乙型脑炎病毒IgM ELISA法测定.ppt

IgM ELISA for Japanese encephalitis virus乙型脑炎病毒 IgM ELISA法测定,1,CHIK,SIN,DEN1-4,TBE(POW),SSH,EEE,WEE,VEE,MAY,DEN1-4,YF,BF,RR,SIN,MVE,DEN1-4,CHIK,SIN,YF,DEN1-4,TAH,SIN,TBE(POW),WN,(TAH,INK),LAC,SLE,POW,EEE,WEE,CTF,DEN1-4,EVE,EEE,VEE,WEE,MAY,DEN1-4,JE,(VEE),SLE,LAC,(VEE),DEN1-4,Most common arboviruses World-wide 全球常见虫媒病毒分布,WN,WN,WN,WN,WN,SLE,SLE,2,uu,Flaviviruses in China 中国黄病毒Japanese encephalitis virus(JEV)乙型脑炎病毒Dengue viruses(DEN)登革热病毒Tick Borne encephalitis virus(TBE)(rare,near Russian border)蜱传脑炎病毒（TBE）（罕见，与俄罗斯边境处）There are 10 members of the JEV serocomplex,the main human pathogens of which are JEV,West Nile(WNV),Murray Valley encephalitis virus(MVE)and St.Louis encephalitis virus(SLEV).Of these,only JEV is found in China乙脑病毒有10个成员，对人类致病的主要有乙脑病毒、西尼罗河病毒（WNV）、默里谷脑炎病毒（MVE）和圣路易斯脑炎病毒（SLEV）。其中，中国只发现乙型脑炎病毒,3,Flavivirus Virion Structure 黄病毒病毒体的结构,Virus particle 40-60 nmSingle-stranded positive-sense RNA virus病毒颗粒4060nm单链正股RNA病毒,4,The Cross-Reactivity Problem 交叉反应问题,Human infections by flaviviruses elicitimmune responses producing 人感染黄病毒产生免疫反应,virus species-specific antibodies 病毒种特异性抗体 flavivirus cross-reactive antibodies 黄病毒交叉反应抗体,5,E Glycoprotein Homodimer E糖蛋白同型二聚体Top View 顶视图,Yellow residues are domain II of E glycoprotein;purple are conserved regions among the flaviviruses黄色残基是E糖蛋白的结构域II；紫色为黄病毒的保守区域,6,Serologic cross-reactivity among the flaviviruses 黄病毒间血清交叉反应性Both IgM and IgG antibodies exhibit significant cross-reactivity among the flaviviruses.For instance,anti-DEN IgM will react with JE virus antigen,even though DEN is not in the JE serocomplex.黄病毒之间IgM和IgG抗体都有交叉反应。例如抗-登革热病毒IgM与乙脑病毒抗原反应，尽管登革热病毒不是乙脑病毒血清群的。IgM tends to be less cross-reactive than IgGThis can be useful in diagnosis of unexpected agents(eg when WNV entered the US it was first thought to be the endemic SLE)However,cross-reactivity can produce false-positivesIgM通常比IgG交叉反应少些。可以用于非预期病原的诊断（如，当西尼罗河病毒进入美国时，最先认为是地方圣路易斯病毒脑炎）。然而，交叉反应可以产生假阳性。,7,Cross-reactivity continued交叉反应续IgM to DEN and TBE(flaviviruses in China)will cross-react in a JEV IgM test.登革热病毒和蜱传脑炎病毒（中国的黄病毒）IgM，在乙脑病毒IgM检测中可以发生交叉反应。TBE is rare in China,therefore false-positives due to TBE are unlikely.蜱传脑炎在中国罕见，因此因蜱传脑炎病毒造成的假阳性可能性不大。DEN does not cause encephalitis but does cause occasional encephalopathy(disturbances in mental status),and therefore a specimen from a DEN patient could be submitted,and it is likely to test positive.登革热病毒不引起脑炎，但是偶尔可以造成脑病(精神障碍)，因此如果对登革热病人的标本进行检测，结果很可能阳性。,8,SHIPMENTS 运输,SERUM SPECIMENS:Acute and/or convalescent血清标本：急性期和/或恢复期Sent on wet,or dry ice or ambient temperature 在冰或干冰或常温环境运送CEREBROSPINAL FLUID 脑脊液Sent on wet or dry ice or ambient temperature 在冰或干冰或常温环境运送Store specimens at 4 C if intended for immediate use;otherwise freeze at-20 C or below.如果准备立即检测，在4 C条件下保存标本；否则在-20 C或以下冻存,9,Information needed for serological testing and interpretation血清学检测和结果解释需要的资料,Dates of sample collection 样品采集日期Evidence of clinical encephalitis 临床脑炎证据Vaccination history 接种史Date of onset of symptoms症状出现日期,10,Safety安全性,Always wear latex or nitrile gloves,safety glasses and a lab coat when handling specimens and during all stages of the test.处理样本和测试各个环节都要带乳胶和硅胶手套、安全眼镜和白大衣Use of a Biosafety cabinet(BSC)Class 2 Type A2 is recommended because acute specimens may contain infectious JE virus or other infectious agents.由于急性期标本可能含传染性乙脑病毒或其它致病因子，建议用II类A2型生物安全柜Class 2 is a ventilated cabinet that provides personnel,product and environmental protection.It has a limited fixed inward air flow access opening,a ventilated downward HEPA filtered exhaust.II级生物安全柜有通风，可以提供人员、产品和环境保护。带有有限的、向内的固定气流，并使用了向下的HEPA过滤通气装置。Type 2 recirculates 70%of the air II型有70的空气循环。Do not use laminar flow hoods intended for cell culture!细胞培养不要用层流罩。If a BSC is unavailable,care should be taken especially when using the undiluted specimens,and during wash steps.Bleach should be added to the discard pan to inactivate viruses.如果没有生物安全柜，操作要特别认真，特别是用未稀释的样品以及各洗涤步骤。在废物盘中加漂白粉灭活病毒。,11,Several kits are available to test for anti-JE IgM testing;the B B&C Beixi试剂盒将用于本项目,B&C(Beixi)EEB-IgM(48T)Cat No.IBCB350P,12,The kits use wells precoated with anti-human IgM antibody.本试剂盒利用抗人IgM抗体包被板。,The JEV IgM ELISA B&C Beixi kit works in the following way:B&C Beixi乙脑病毒IgMELISA检测试剂盒检测原理如下：,2.Diluted patient serum or CSF is added.If any IgM antibody is present it will be captured by the anti-IgM antibody.加入稀释的病人血清或脑脊液。如果存在IgM抗体，就能被抗IgM抗体捕获。,3.A mixture of JEV antigen()and JEV-specific monoclonal antibody conjugated to horseradish peroxidase()is added to the well.If the IgM captured in the last step has been made to JEV,the antigen-conjugate pair will attach to the IgM antibody.3.加入乙脑病毒抗原()和标记辣根过氧化物酶乙脑病毒特异单克隆抗体()的混合物于孔中。如果上一步捕获的IgM是乙脑病毒的，抗原耦合将与IgM抗体结合。,4.A substrate is added which causes color to develop.The reaction is stopped and read at 450 nm4.加底物现色。终止反应在450nm波长条件下读数。,13,Test interpretation检测结果解释,14,Theoretical Depiction of Human JE Viremia and Immune Response人乙脑病毒血症和免疫反应理论曲线,DAYS POST ONSET 发病后天数,1 2 3 4 5 6 7 8 9 10,-14 to-2 0,IgM,IgG,ELISAP/N(depends on test used)依赖于测试方法,#pfu/ml,WNViremia病毒血症,250,CNS illness 中枢神经系统疾病RNA difficult to detect in CSF at onset of encephalitis在脑炎发病时难以在脑脊液中检测到RNA,2,20,Serologic Assays血清学检测方法,Virus Assays病毒检测方法,15,SerumUsed at 1:100血清，1:100稀释,Negative result:阴性结果 Interpreted as either NEG or IgM not yet present 解释为阴性或IgM尚未出现,Positive result:阳性结果IgM to JE or to vaccine if given 1 month prior to symptoms 乙脑IgM或出现症状前一个月内注射过疫苗,Action:Report as negative,or test convalescent specimen if available措施：报告阴性结果，如果可能测定恢复期血清,Action:Report as positive;clinical correlation/vaccine history required措施：报告阳性结果，需要临床相关/疫苗接种史,CSF(preferred specimen)Used at 1:10脑脊液（最佳）1:10稀释,Acute specimens急性期标本9 days after onset of symptoms症状出现后 9天,Positive result:IgM to JE 阳性结果：乙脑病毒IgM阳性,16,Negative result:阴性结果 Interpreted as either NEG or IgM not yet present 解释为阴性和IgM尚未出现,Action:Report as negative,or test convalescent specimen if available措施：报告阴性结果，如果可能测定恢复期血清,Action:Report as positive;clinical correlation required措施：报告阳性结果，需要临床相关资料,Negative result:Interpreted as NEG阴性结果：解释为阴性,Action:Report as negative措施：报告阴性结果,Convalescent specimens 恢复期样本9 days after onset of symptoms 症状出现后 9天后,Positive result:IgM to JE 阳性结果：乙脑病毒IgM阳性,Note:The majority of specimens will probably be single,acute CSF 注：多数样本是单份急性期脑脊液,17,SerumUsed at 1:100血清，1:100稀释,CSF(preferred specimen)Used at 1:10脑脊液（最佳）1:10稀释,Positive result:阳性结果IgM to JE or to vaccine if given 1 month prior to symptoms 乙脑IgM和出现症状前一个月内注射过疫苗,Action:Report as positive;clinical correlation/vaccine history required措施：报告阳性结果，需要临床相关/疫苗接种史,Action:Report as negative措施：报告阴性结果,Negative result:Interpreted as NEG阴性结果：解释为阴性,Action:Report as positive;clinical correlation required措施：报告阳性结果，需要临床相关资料,Performance of test kit试剂盒操作,18,Results of a limited analysis of B&C Beixi kit using serum samples:用血清样品测试B&Cbeixi试剂盒的初步分析结果,19,Analysis of B&C JEV IgM kit,20,Test limitations 测试方法的局限性,21,Recommendations 建议If the B dilute as appropriate before use and use these negatives in your calculations for test samples(not for positive control).The kit negative isnt necessarily representative of your population,and it is unclear if it is serum or CSF.除了试剂的对照孔外，应另外加一个对照孔。准备已知当地病人的阴性血清和阴性脑脊液库，分装并冻存；用前适当稀释，用这些阴性样品做计算测试样品（不用于阳性对照）。试剂盒的阴性对照不一定代表当地人群，也不知是血清还是脑脊液。If the kit positive control does not give a positive:negative(P/N)OD ratio of 5,consider rerunning the test.如果试剂盒的阳性对照计算出的阳性/阴性（P/N)OD比值没有5，考虑重新测试。,22,A more extensive assessment of the B&C(Beixi)kit was performed by a student of Dr.Liang.梁博士的学生对B&C(Beixi)试剂盒进行了进一步评价The following slide reports his data 以下的幻灯片报告他的数据,23,Analysis of B&C Beixi kit compared to IFA as a gold standard using 121 samples B&C Beixi试剂盒与作为金标准的间接荧光法测定121份样品的比较分析,Agreement with IFA 与IFA一致的比例False positive rate 假阳性率False negative rate 假阴性率Sensitivity 灵敏度Specificity 特异度Reproducibility 重复性(297 samples)（297份样品）,B&C Beixi IgM capture 捕获 93.8%13.33%4.04%95.96%86.67%97.31%,24,