而作出标准数据为0.99毫克/升，就是说你的回收率是99%，这个与真实成分有密切的关系，说明方法的准确度。比如水中总无机氯含量测定，样品水中含有无机氯20mg/L，取100mL被测水样品，加入0.1mL浓度为10mg/mL的含无机氯标准样品，测定时忽略体积变化，如果测定出样品中无机氯为2.98mg/L，则认为回收率为99%。向左转|向右转
2、规范包被操作，吸附是否均匀
3、重复性、可靠性
6、是否提供技术
7、适用于血浆、血清、组织匀浆液、细胞培养上清液、尿液等多种类型的样本
8、可检测动物类型是否丰富
9、可检测指标是否齐全
