Improved detection of citrus psorosis virus using polyclonal and monoclonal antibodies

Citrus psorosis is a serious and widespread disease associated with citrus psorosis virus (CPsV), a novel filamentous negative-stranded virus in the genus Ophiovirus . Laborious and costly indexing on test plants has been the only routine diagnostic method available, but recently an antiserum usable in double antibody sandwich (DAS) ELISA has been prepared. Here, major improvements to the DAS-ELISA protocol, a new purification method, and production of two monoclonal antibodies (mabs) to CPsV, an IgG and an IgM are reported. A highly sensitive triple antibody sandwich (TAS) ELISA making use of the mabs is described. In glasshouse citrus the homologous virus was still detectable at a tissue dilution of 1/6250 in DAS and at 1/31250 in TAS-ELISA. Both the DAS and IgG mab-TAS formats detected all CPsV isolates so far tested (from Argentina, Italy, Lebanon, Spain and the USA). A few isolates were not detected by the IgM mab.     

烟草不同时期接种烟草花叶病毒后的种子带毒率比较

烟草种子带毒一直对烟草生产危害较大.烟草品种K326、云烟85和云烟87在不同时期分别接种烟草花叶病毒(TMV)后收集烟株种子,采用三抗体夹心法(TAS-EKLISA)检测TMV带毒情况,结果表明,接种TMV后病株上采收的种子的平均带毒率超过50%,但不同时期接种和不同品种间烟草种子带毒率存在明显差异.     

从海南省杂草胜红蓟和假马鞭上检测到粉虱传双生病毒

 利用三抗体夹心ELISA(TAS-ELISA)及PCR检测的方法对采自海南的胜红蓟(Ageratum conyzoides)和假马鞭(Stachytarphetajamaicensis)的5个病样进行了检测,表明均为粉虱传双生病毒。PCR扩增产物克隆后进行序列测定,结果表明存在2类双生病毒,其中样品Hn2存在2类病毒的复合侵染。这是在海南首次报道存在有粉虱传双生病毒。     

罗氏沼虾诺达病毒单克隆抗体的制备及应用

罗氏沼虾肌肉白浊病（whitish muscle disease of Macrobrachium rosenbergii）是一种发生在罗氏沼虾苗种阶段的流行病，发病虾苗出现肌肉白浊、白斑或白尾症状，死亡率高达60％以上，作者所在实验室在确定其病原是罗氏沼虾诺达病毒（Macrobrachiium rosenbergii Nodavirus，MrNV）的基础上，用MrNV免疫BALB／c小鼠，取小鼠脾细胞与SP2／0鼠骨髓瘤细胞融合，用间接ELISA筛选阳性孔，经有限稀释法克隆，得到12株能特异分泌抗MrNV的单兜隆抗体（Mab）的杂交瘤细胞。注射小鼠，制备腹水单克隆抗体，ELISA效价为1：10^5～10^6。亚型鉴定结果表明，有6株单抗为IgG1型，4株甲抗为IgG2a型，2株单抗为IgG2b型。12株单抗与对虾白斑综合征病毒（WSSV）和桃拉综合征病毒（TSV）均无交叉反应：挑选效价高的腹水单抗2B5，建立了检测罗氏沼虾诺达病毒的三抗体夹心酶联免疫吸附测定（TAS—ELISA）法，该方法检测灵敏度达0．98ng左右。Wesrtem blot分析表明，2B5能与MrNV 43kD的外膜蛋白特异性结合。     

利用ELISA检测两种兰花病毒的研究

建立了三抗夹心酶联免疫吸附法(TAS-ELISA)检测建兰花叶病毒(Cymbidium mosaic virus,CymMV)和双抗夹心酶联免疫吸附法(DAS-ELISA)检测齿兰环斑病毒(Odontoglossum ringspot virus,ORSV)的方法.共检测了浙江省内139个兰科样品和8个百合科样品,结果表明有31.7%的兰花检出CymMV,有28.8%的兰花检出ORSV.根据病毒外壳蛋白基因上下游保守序列设计了检测ORSV的引物,参照周国辉报道设计了检测CymMV的引物并对人工接种两种病毒的兰花进行了逆转录聚合酶链式反应(RT-PCR)检测,结果表明,RT-PCR检测结果与ELISA结果相一致,进一步证实本研究建立的TAS-ELISA,DAS-ELISA方法稳定可靠,重复性好,可应用于兰花上CymMV和ORSV两种病毒的早期诊断和检测.     

单抗I-ELISA和TAS-ELISA检测百合无症病毒的研究

 以抗百合无症病毒(Lily symptom less virus,LSV)的单克隆抗体为核心,建立了间接ELISA (I-ELISA)和三抗体夹心ELISA (TAS-ELISA)的检测方法。I-ELISA检测体系中,病叶汁液和单克隆抗体腹水的工作浓度分别为1:20和1:6 000,对病叶汁液的检测灵敏度达到了1:2 560,可检测到提纯病毒绝对量为1.35 ng。TAS-ELISA检测体系中捕获抗体和单克隆抗体腹水的工作浓度分别为1:200和1:6 000,检测病叶汁液的灵敏度达到了1:5 120,对于提纯病毒可检测到0.68 ng。使用美国Agdia公司双抗体夹心ELISA (DAS-ELISA)检测试剂盒对病叶汁液的检测灵敏度为1:2 560,提纯病毒检出量1.35 ng。用3种ELISA方法检测了采自浙江省丽水市的46个田间样品,I-ELISA、TAS-ELISA和DAS-ELISA测出的阳性样品数分别为19、21和18个,阳性率为41%、46%和39%。灵敏度检测和田间样品检测结果显示,TAS-ELISA的灵敏度高于DAS-ELISA和I-ELISA。相同样品I-ELISA所测出的OD₄₀₅值和P/N值普遍高于DAS-ELISA,表明LSV单抗比多抗具有更强的特异性和更高的灵敏度。     

罗氏沼虾诺达病毒TAS-ELISA检测法的建立及应用研究

罗氏沼虾肌肉白浊病(Whitish muscle diseases of Macrobrachium rosenbergii)是近年来流行于罗氏沼虾苗种阶段的严重疾病,该病病原已确定为罗氏沼虾诺达病毒(Macrobrachium rosenbergii Nodavirus,MrNV).作者在病毒超离提纯的基础上,制备了罗氏沼虾诺达病毒兔抗血清,并应用抗罗氏沼虾诺达病毒单克隆抗体2B5,建立了三抗体夹心酶联免疫检测方法(TAS-ELISA).TAS-ELISA最适反应条件为:兔抗体以1μg/mL包板,小鼠单抗2B5的工作浓度为1:50000,该法检测MrNV的灵敏度达0.98ng,对WSSV、TSV及其他细菌感染死亡的罗氏沼虾苗没有非特异性反应.通过对2000-2002年病虾及疑似病虾的病毒检测,表明TAS-ELISA法比间接ELISA法和核酸电泳法有更高的阳性检出率和符合率;此外还摸索了降低孵育温度和缩短孵育时间对病毒检测的影响,表明各步孵育温度和时间分别为25℃和20 min对于白浊症状的病苗检测无明显影响,使得本法更加适合于生产实践.     

Preparation of monoclonal antibody against Macrobrachium rosenbergii Nodavirus and application of TAS-ELISA for virus diagnosis in post-larvae hatcheries in east China during 2000-2004

“Whitish muscle disease” of Macrobrachium rosenbergii, also called “whitish disease” or “white tail disease”, is a new serious epizootic disease that has occurred in recent years in giant freshwater prawn culture regions, mainly in southern China. This disease occurred in post-larvae 3-5 days to 3 weeks after desalting. Clinical signs include the development of white spot in muscles or milky muscles throughout the body, causing serious loss in few days, with a mortality rate of 40-90%. A 26-27 nm icosahedral non-enveloped virus, identified as M. rosenbergii Nodavirus (MrNV), was confirmed as the aetiological agent. Twelve hybridomas strongly secreting monoclonal antibodies (Mabs) against MrNV were shown to be specific for MrNV and reacted with MrNV 42 kDa coat protein by Western blot. A triple antibody enzyme-linked immunosorbent assay (TAS-ELISA) was developed and shown to be a useful diagnostic tool for MrNV infection.     

浙江宁波雪里蕻花叶病病原鉴定及雪里蕻品种的抗病性测定

 从浙江宁波雪里蕻上获得97株呈花叶症状的病毒样品,利用三抗体夹心酶联免疫吸附测定(TAS-ELISA)在97株雪里蕻花叶样品中均检测到芜菁花叶病毒(Turnip mosaic virus,TuMV),所有的样品都没有检测到黄瓜花叶病毒(Cucumber mosaic virus,CMV)和烟草花叶病毒(Tobacco mosaic virus,TMV);利用免疫捕捉反转录PCR (IC-RT-PCR)对部分TuMV样品中的CP和HC-Pro基因进行了扩增,所有样品都得到约0.8kb和1.4kb的2条特异条带,因此宁波雪里蕻花叶病的主要病原是TuMV。对53个雪里蕻栽培品种在温室和大田进行了人工接种,共鉴定出抗病品种7个,耐病品种22个,感病品种24个,未发现高抗品种。总的来说,细叶型品种比花叶型和板叶型品种抗病。利用TAS-ELISA方法对接种的53个雪里蕻品种中的TuMV浓度进行测定,在接种后10、15、20和30d,TuMV检出率分别为45.28%、90.57%、100%和100%,大多数雪里蕻OD405值随接种后天数的增加而呈上升趋势,说明TuMV可以在雪里蕻抗性品种内繁殖,抗性品种的抗性主要表现为耐病。