Kitsize | 12x8 |
Method | ELISA |
Incubationtime | 1x1h,1x30min,1x15min |
Standardrange | cut-offindex |
Specimen/Volumes | 10µLserum,plasma |
Substrate/isotope | TMB450nm |
RegulatoryStatus: | EU:CE |
SEROlogicalantibodytestingisincreasinglyrecognizedasavaluabletoolforthediagnosisoflegionellosis.ThedetectionofantibodiesusingELISAmethodshowshighersensitivityandbettercharacteristicsintermsofautomationforroutineapplicationandobjectivemeasurementthanimmunofluorescence.Moreoverithasbeenshowntobeavaluabletoolinepidemiologicalstudies.
IBLInternationalintroducesitsnewLegionellapneumophilaIgG-andIgMELISAsforthequalitativedeterminationofantibodiesagainstLegionellapneumophilaserovars1-7inhumanserumorplasma(citrate).
Diseasebackground
LegionellosisisalungdiseasemostcommonlycausedbyLegionellapneumophilaserogroup1andwithtwodistinctclinicalpresentations:Legionnaires"disease,asevereformofpneumoniathatcanbefatal.Pontiacfeverisamilderflulikeillness.
Legionellaisnaturallyfoundinstagnantwater,especiallywarmwater.Alsospas(hottubs)canbecomecontaminatedwithLegionella.Legionellosiscanbeacquiredbytheinhalationofcontaminatedaerosols,butitisnotcontagious(itcannotbepassedfrompersontoperson).
Detectionmethods
CommonlyusedmethodsfordetectionofaLegionellainfectioninvolvedirectdetectionmethodsusingculturing,directfluorescentstainingorantigendetectioninurineandindirectmethodswhichmeanstheassessmentofhumoralimmuneresponsebyantibodydetection.
Althoughculturingremainsthegoldstandardforthediagnosisoflegionellosis,itssensitivitymaybelimitedforclinicalroutineapplication.Directfluorescentstainingishamperedbylowandvariablesensitivity.
Directcomparisonstudies
DirectcomparisonstudiesshowedthatthediagnosticperformanceofIBL´sLegionellapneumophilaIgG-andIgMELISAsissimilartothatofcurrentlyavailableLegionellapneumophilaIgG-andIgMassays(Tables1and2),andcanbesummarizedasfollows:
IBLInternationalLegionellapneumophilaELISAs | ||
IgG | IgM | |
Diagnosticsensitivity | 90.0% | 100% |
Diagnosticspecificity | 100% | 95.7% |
Agreement | 96.1% | 96.4% |
Table1
Diagnosticsensitivityandspecificity(internal+external)of
IBL´sLegionellapneumophilaIgGELISAissimilartothatofacommerciallyavailableLegionellapneumophilaIgGassay.
SerionIgGELISA | ||||
positive | negative | ∑ | ||
IBLInternationalIgGELISA | positive | 18 | 0 | 18 |
negative | 2 | 31 | 33 | |
∑ | 20 | 31 | 51 |
Table2
Diagnosticsensitivityandspecificity(internal+external)of
IBL´sLegionellapneumophilaIgMELISAissimilartothatofacommerciallyavailableLegionellapneumophilaIgMassay.
SerionIgMELISA | ||||
positive | negative | ∑ | ||
IBLInternationalIgMELISA | positive | 9 | 2 | 11 |
negative | 0 | 44 | 44 | |
∑ | 9 | 46 | 55 |
ExerptfromtheInstructionsforUse
Legionellaeareaerobicgram-negativefacultativeintracellularparasitesofcertainprotozoa.Theyarefoundinfreshwaterenvironmentsworldwideandcancauserespiratorydisease(legionellosis)inhumans.Legionellawasfirstidentifiedafteranoutbreakofpneumoniainvolvingdelegatesofthe1976AmericanLegionConventionataPhiladelphiahotel.ThegenusLegionellacurrentlyhasatleast50speciescomprising70distinctserogroups.OnespeciesofLegionella,L.pneumophila,istheaetiologicalagentofapproximately90%oflegionellosiscases,andserogroup1(Sg1)accountsforabout84%ofthesecases.
L.pneumophilamultipliesitselfattemperaturesbetween25and42C,withanoptimalgrowthtemperatureof35°C.Legionellathrivesinwarm,stagnantwaterintheenvironmentandinartificialsystemssuchascoolingtowers,evaporativecondensers,hotandcoldwatersystemsandspapoolsthatmimicthenaturalenvironmentinwhichtheorganismthrives.Thesesystemsalsoprovidethemeansbywhichaerosols/dropletsaregeneratedandtheorganismdispersedintotheatmosphere.LegionellosiscanbeacquiredbytheinhalationofaerosolscontainingLegionellabacteriaorbymicro-aspirationofingestedwatercontaminatedwithLegionella.Person-to-persontransmissionisnotthoughttobearisk.Legionellosiscanappearintwodistinctclinicalpresentations:Legionellapneumonia(Legionnaires’disease)withanincubationperiodofapprox.2-10days(mayextendupto16-20days)andPontiacfever(incubationperiod:normally12-48hours).
Legionellapneumonia(Legionnaires’disease)isaseriousformofpneumoniathatcarrieswithitacase-fatalityratioof10-15%.Legionnaires’diseasepatientsinitiallypresentwithcough,feverandnonspecificsymptomsincludingmalaise,myalgiaandheadache.Somepatientsdevelopshakingchills,chestpain,diarrhea,deliriumorotherneurologicsymptoms.Extrapulmonaryinvolvementisrare.
Thepresenceofbacteriaresp.infectionmaybeidentifiedby:
- Culture
- Urinaryantigendetection
- PCR
- Serology:DetectionofantibodiesbyIF,ELISA
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1.标准曲线的标准品是否一定要梯度稀释,为什么?我试过非梯度稀释的,也可以达到线性R2=0.99.
2.我用了CurveExpert做标曲,自动搜索后发现有10种提供的方程,各种形式的,其中一个十分适合我的实验结果(LogisticModel),而其他的感觉又不适合,因为结果常常为负值。这又是为啥捏?
3.实验的酶标仪最大OD值可以测到4,如果我的测量结果在1.3,是否像其他人所说的>1了就不准确了。
4.利用夹心法进行定量分析是否一定要使用线性方程?
不好意思啊,一下问了这么多问题,最近做了一个月的ELISA,完全摸不清头脑啊。谢谢各位了
我现在用商品化的试剂盒进行夹心法ELISA测定某抗体的浓度,盒子给定了4个标准品,标准品抗体浓度为70000~1000单位,测定步骤中要求对样品进行1:1000稀释后测定。
但是有的样本按照1:1000稀释后,最终OD值大于70000浓度的标准品的OD值,然后使用ELISACALC软件进行四参数拟合,超过了标准曲线的范围,就算不出来这个样品的浓度,但是如果在1000-70000之间的OD值就可以算出相应浓度。请教大神们,接下来我是否可以对样品进行1:2000,1:4000浓度稀释,算出结果后再×2、×4,算作此样品的浓度呢?还是直接给一个>70000的定性结果?或者可以有能算出来的其他软件?
Ps试剂盒中提及Dilutionlinearity(稀释线性?)为141%,这个是什么意思呢?
多谢!!
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请大家帮帮忙,第一步加完抗原后必须封闭吗
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