Description
m-Clodrosome®isamultilamellarliposomesUSPensioninwhichClodronateisencapsulatedintheaqueouscompartmentsofthemannosylatedliposomes.m-Encapsome®isformulatedandpreparedidenticallytom-Clodrosome®exceptthatclodronateisnotaddedtotheliposomes.Theliposomesarefilteredthrough2μmpolycarbonatemembranestoensurethatlargerparticles,whichmaybetoxictoanimals,areremovedfromthesuspension.Botharepreparedandpackagedundersterileconditions.Whenanimalsorcellsaretreatedwithm-Clodrosome®,phagocyticcellsrecognizetheliposomesasinvADIngforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Non-encapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.m-Encapsome®-controlliposomes-arerecognizedandphagocytosedbythesamemechanismasm-Clodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofcontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
Mannosereceptortargetingbymannosylatedliposomeshasbeendemonstratedforavarietyofmannosylatedlipidconjugatesinavarietyofliposomemorphologiesandcompositionsinseveraldifferentinvitroandinvivomodels.Thereareseveralpublicationsaboutusingahydrophobicderivativeofmannose(4-aminophenylalpha-D-mannopyranoside)ratherthanusingamannosylatedlipidinclodronateliposomes.Thisismainlyduetothehighcostandcomplexityofsynthesizingandconjugatingmannosetolipid.4-aminophenylalpha-D-mannopyranosideiscommerciallyavailableandfarlessexpensivethansynthesizingmannoseconjugatedlipid.
Whymannose?Mannoseisoneofthecarbohydratecomponentsofmanybacterialandviralcellsurfaces,thereforetheever-efficient,highlyredundantimmunesystemhasevolvedmultiplemechanismsforidentifyingpathogensbasedonmannoserecognition.Theanimalandplantkingdomslikewiseutilizecarbohydraterecognitionsignalingmechanismsincludingmannoseresidues.Manypublicationsevaluateothercarbohydratesastargetingmechanismsforvariouscelltypes,howevermannosetargetingtophagocytesappearstobeoneofthemorespecificmechanismsidentifiedtodate.Mammaliancellsurfaceidentificationmoleculesbasedonmannosebinding,suchastheICAMfamilyofleukocyteadhesionmolecules,targettheSIGNfamilyofmannosereceptorstoaccomplishself-recognitioninvivo.
Awell-knownandcitedstudybyUmezawa&Eto [1]demonstratesthatliposomescontainingaminophenylmannosideweremostefficientlyincorporatedintothemousebrainacrossthebloodbrainbarrier.Theradiolabeledliposomesbearingaminophenyl-alpha-D-mannopyranosideweremaximallyincorporatedintothemousebrainafter48hours,whereasinthespleenandliver,theseradioactivitiesweremaximumafter12hours.Thestudiesalsoshowedthatliposomesweremostincorporatedwasglialcellsratherthanneuronalcell.Thesubcellularfractionationstudyindicatesthatmannoselabeledliposomesareincorporatedintolysosomesrichfractionbothinliverandbrain.
Mannosylatedfluorescentliposomes(m-Fluoroliposome®)suitableformacrophagetargetingandtrackingareavailablecontainingfivedifferentfluorescentdyes(DiI,DiO,DiD,DiAandDiR)thatcoverstheentirespectrum.Fluorescentliposomescomeinstandardandmannosylatedform.Formoreinformationsee here.
Clodrosome®andEncapsome®arestandardandnon-mannosylatedreagentsthatareusedfortargetingmacrophagesinorgansandtissuesotherthancentralnervoussystems.Formoreinformationaboutthesereagentseehere.
FormulationInformation
m-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. | |
| Mannosylation | Concentration |
|---|---|
| 4-Aminophenyl-alpha-D-mannopyranoside | 9.53mol% |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
TechnicalNotes
- Toreachbloodstream-accessIBLe,mannose-receptorpositivecellsoutsidetheliver,asignificantnumberofliposomeswillhavetoescapefirst-passuptakebytheliverandspleen,sothatthetargetcellsareexposedtoahigherconcentrationofmannosylatedliposomesfromtheblood.Onestrategythathasbeenusedtoensurethatliposomesescapetheliverandspleenisknownasreticuloendothelialsystem(RES)blockadeinwhichanimalsarepre-dosedwithasufficientquantityofliposomestotemporarilysaturatethephagocyticcellsoftheblood,liverandspleen,alsoknownasthereticuloendothelialsystem(RES)orthemononuclearphagocytesystem(MPS).Thissufficientquantityisdependentupontheliposometypeandcompositionaswellasthespeciesbeingdosed;thepre-dosedliposomesdonotnecessarilyneedtobethesametypeorcompositionasthetherapeuticordiagnosticliposomesavoidingtheRES.Soonafterthispre-doseisclearedfromthebloodstream(usuallywithinacoupleofhours),theliposomesofinterestaredosed.SincetheRESisinvolvedindigestingthepreviousdoseofliposomes,thesubsequentlydosedliposomeswillremaininthecirculationmuchlongerthusbemuchmorelikelytobindtotheirtargetsiteoutsidetheRESincludingthosephagocyticcellswhichareaccessible,butarenotusuallyexposedtoahigherconcentrationofliposomes.
- WhileRESblockadeisusuallythoughtofassaturatingphagocyticcells,ithasbeenshownthatopsonin-bindingbyliposomesisasaturablephenomenon.Therefore,partofRESblockademayinvolveserumdepletionofcomplementandotheropsoninsknowntocoatliposomes.Inthecurrentapplication,removalorreductionintheconcentrationofsolublemannose-receptorsmayfurtherincreasetheprobABIlityofamannosylatedliposomebeingabletointeractwithmannosereceptorsonthetargetcell.Therefore,ifthegoalistodepleteatargetsubsetofmannose-receptor+cellswhichmaynotnormallybeexposedtoasubstantialnumberofmannosylatedliposomes,pre-dosingwithmannosylatedclodronateliposomes,inordertobothsaturatetheblood,liverandspleenphagocytesandreducetheconcentrationofopsoninsincludingsolublemannosereceptors,shouldincreasethenumberofsubsequentlydosedmannosylatedclodronateliposomesavailabletothistargetsubsethypotheticallyresultinginincreaseduptakeanddepletionbythesetargetedcells.
- 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
m-Clodrosome®andm-Encapsome®arebothwhitemilkysuspensionsmadeoflargemicrosizemultilamellarliposomes.Duetotheirlargesizesomeliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,m-Encapsome®willphaseseparateandformpelletsinthebottomofthevialleavingaclearsolutionontop.m-Clodrosome®mightdothesameonlynotasseverely.Therefore,bothshouldbegentlyshakennottoformbubblesbuttoformahomogeneoussolutionpriortouse.
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
m-Clodrosome®andm-Encapsome®shouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.Ifthesuspensionisfrozen,clodronatecanbereleasedfromtheliposomesthuslimitingitseffectivenessindepletingmacrophages.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.
ShelfLife
m-Clodrosome®andm-Encapsome®aremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.
Referencesandbackgroundreading
1.UmezawaFA,EtoY.Liposometargetingtomousebrain:mannoseasarecognitionMarker.Biochemicalandbiophysicalresearchcommunications.1988Jun30;153(3):1038-44.
2.ItoT,IshigamiM,MatsushitaY,HirataM,MatsubaraK,IshikawaT,HibiH,UedaM,HirookaY,GotoH,YamamotoA.SecretedEctodomainofSIGLEC-9andMCP-1SynergisticallyImproveAcuteLiverFailureinRatsbyAlteringMacrophagePolarity.Scientificreports.2017Mar8;7:44043.
3.MironVE,BoydA,ZhaoJW,YuenTJ,RuckhJM,ShadrachJL,vanWijngaardenP,WagersAJ,WilliamsA,FranklinRJ.M2microgliaandmacrophagesdriveoligodendrocytedifferentiationduringCNSremyelination.Natureneuroscience.2013Sep;16(9):1211.
4.AndreouK,SarmientoSotoM,AllenD,EconomopoulosV,deBernardiA,LarkinJ,SibsonNR.Anti-InflammatoryMicroglia/MacrophagesasaPotentialTherapeuticTargetinBrainMetastasis.Frontiersinoncology.2017;7:251.
5.AlishekevitzD,Gingis-VelitskiS,Kaidar-PersonO,Gutter-KaponL,SchererSD,RavivZ,MerquiolE,Ben-NunY,MillerV,Rachman-TzemahC,TimanerM.Macrophage-inducedlymphangiogenesisandmetastasisfollowingpaclitaxelchemotherapyisregulatedbyVEGFR3.Cellreports.2016Oct25;17(5):1344-56.
6.OhSH,KimHN,ParkHJ,ShinJY,BaeEJ,SunwooMK,LeeSJ,LeePH.Mesenchymalstemcellsinhibittransmissionofalpha-synucleinbymodulatingclathrin-mediatedendocytosisinaParkinsonianmodel.Cellreports.2016Feb2;14(4):835-49.
7.KanoF,MatsubaraK,UedaM,HibiH,YamamotoA.SecretedEctodomainofSialicAcid‐BindingIg‐LikeLectin‐9andMonocyteChemoattractantProtein‐1SynergisticallyRegenerateTransectedRatPeripheralNervesbyAlteringMacrophagePolarity.STEMCELLS.2017Mar1;35(3):641-53.
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人FSH放免测试单位浓度为mIU/ml,可否换算成ng/ml,如何换算
非常感谢
一般来说，试剂盒曲线上的最小浓度是比较准确的，低于这个值因为与曲线是个“S"型结构有关，所以结果是有偏差的，是一种估算。再加上实验误差，所以达不到理想的效果。建议选择试剂盒时，应该看曲线上最小的浓度值，而不是试剂盒上写的灵敏度。
虽然酶活性调节ELISA方法的灵敏度目前并不十分理想，但在酶活性放大ELISA中，检测的灵敏度远比RIA高。根据质量作用定律。即免疫反应所形成的免疫复合物量与反应物浓度成正比。推测所检测的待测物分子数为1。已知1个摩尔浓度含6.02×1023个分子，那么理论推测酶活性放大ELISA方法的最低检测限可达1.7×10-24mol/L。虽然在实际应用中由于反应条件和试剂纯度以及仪器精度等因素的影响，往往达不到这个水平(大于104个分子)，但表明ELISA在灵敏度方面的改进潜力是很大的。
基本原理：
在放射免疫分析的实验中，加入超量的标记抗原*Ag与未标记抗原Ag（即：待测抗原）与较少量的抗体（Ab）竞争性结合。
如果实验结果所计量到的结合物（*Ag-Ab）放射活性较高，表示待测物的浓度较低。
如果所计量到的结合物放射活性较低，则表示待测物的浓度较高。 藉由标准 曲线图的分析，可以推算出待测物的浓度。
1960年，美国学者Yalow 和Berson 创立了放射免疫分析（Radioimmunoassay，RIA），并首先用于糖尿病人血浆中胰岛素含量的测定。这是医学和生物学领域中方法学的一项重大突破，开辟了医学检测史上的一个新纪元。它使得那些原先认为是无法测定的极微量而又具有重要生物学意义的物质得以精确定量，从而为进一步揭开生命奥秘打开了一条新的道路，使人们有可能在分子水平上重新认识某些生命现象的生化生理基础。其后30年中，内分泌科学的飞速进展，充分证明了这一超微量分析技术的巨大推动力。1977年，这项技术的发明者荣获诺贝尔生物医学奖。随后这一崭新的技术迅速渗透到医学科学的其它领域，如病毒学、药理学、血液学、免疫学、法医学、肿瘤学等，以及与医学生物学相关的学科，如农业科学、生态学及环境科学等。放射免疫分析的物质，由激素扩大到几乎一切生物活性物质。我们放射免疫分析研究起步于1962年，并迅速发展与普及，对我国生物医学的进展起着很大的促进作用。 （一）RIA的优点
放射免疫分析具有许多其它分析方法无可比拟的优点。它既具有免疫反应的高特异性，又具有放射性测量的高灵敏度，因此能精确测定各种具有免疫活性的极微量的物质。
1．灵敏度高一般化学分析法的检出极限为10～10g,而RIA通常为10（毫微克，ng）、10g（微微克,pg），甚至10g(毫微微克，fg)、10g(微微微克，ag)。
2．特异性强由于抗原—抗体免疫反应专一性强，所被测物一定是相应的抗原。良好的特异性抗体，能识别化学结构上非常相似的物质，甚至能识别立体异构体。
3．应用范围广据不完全统计，目前至少已有300多种生物活性物质已建立了RIA。它几乎能应用于所有激素的分析（包括多肽类和固醇类激素），还能用于各种蛋白质、肿瘤抗原、病毒抗原、细菌抗原、寄生虫抗原以及一些小分子物质（如环型核苷酸等）和药物（如地高辛、毛地黄甙等）的分析，应用范围还在不断扩展。近年来由于小分子半抗原制备抗体的技术有很大的发展，有人预测几乎所有的生物活性物质，只要其含量不低于RIA的探测极限，都可建立适当的RIA法。
4．操作简便RIA所需试剂品种不多，可制成配套试剂盒；加样程序简单一次能分析大量标本，标本用量也少；反应时间不长；测量和数据处理易于实现自动化；RIA属体外分析技术，对患者无任何辐射危害。
（二）RIA的缺点
1．只能以免疫反应测得具有免疫活性的物质，对具有生物活性百失去免疫活性的物质是测不出的。因此RIA结果与生物测定结果可能不一致。
2．由于使用了生物试剂，其稳定性受多种因素影响，需要有一整套质量控制措施来确保结果的可靠性。
3．灵敏度受方法本身工作原理的限制，对体内某些含量特别低的物质尚不能测定。
4．由于放射免疫分析是竞争性的反应，被测物和标准物都不能全部参与反应，测得的值是相对量而非绝对量。
5．存在放射线辐射和污染等问题。
尽管RIA存在以上缺点，但它毕竟是定量分析方法的先进技术。随着科学技术的进步，放射免疫分析技术将会得到更加广泛、更加深入的发展。向左转|向右转
我使用的是北京北方生物技术研究所提供的KIT.盒子里面有睾酮标准品，兔抗睾酮抗体，带碘标记的睾酮，和驴抗兔免疫分离剂。实验过后我查了一下生物化学书，发现睾酮这种激素的化学本质非蛋白质，而是缁醇。这样问题就出来了：据我所知，产生免疫反应的是蛋白质，然而性类固醇激素的化学本质是缁醇。请问各位，缁醇也能产生免疫反应吗？
