Description:
REAADSProteinCAntigenisanenzyme-linkedimmunosorbentassay(ELISA)forthequantitativedeterminationofProteinCAntigenincitratedhumanplasma.
KitComposition:
Reagents
- 12x8anti-humanProteinCantibodycoatedmicrowells.
- 60mlSampleDiluent(blue-greensolution);containssodiumazide.
- 3vialsx0.5mllyophilizedReferencePlasma,withassaysheet
- 12mlanti-humanProteinCHRPConjugate(bluesolution).
- 13mlSubstrate(TMBandH2O2).
- 15mlStoppingSolution(0.36Nsulfuricacid).
- 30mlWashConcentrate(33Xphosphatebufferedsalinewith0.01%Tween20).Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.
Storeat2–8°C.DoNotFreeze.
Materialsrequiredbutnotsupplied
- ProteinCControlPlasma.ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.
- Reagentgradewater(1L)topreparePBS/Tween20washsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
- Graduatedcylinders
- Precisionpipettorscapableofdeliveringbetween5and1000microliters,withappropriatetips
- Miscellaneousglasswareappropriateforsmallvolumehandling
- Flaskorbottle,1liter
- Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
- Disposablegloves,powder-freerecommended
- PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreferenceifavailable)
- Multichannelpipettorscapableofdeliveringto8wellssimultaneously
- Microdilutiontubesforpatientsamplepreparation
MeasurementPrinciple:
Principle
TheProteinCAntigenassayisasandwichELISA.AcaptureantibodyspecificforhumanProteinCiscoatedto96-microwellpolystyreneplates.Dilutedpatientplasmaisincubatedinthewells,allowinganyavailableProteinCtobindtotheanti-humanProteinCantibodyonthemicrowellsurface.Theplatesarewashedtoremoveunboundproteinsandotherplasmamolecules.BoundProteinCisquantitatedusinghorseradishperoxidase(HRP)conjugatedanti-humanProteinCdetectionantibody.Followingincubation,unboundconjugateisremovedbywashing.Achromogenicsubstrateoftetramethylbenzidine(TMB)andhydrogenperoxide(H2O2)isaddedtodevelopacoloredreaction.Theintensityofthecolorismeasuredinopticaldensity(O.D.)unitswithaspectrophotometerat450nm.ProteinCAntigenrelativepercentconcentrationsinpatientplasmaaredeterminedagainstacurvepreparedfromthereferenceplasmaprovidedwiththekit.
Procedure
DilutedcitratedpatientplasmaandcontrolsareincubatedinmicrowellscoatedwithcaptureantibodyspecificforhumanProteinC.Duringanincubationperiod,patientProteinCisallowedtobindtothesurface.
Followingawashtoremoveanyunboundplasma,horseradishperoxidase(HRP)conjugatedanti-humanProteinCdetectionantibodyisaddedtothewells.Afterwashingtoremoveunboundconjugate,achromogenicsubstrateisadded,resultinginasolublecoloredproductthatismeasuredinaspectrophotometerat450nmfollowingtheadditionofastopsolution.
PatientProteinClevelsaredeterminedfromasixpointcurvepreparedfromthereferenceplasmaprovidedinthekit.Totalincubationtimeis60minutes.
AssayProcedure:
1.Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebag provided.
2.Assayeachreferenceplasmadilutioninduplicate.Duplicatedeterminationsarealsorecommended forpatientandcontrolsamples.Onewellshouldberunasareagentblank;samplediluentwithout serumisaddedtothewellasexplainedinstep7ofthissection.Thiswellistreatedthesameasa controlorpatientsampleinsubsequentassaysteps.Awaterblankwellshouldbeincludedwith eachplate;itistoremainemptyuntil200µlofreagentgradewaterisaddedatthecompletionof theassay,immediatelypriortoreadingtheplate.Thewaterblankwellistobeusedtozerotheplate reader.
3.Pre-diluteallplasmas(1:2dilutioninSampleDiluent)asfollows: Referenceplasma:add100µlreferenceplasmato100µlSampleDiluent Controlandpatientsamples:add20µlplasmato20µlSampleDiluent Mixwell.Thesepre-dilutionsareutilizedinpreparingtheworkingdilutionsinsteps4and5.
4.Usingthe1:2referenceplasmadilutionfromstep3,preparesixworkingreferencedilutionsas describedbelow.
| VolumeReference Plasma(1:2) | VolumeSample Diluent | *ReferenceLevel | ||
| 30μl | + | 500μl | = | 150 |
| 20μl | + | 500μl | = | 100 |
| 15μl | + | 500μl | = | 75 |
| 10μl | + | 500μl | = | 50 |
| 10μl | + | 1000μl | = | 25 |
| 10μl | + | 2000μl | = | 12.5 |
| *Referencelevelvaluetobeusedforconstructingreferencecurveonly | ||||
5.Prepareworkingdilutionsofcontrolandpatientsamplesbyadding20µlofpredilutedplasma(1:2 dilutionfromstep3)to500µlSampleDiluent.(Note:thesedilutionscorrespondtothe100% referenceplasmadilution.)
6.Mixthoroughly,andadd100µloftheworkingdilutions(referenceplasmas,controlsandpatient samples)totheappropriatemicrowells.
7.Add100µlofSampleDiluenttothereagentblankwell.Leavethewaterblankwellempty.
8.Incubate40minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsanddumpthesamplefluid.Donotallowsamplestocontaminateothermicrowells.
9.Wash4timeswithworkingwashsolution(PBS/Tween20).Eachwellshouldbefilledwithwash solutionperwash.Washsolutionintheemptywellintendedtoserveasawaterblankwillnot interferewiththeprocedure.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnapping motionofthewristtoshaketheliquidfromthewells.Theframemustbesqueezedatthecenteron thetopandbottomtoretainmicrowellmodulesduringwashing.Blotonabsorbentpapertoremove residualwashfluid.Donotallowwellstodryoutbetweensteps.
10.Add100µlConjugate(blue)toeachwell(exceptthewaterblankwell).
11.Incubatefor10minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsanddumptheconjugatesolution.
12.Wash4timeswithworkingwashsolution(PBS/Tween20)asinstep9.Washsolutioninthewater blankwelldoesnotinterferewiththeprocedure.Useasnappingmotiontodraintheliquid,andblot onabsorbentpaperafterthefinalwash.Donotallowthewellstodryout.
13.Add100µlSubstratetoeachwell(exceptforthewaterblankwell)andincubatefor10minutesat roomtemperature.Addthesubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwells withpositivesamples.
14.Add100µlStoppingSolution(0.36Nsulfuricacid)toeachwell(exceptforthewaterblankwell)to stoptheenzymereaction.BesuretoaddStoppingSolutiontothewellsinthesameorderandatthe samerateastheSubstrateSolutionwasadded.BlueSubstratewillturnyellowandcolorlesssubstrate willremaincolorless.DonotaddStoppingSolutiontothewaterblankwell.Instead,add200µlof reagentgradewatertothewaterblankwell.Blankorzerotheplatereaderagainstthewaterblank well.ReadtheO.D.ofeachwellat 450nm,againsta650nmreferencefilter(ifavailable).Forbestresults,theO.D.valuesshouldbemeasuredwithin30minutesaftertheadditionofStoppingSolution.
Performance:
ClinicalPerformance
PlasmasamplesfromhealthyblooddonorsandfrompatientswithahistoryofthrombosisweretestedtodefineandcomparetheclinicalperformanceofREAADSProteinCELISAwithawellestablished,commerciallyavailableProteinCAntigenRocketEIDmethod.Asshowninthetable,theresultscorrelatedwell,andwereshowntobestatisticallysimilarbysinglefactorAnova.
TechnicalPerformance
Intra-assayprecisionofREAADSProteinCELISAis7.0%whileintra-assayprecisionis7.5%Linearity,expressedasthecoefficientofdetermination(r2)is0.992withameanaccuracyof99.4%REAADSProteinCELISAisarapid,convenient,highlyaccurateandprecisemethodforthequantitativedeterminationofProteinClevelsinhumanplasma.
Background:
ProteinCisavitaminK-dependentproteinsynthesizedprimarilybyhepatocytesintheliverandplaysanimportantphysiologicroleintheProteinCAnticoagulantSystem.ProteinC,thrombinfrombloodclots,andendothelialcells,throughcomplexinteractionswithotherfactorsofthecoagulationcascade,contributetothemaintenanceofnormalhemostaticmechanismsbydown-regulatingclotformationandbypromotingfibrinolysis.TheProteinCAnticoagulantSystemisactivatedbythebindingofthrombintothrombomodulin,atransmembraneproteinreceptoronendothelialcells.Thethrombin-thrombomodulinbindingonendothelialcellmembranesactivatescirculatingProteinC.ActivatedProteinCbindstoProteinSonthemembraneofendothelialcellsorplatelets.InthisProteinC-ProteinScomplex,activatedProteinCisnowcapableofinactivatingcoagulationfactorsVaandVIIIa,down-regulatingclotformation.ActivatedProteinCalsoenhancesthefunctionoftissueplasminogenactivator(TPA)bydissociatingthismoleculefromitsinhibitor,plasminogenactivatorinhibitor-1(PAI-1),therebyfacilitatingclotdissolutionorfibrinolysis.
ProteinCdeficiency,eithercongenitaloracquired,mayleadtoseriousthromboticeventssuchasthrombophlebitis,deepveinthrombosis,orpulmonaryembolism.Patientswithacongenitalheterozygousdeficiencymaypresentwithvenousthrombosisinyoungadulthood,whilepatientswiththerarehomozygousdeficiencypresentwithmassivethrombosis(purpurafulminans)duringtheneonatalperiod.TheprevalenceofProteinCdeficiencyinthegeneralpopulationhasbeenestimatedat1in300.Inyoungerpatients(<40-45years)withrecurrentvenousthrombosis,thefrequencyofProteinCdeficienciesmaybeashighas10to15%.AcquiredProteinCdeficiencymaybeseeninliverdisease,extensivethromboticepisodes,surgery,oralanticoagulanttherapy,antiphospholipidsyndrome,etc.AdecreasedProteinCactivityinplasmamaybetheresultoflowconcentrationsandfunction(typeI)oronlylowfunction(typeII).
ThelaboratorydiagnosisofProteinCdeficiencymayrequirebothquantitativeandqualitative(functional)determinations.QuantitativedeterminationsofProteinCAntigenarebasedonimmunologicproceduressuchasradialimmunodiffusioningel,Laurellrocketimmunoelectrophoresisandenzyme-linkedimmunosorbentassay(ELISA).ELISAproceduresarelesslaborintensiveandofferseveraladvantagesincludingmoreobjective,accurateandreproducIBLeresults.Inaddition,ELISAallowsautomationwithcommonlyavailablelaboratoryinstruments.
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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。
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试剂盒在全球市场上的研发与销售呈快速上升趋势,2005年全球市场销售额超过200亿美元,且以15%左右的速度逐年增长.一方面是试剂盒的迅猛发展,而另一方面试剂盒市场良莠不齐的现象愈加明显,试剂盒的生产、销售及认证认可体制尚不完善,没有相应的标准或质量评价政策.且其灵敏度,稳定性及假阴/阳性控制尚不能满足检测需要,采用试剂盒进行检测的公信度受到质疑.
同时,食品安全领域是当前问题最多、最受关注的领域,这个领域的检测包括了物理、化学、微生物及分子生物学基础理论,无论是按检测原理、用途还是其它分类方式,涉及食品安全检测项目的试剂盒的品种是最全最多的.因此,从该领域着手从事评价制度的研究,便于获得基础性数据结果,并由此推广至动植物检疫及其它领域.
随着H7N9禽流感疫情的不断发散,国家流感中心已经多次发放人感染H7N9禽流感检测试剂,覆盖了全国31个流感网络实验室,并表示,诊断试剂的广泛发放是实现关口前移,控制疫情传播、蔓延的重要手段.而一旦H7N9监测关口继续前移,主动监测范围扩大,病毒检测试剂的需求量将进一步加大.
可采用H7N9亚型禽流感病毒RNA检测试剂盒（荧光PCR法）和H7N9禽流感病毒核酸检测试剂盒（PCR-荧光探针法）,定价分别为48人份/盒和48反应/盒,相比市场此前预期的100-200元之间的价格定位低了很多.在检验方法上,卫纪委提醒,前者需要配备全自动荧光PCR检测仪专用PCR扩增管和核酸分离试剂盒(硅胶膜吸附法)等必须设备及咽拭子样本,后者卫计委推荐采用达安基因生产的核酸提取试剂盒进行检验.
我用Promega公司的双荧光素酶检测试剂盒（E2920）检测到的firefly萤光素酶活性很低，只有4*10的3次方；海肾萤光素酶活性有10的6次方。
我用Ad293细胞做了转染，fireflyluciferase质粒：RanillaLuciferase质粒=0.1ug：0.025ug/一个孔（96孔板），共转染了二天，再进行双萤光素酶检测。
我想了解fireflyluciferase活性用promega的这个E2920-双萤光素酶试剂盒检测得到10的3次方，这种数值正常吗？
我做了3次重复实验，每次firefly萤光素酶活性很低，只有4*10的3次方；而海肾萤光素酶活性有10的6次方。
求指教？fireflyluciferase活性低？会是什么原因呢？
牛胰岛素,是一种多肽
在1965年9月17日我国完成了结晶牛胰岛素的全合成。经过严格鉴定，它的结构、生物活力、物理化学性质、结晶形状都和天然的牛胰岛素完全一样。这是世界上第一个人工合成的蛋白质，为人类认识生命、揭开生命奥秘迈出了可喜的一大步。这项成果获1982年中国自然科学一等奖。
1953年，英国人F. SangerSanger由于测定了牛胰岛素的一级结构而获得1958年诺贝尔化学奖。
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
