Description:
REAADSMonoclonalFreeProteinS usesamonoclonalantibodyspecificforfreeProteinStomeasurementfreeProteinSlevelsincitratedhumanplasma.Nopretreatmentofsampleswithpolyethyleneglycol(PEG)isrequired.Resultsarereportedinpercent(%)ofnormal,relativetoareferenceplasmathathasbeenstandardizedagainsttheSecondaryStandardforCoagulation/InternationalSocietyonThrombosisandHemostasis (SSC/ISTH)preparation,whichiscalibratedtoWorldHealthOrganization(WHO)standards.
Advantages:
- UtilizesamonoclonalantibodyspecificforFreeProteinS
- ConvenientELISAprocedure
- Objective,accurateandreproducIBLe
- Reagentcompletekit
- Totalincubatetime:60minutesatroomtemperature
KitComposition:
Reagents
- 12x8MouseMonoclonalantibodytohumanFreeProteinScoatedmicrowells.
- 60mlSampleDiluent(blue-greensolution);containssodiumazide.
- 3x0.5mlLyophilizedReferencePlasma,withassaysheet.
- 12mlRabbitAnti-humanProteinSHRPConjugate(redsolution).
- 13mlSubstrate(TMBandH2O2).
- 15mlStoppingSolution(0.36Nsulfuricacid).
- 30mlWashConcentrate(33XPBSwith0.01%Tween20).Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.
Storeat2–8°C.DoNotFreeze.
MaterialsRequiredbutnotSupplied
- FreeProteinSControlPlasma.ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.
- Reagentgradewater(1L)topreparePBS/Tweenwashsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
- Graduatedcylinders
- Precisionpipettorscapableofdeliveringbetween5and1000microliters,withappropriatetips
- Miscellaneousglasswareappropriateforsmallvolumehandling
- Flaskorbottle,1liter
- Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
- Disposablegloves,powder-freerecommended
- PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreference,ifavailable)
- Multichannelpipettorscapableofdeliveringto8wellssimultaneously
- Microdilutiontubesforpatientsamplepreparation
- Centrifuge
PrincipleandProcedure:
Principle
REAADSMonoclonalFreeProteinSELISAusesamonoclonalantibodyspecificforfreeProteinStomeasurementfreeProteinSlevelsincitratedhumanplasma.Nopretreatmentofsampleswithpolyethyleneglycol(PEG)isrequired.Resultsarereportedinpercent(%)ofnormal,relativetoareferenceplasmathathasbeenstandardizedagainsttheSecondaryStandardforCoagulation/InternationalSocietyonThrombosisandHemostasis (SSC/ISTH)preparation,whichiscalibratedtoWorldHealthOrganization(WHO)standards.
Procedure
DilutedcitratedpatientplasmaisincubatedinmicrowellscoatedwithamonoclonalcaptureantibodyspecificforfreeProteinS,allowingpatientfreeProteinStobindtothesurface.Afterwashingtoremoveunboundplasmaproteins,HRP-conjugatedpolyclonalanti-humanProteinSdetectionantibodyisadded,whichattachestothesurfaceboundfreeProteinSantigenduringasecondincubation.Thewellsarewashed,andachromogenicsubstrateisadded,resultinginasolublecoloredproductthatismeasuredinaspectrophotometerat450nmaftertheadditionofstopsolution.TheconcentrationoffreeProteinSinthetestsampleisdeterminedfromastandardcurvepreparedfromthereferenceplasmaprovidedinthekit.Totalassayincubationtimeis60minutesatroomtemperature.
Performance:
ClinicalPerformance
Theclinicalperformancewasdeterminedbytestingplasmasamplesfrom35healthyindividualsand20patientswithknownProteinSdeficiencywithREAADSMonoclonalFreeProteinSassayandREAADSProteinSAntigentestkit(PEGmethod).Asshowninthetable,agoodcorrelationwasseenbetweenthetwomethodsforthecombinedtestpopulation(r=0.980),withaPvalueof0.739bysinglefactorAnova.
TechnicalPerformance
Intra-assayprecision,expressedin%CV,was4.7%whensamplesranginginvaluefrom6–150%weretestedinduplicatewithREAADSMonoclonalFreeProteinSassay.Inter-assayprecisionwasshowntobe5.2%.AccuracywasdemonstratedbytestingtherecoveryofplasmasamplesspikedwithknownlevelsoffreeProteinS;themean%recoverywas101.2%acrossthreeproductionlots.Linearitywasdeterminedbylinearregressionofthelog-logcurveexpressedasthecoefficientofdetermination(r2)=0.994.
REAADSMonoclonalFreeProteinSAssayisarapid,accurateandprecisemethodforthedeterminationoffreeProteinSlevelsinhumanplasma,offeringimprovedspecificity,convenienceandsignificanttimesavingsovertraditionalmethodthatrequirePEGprecipitation.
REAADSMonoclonalFreeProteinS | REAADSProteinSAssay(PEGMethod) | ||
Normals | Mean | 105% | 100% |
Range | 65–144% | 61–130% | |
Deficients | Mean | 20% | 22% |
Range | 8–40% | 12–34% | |
Correlation(r)=0.980;Pvalue=0.739 |
Background:
ProteinSisavitaminK-dependentproteinsynthesizedintheliver,vascularendothelium,andmegakaryocytes,whichplaysanimportantphysiologicroleintheProteinCAnticoagulantSystem.Thisanticoagulantsystemisoneofthemajorregulatorsofhemostasisbyinhibitingclotformationandbypromotingfibrinolysis.ProteinSfunctionsasacofactorforactivatedProteinConthevascularmembranetofacilitatethedegradationofclottingfactorsVaandVIIIa,down-regulatingclotformation.Innormalplasmaapproximately40%ofProteinScirculatesasafreemolecule,while60%iscomplexedwithC4b,aplasmaproteinoftheclassicalcomplementpathway.OnlyFreeProteinSisfunctionallyactiveandabletobindtoactivatedProteinC,whilethecomplexedformofProteinSisnot.
ProteinSdeficiency,eithercongenitaloracquired,mayleadtoseriousthromboticeventssuchasthrombophlebitis,deepveinthrombosis,orpulmonaryembolism.TheprevalenceofProteinSdeficiencyhasbeenestimatedtobelessthan1caseper300inthegeneralpopulation.Two-thirdsofpatientswithacongenitaldeficiencyofProteinS(levelslessthan50%ofnormal)maypresentwithvenousthrombosisinyoungadulthood.Inyoungpatients(<35years)withahistoryofthrombosis,theprevalencemaybeashighas15to18%.AcquiredProteinSdeficiencymaybeseenduringpregnancy,oralcontraceptiveororalanticoagulanttherapy,liverdisease,diabetesmellitus,postoperativecomplications,septicemia,andvariousinflammatorysyndromes.AdecreasedProteinSactivityinplasmamaybetheresultoflowconcentrationsorabnormalfunctionoftheProteinSmolecule.
ThelaboratorydiagnosisofProteinSdeficiencymayrequirebothquantitativeandqualitative(functional)determinations.QuantitativedeterminationsofProteinSAntigenarebasedonimmunologicproceduressuchasradialimmunodiffusioningel,Laurellrocketimmunoelectrophoresis,andenzyme-linkedimmunosorbentassay(ELISA).ELISAproceduresarelesslaborintensiveandofferseveraladvantagesincludingmoreobjective,accurate,andreproducibleresults.Inaddition,theELISAformatallowsautomationwithcommonlyavailablelaboratoryinstrumentation.
MeasurementofplasmalevelsofbothTotalandFreeProteinSareusefulindeterminingthetypeofdefectinpatientswithProteinSdeficiency.Historically,ELISAproceduresmeasuringProteinSusedapolyclonalantibodyspecifictoboththefreeandboundformsofProteinS.Theadditionofpolyethyleneglycol(PEG)toprecipitatetheboundProteinSinthepatientsamplealloweddeterminationoflevelsoffreeProteinS.WhilethePEGprecipitationprocedureallowsthemeasurementofFreeProteinS,itisnon-specific,timeconsuming,anddifficulttoperformaccurately.ThisassayutilizesamonoclonalantibodyspecificforFreeProteinSinanELISAformattomeasureFreeProteinSdirectly,withoutPEGprecipitation.
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实验室要开展支原体检测,方法是PCR法,先要采购试剂盒,用过的同学给推荐一下好用的品牌呗
前一段时间,客户让我推荐大鼠的褪黑素检测试剂盒,我对这个指标也是很慎重,因为这个指标不一般。我也查了一些文献,从检测方法上,首选高效液相色谱法,但问题是检测系统最好是电化学检测,这个检测系统,我问了广州好多实验室,都没有配备;另外就是查到了Raybiotech公司的放免试剂盒,价格也不贵,3200多,但是厂家需要现订做这个试剂盒,生产周期是6个月左右,很少有客户能接受这个到货周期;第三,我查到了Millipore公司的Luminex检测系统,有这个DIY套装,突然眼睛一亮,毕竟是millipore是大品牌,而且液相芯片技术也越来越普及,权衡所有利弊,我给客户推荐了millipore的这个试剂盒。虽然客户后面因为价格问题,没有通过我采购,但是我还是感到很欣慰,因为自己的推荐还是扎根于客户的心里了。
接下来就是客户通过其他的供货商采购到了Millipore的大鼠褪黑素检测试剂盒,采用的方法是液相芯片法。然后就是在技术员的指导下进行实验,后来结果让我分析了一下,标准曲线是invalid,我发现了标准曲线中一个点偏差太大,建议客户删掉这个点,一切就OK了,试剂盒自带的control,通过计算也落在了厂家说明书的范围内。从标准曲线和control看,整个盒子没有一点问题。但是真正的问题来了,样本所计算出来的浓度大部分在20000pg/ml,这个和客户及我手头查到的文献,差别不是一个数量级了,可以认定为差了3个数量级了。Millipore的国外技术很快有了回复,经过他们的检测正常大鼠血清标本褪黑素的范围的确落在了他们试剂盒的标准曲线范围内,16000-400000pg/ml的范围,但是对于客户的疑问,他们会采用其他的方法去验证,2周后会给客户一个答复。
现在时间没有到2周,我也不知道最终的答复是什么。从这件事情上,我想了很多,很多客户做的很多指标的检测都是希望定性加定量,但是在定量的过程中,出现了各个厂家有各自的标准,整个行业没有统一的标准了,这就决定在整个试剂盒研发的过程中,任重而道远。大家过多的去相信权威,而不知道权威下面是不是掩饰了什么。
目前,我们去评价国产的ELISA试剂盒,不管你认定它是假货也好,是真实的质量有保障的也好。你去评价这些的标准是什么,认定的理由是什么,是靠经验,靠周围同学、试剂商的推荐,还是靠这些厂家的权威?目前没有统一的质控标准品或者血清样品,这些评价都是非常苍白无力的。目前为什么假的ELISA试剂盒这么猖狂,质量差的试剂盒也可以占据市场,就是因为所有盒子的标准品都是自己提供的,标准都是自己定的,自己的标准就是自己卖出试剂盒的权威认证。
版主鱼小留言:
很不错的商家感想。赞一个。
ldh试剂盒指乳酸脱氢酶检测试剂盒,是一种基于diaphorase催化的INT显色反应,通过比色法检测细胞毒性时释放的乳酸脱氢酶活性或检测其它样品中的乳酸脱氢酶活性的试剂盒。可以用于常规的乳酸脱氢酶活性的检测,更常用于以LDH释放为指标的细胞毒性检测。
ctl毒性杀伤检测试剂盒是基于LDH在胞浆内含量丰富,正常时不能通过细胞膜,当细胞受损伤或死亡时可释放到细胞外,此时细胞培养液中LDH活性与细胞死亡数目成正比,用比色法测定并与靶细胞对照孔LDH活性比较,可计算效应细胞对靶细胞的杀伤。
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