Description
Clodrosome®isamultilamellarliposomesUSPensioninwhichClodronateisencapsulatedintheaqueouscompartmentsoftheliposomes.Encapsome®isformulatedandpreparedidenticallytoClodrosome®exceptthatclodronateisnotaddedtotheliposomes.Theliposomesarefilteredthrough2μmpolycarbonatemembranestoensurethatthelargerparticles,whichmaybetoxictoanimals,areremovedfromthesuspension.Botharepreparedandpackagedundersterileconditions.WhenanimalsorcellsaretreatedwithClodrosome®,phagocyticcellsrecognizetheliposomesasinvADIngforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosol,resultingincelldeath.Non-encapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.
Controlliposomes(Encapsome®)arerecognizedandphagocytosedbythesamemechanismasClodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofcontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
Fluorescentliposomes(Fluoroliposome®)suitableformacrophagetargetingandtrackingareavailablecontainingfivedifferentfluorescentdyes(DiI,DiO,DiD,DiAandDiR)thatcoverstheentirespectrum.Fluorescentliposomescomeinstandardandmannosylatedform.Formoreinformationsee here.
m-Clodrosome®andm-Encapsome®aremannosylatedreagentsthatarespecificallyformulatedtoefficientlytargetmacrophagesincentralnervoussystemsandmacrophagesthatcontainmoremannosereceptors.Formoreinformationaboutthesereagentseehere.
FormulationInformation
Clodrosome®LiposomalClodronateSuspension
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 23mg/ml | 35.1mM | 100 |
| L-alpha-Phosphatidylcholine | 18.8 | 24.3 | 70 |
| Cholesterol | 4.2 | 10.9 | 30 |
| EncapsulatedDrug | Concentration |
|---|---|
| Clodronate((Dichloro-phosphono-methyl)phosphonate),DisodiumSalt | 18.4*mM |
| *Dependingonthetypeoftheclodronatesalt,itsconcentration(mg/ml)varies.Iftetrahydratesaltisused,theconcentrationoftheencapsulateddrugwillbe~7mg/ml,andifanon-hydratedsaltisused,theconcentrationwillbe~5mg/ml. | |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
TechnicalNotes
- WhenanimalsorcellsaretreatedwithClodrosome®,phagocyticcellsrecognizetheliposomesasinvadingforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Unencapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.
- Encapsome®controlliposomesarerecognizedandphagocytosedbythesamemechanismasClodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofthecontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
- Theproductmustberemovedfromthevialusingsteriletechnique.Donotuseifsterilityiscompromised.Thisisparticularlyimportantifasinglevialisaccessedmultipletimesoverseveralweeks.Theproductshouldnotbeusedmorethan60daysafterreceipt,evenifunopened.
- Liposomesmaysettlewhenleftundisturbedformorethanafewhours.Immediatelypriortouse,inordertoensureahomogeneousliposomesuspension,slowlyinvertthevialseveraltimesuntilthesuspensionappearshomogeneousbyvisualinspection.Vigorousorerraticshakingwillnotdamagetheliposomesbutmayinducefoamingandbubbleformationmakingitmoredifficulttoaccuratelymeasurethedesireddosage.
- Ifthepersonnelperformingintravenousinjectionsarenotexperiencedinorfamiliarwith,precautionsforinjectinglargervolumes(~10%animalweightinml),viscousliquidsorparticulatesuspensions,considerhavingextraanimalsavailableincaseseriousinjection-relatedadverseeventsoccur.Dosecontrolanimalsfirsttobecomefamiliarwithlargevolumeinjections.
- WithinhoursaftersystemicadmiNISTrationofClodrosome®,animalsbegintoloseimportantcomponentsoftheirimmunesystem.Standardanimalhandlingandhousingprotocolsarenotsuitableforimmunocompromisedanimals.Evenwhensuchprecautionsaretaken,monitorthegeneralhealthofeachanimalforopportunisticinfectionsunrelatedtotheexperimentalprotocol.Thereisnoinherenttoxicitytotheproductattherecommendeddoselevels.
- Whendosingintravenously,usestandardprecautionsfordosinglargervolumestoanimalsincludingthefollowing:a)warmproducttoroomtemperaturepriortodosing;b)ensurethatallairbubblesareremovedfromthesyringepriortodosing.Intravenousinjectionofairbubblesmayresultinairemboliwhichcankillorseriouslyinjureanimals;c)injectproductataslow,steadyrateofnomorethan1ml/min;d)decreaseinfusionrateifanimalsdisplayanyatypicalreactionssuchasunusualagitation.
- Infusion-relatedadversereactionsusuallyinvolvetheanimalgaspingforairorotherseizure-likemovements.Animalsoftenrecoverwithnoapparentpermanentinjury,butanypotentialeffectsonexperimentalresultsmustbeassessedbytheresearcher.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Dosage
Appearance
Clodrosome®andEncapsome®arebothwhitemilkysuspensionsmadeoflargemicrosizemultilamellarliposomes.Duetotheirlargesize,someliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,Encapsome®willphaseseparateandformpelletsinthebottomofthevialleavingaclearsolutionontop.Clodrosome®mightdothesameonlynotasseverely.Therefore,bothshouldbegentlyshakennottoformbubblesbuttoformahomogeneoussolutionpriortouse.
RoutesofAdministration
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EducationalVideos
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
Clodrosome®andEncapsome®shouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.Ifthesuspensionisfrozen,clodronatecanbereleasedfromtheliposomesthuslimitingitseffectivenessindepletingmacrophages.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.
ShelfLife
Clodrosome®andEncapsome®aremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.
Referencesandbackgroundreading
1.HinsonSR,CliftIC,LuoN,KryzerTJ,LennonVA.Autoantibody-inducedinternalizationofCNSAQP4waterchannelandEAAT2glutamatetransporterrequiresastrocyticFcreceptor.ProceedingsoftheNationalAcademyofSciences.2017May23;114(21):5491-6.
2.DhupkarP,GordonN,StewartJ,KleinermanES.Anti‐PD‐1therapyredirectsmacrophagesfromanM2toanM1phenotypeinducingregressionofOSlungmetastases.CancerMedicine.2018May7.
3.XiongY,PageJC,NarayananN,WangC,JiaZ,YueF,ShiX,JinW,HuK,DengM,ShiR.Peripheralneuropathyandhindlimbparalysisinamousemodelofadipocyte-specificknockoutofLkb1.EBioMedicine.2017Oct1;24:127-36.
4.CriderA,FengT,PandyaCD,DavisT,NairA,AhmedAO,BabanB,TureckiG,PillaiA.Complementcomponent3areceptordeficiencyattenuateschronicstress-inducedmonocyteinfiltrationanddepressive-likebehavior.Brain,behavior,andimmunity.2018Mar5.
5.KocherT,AsslaberD,ZaborskyN,FlenadyS,DenkU,ReinthalerP,AblingerM,GeisbergerR,BauerJW,SeiffertM,HartmannTN.CD4+Tcells,butnotnon-classicalmonocytes,aredispensableforthedevelopmentofchroniclymphocyticleukemiaintheTCL1-tgmurinemodel.Leukemia.2016Jun;30(6):1409.
6.ZhuZ,DingJ,MaZ,IwashinaT,TredgetEE.Systemicdepletionofmacrophagesinthesubacutephaseofwoundhealingreduceshypertrophicscarformation.WoundRepairandRegeneration.2016Jul1;24(4):644-56.
7.HaqueMR,LeeDY,AhnCH,JeongJH,ByunY.Localco-deliveryofpancreaticisletsandliposomalclodronateusinginjectablehydrogeltopreventacuteimmunereactionsinatype1diabetes.Pharmaceuticalresearch.2014Sep1;31(9):2453-62.
8.MayoL,CunhaAP,MadiA,BeynonV,YangZ,AlvarezJI,PratA,SobelRA,KobzikL,LassmannH,QuintanaFJ.IL-10-dependentTr1cellsattenuateastrocyteactivationandamelioratechroniccentralnervoussysteminflammation.Brain.2016May31;139(7):1939-57.
9.KermanizadehA,ChauchéC,BalharryD,BrownDM,KanaseN,BoczkowskiJ,LanoneS,StoneV.TheroleofKupffercellsinthehepaticresponsetosilvernanoparticles.Nanotoxicology.2014Aug31;8(sup1):149-54.
10.NandiB,ShapiroM,SamurMK,PaiC,FrankNY,YoonC,PrabhalaRH,MunshiNC,GoldJS.StromalCCR6drivestumorgrowthinamurinetransplantablecoloncancerthroughrecruitmentoftumor-promotingmacrophages.Oncoimmunology.2016Aug2;5(8):e1189052.
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1、直接竞争，标记抗原，与检测样品中的抗原竞争抗体。
2、间接竞争，标记抗体，固相抗原与液相抗原（样品）竞争标记抗体。
3、定义
间接竞争法的模型：包被抗原，用HRP-抗体与样本一起加入。样本中的Ag与Solid-Ag竞争HRP-Ab，固相吸附的HRP-Ab与样本中的Ag浓度成反比。
直接竞争法的模型：包被抗体，用HRP-抗原与样本一起加入。样本中的Ag与HRP-Ag竞争Solid-Ab，固相吸附的HRP-Ag与样本中的Ag浓度成反比。
4、竞争法的理论基础：是限量抗体。只有在限量抗体基础上，两种抗原才会形成竞争关系。
5、、间接竞争法具备较高灵敏度原因。
直接竞争法里，标记抗原与待测抗原均是液相，与抗体的结合机会是一样的，例如有1份标记抗原与1份待测抗原竞争1份抗体，那么有50%的标记抗原能与抗体结合，所以标记抗原的相对结合率为50%。间接法里，固相抗原与抗体的接触面积较小，固相抗原与待测抗原的结合抗体机会是不平等的，接近顺序饱和法，即只有与待测抗原结合剩余的抗体才会与固相抗原结合，同样有1份固相抗原与1份待测抗原竞争1份抗体时，基本上抗体会被待测抗原中和掉，与固相抗原结合的抗体非常少。固相抗原的相对结合率为0%。因此，间接法的抑制曲线斜率会大于竞争法。
因为抑制率越大则斜率越大，从而灵敏度越大。（假设零管变异5%，以两倍SD为灵敏度限，则为90%相对结合率，则间接法可以在较低的待测抗原浓度达到这一相对结合率，因此灵敏度要高。）
在进行系统放大时，间接法一般可以使用酶标二抗。因为二抗可以针对抗体的多个部位，所以存在放大效应，从而能提高间接法的灵敏度。直接法一般难以进行放大，常用的有生物 素化抗原与酶标亲和素，但模式上似乎不存在放大效应。
6、间接法的高灵敏度难以实现的原因：
双抗体 夹心的免放（IRMA）模式刚出现时，也被模型证明灵敏度优于竞争法的放免（RIA），原因也是较大的斜率，但是IRMA的高灵敏度一直到单抗发展后才得以实现。
GM实验：试剂说明书上要求20分钟显色，10分钟的时候看一下，差不多就可以加终止液，但是我的标曲显色特别快，2分钟最低浓度蓝色就就深了，此时样本颜色还很浅很浅，我只能一块儿加终止液。所以，标曲显色过快，可能是什么原因？
（ps:第一列是标曲，后面是样本）
采用抗-HBe抗体包被反应板，加入校准品及被测样本，同时加入定量HBeAg中和抗原，经过振荡孵育,洗板后再加入铕标记的抗-HBe，若标本中抗-HBe浓度高，HBeAg将被大量中和，使最后形成的抗-HBe-HBeAg-铕标记抗-HBe复合物减少。增强液（β－NTA）将标记在抗体上的Eu3+解离到溶液中，Eu3+和增强液中的有效成分形成高荧光强度的螯合物，荧光强度和样本中的抗-HBe浓度成反比。
