赤羽病琼脂免疫扩散试验诊断方法的研究

应用引自美国和日本的羽病毒和标准阳性血甭,制备了琼脂免疫扩散试验（ＡＧＩＤ）抗原和高免阳性血清,建立了赤羽病ＡＧＩＤ诊断方法。应用此方法对上海、杭州、广州等地的１３８３头牛进行了检疫,ＡＧＩＤ抗体阳性牛７４６头,阳笥率５４．０％,与流行情况相符。同时对从澳大利亚、美国、新西兰和加拿大等国进口的牛、羊、猪血清１６２头份进行了检疫,全部为ＡＧＩＤ抗体阴性。     

琼脂免疫扩散试验检测鸭肝炎病毒抗体的研究

自Levine和Fabricant（1950）首次在鸭胚中分离到鸭肝炎病毒（DHV）以来[1]，各国学者对鸭病毒性肝炎的血清学诊断和免疫学监测等方面进行了大量研究。本病的琼扩试验虽有报道，但被认为特异性、敏感性存在欠缺。然而建立一快速简易、比较可靠且易被基层推广应用的检测手段是非常必要的。鉴于此，我们进行了琼扩（AGID）试验的研究，并取得较好结果。1材料与方法1．1材料1．1．1种毒：DHVC81弱毒株、ATCC（C9／D2）标准毒株、E52弱毒疫苗由南京农业大学动物医学院提供。1．1．2鸡胚：购自上海市农科院畜牧所。1．1．3试验鸭：来源于…     

应用琼脂免疫扩散试验检测番鸭细小病毒抗体

将分离的番鸭细小病毒通过正常发育的番胚传代,分别取不同代次的病毒尿囊液经氯仿、聚乙二醇浓缩处理后制成不同代次的抗原。该抗原与抗番鸭细小病毒抗血清在含有２０ｇ／ＬＰＥＧ６０００的琼脂平板上作用,可见有明显的沉淀线。     

应用琼脂免疫扩散试验检测猪伪狂犬病抗体的研究

建立了ＡＧＩＤ用于猪伪狂犬病（Ｐｓｅｕｄｏｒａｂｉｅｓ,Ｐｒ）的诊断。对９８份被俭血清的ＡＧＩＤ结果与ＳＮ结果比较,当ＳＮ滴度大于１：８时两者的阳性检出符合率为１００％,ＳＮ滴度小于或等于１：８时,两者的阳性检出符合率为８７．５％,ＡＧＩＤ和ＳＮ总的阳性符合率为９４．４％,结果表明,ＡＧＩＤ对Ｐｒ可进行特异性诊断,流行病学调查和免疫动态监测。     

应用琼脂免疫扩散试验检测猪伪狂犬病

应用琼脂免疫扩散试验对5个猪场共175份样进行猪伪狂犬病血清学检测，共检出阳性血清90份，阳性率51.4%，血清阳性猪场有4个，占所测猪场80.0%。说明猪伪狂犬病在黑龙江省呼玛县呈地方性流行趋势。     

琼脂免疫扩散试验检测鸭病毒性肝炎抗原

琼脂免疫扩散试验检测鸭病毒性肝炎抗原孙泉云刘红（上海市畜牧兽医站,闵行２０１１０３）李劲松（南京农业大学动物医学院,江苏２１００９５）鸭病毒性肝炎（ＤＶＨ）已成为蔓延地区广、流行趋势快、危害严重的鸭传染病之一,因此,正确的诊断有助于本病的有效防治。对...     

禽网状内皮组织增殖病琼脂免疫扩散抗原的研制及应用

禽网状内皮组织增殖病（ＲＥ）琼脂免疫扩散抗原为淡粉以液体，具有良好的特异性，与抗鸡马立克氏病，鸡传染性氏囊病，禽白血病，鸡传染性贫血病，鸡新城疫病毒标准阳性清不发生交叉反应，抗还具有良好的敏感性，在实验室对人工感染ＲＥＶ和ＳＰＦ鸡７天即可检出抗体，检出率为４０％，１４天检出率为１００％，抗原在３７℃保存７天，在１５℃保存１５天，在４℃保存３０天，在一－２０℃至少保存１８０天，未见效价下降，用本抗原     

Development of a disperse dye immunochromatographic test for the detection of antibodies against infectious bursal disease virus

For investigating the feasibility of using disperse dyes as an immunoassay chromogenic marker, a disperse dye, DADISPERSE NAVY BLUE SP, was selected in analyzing antibody against infectious bursal disease virus (anti-IBDV). With the color intensity revealed in the disperse dye immunochromatographic test (DICT) strip as the objective function, the optimal dyeing conditions were found as follows: dye concentration absorbance (at lambda(max)=587nm)=3, pH 7, 50 degrees C, for 10min. Under these conditions, the resultant dyed-antibody (rabbit anti-chicken) can produce an optimal color intensity reading of 55,054 on the strip. For performing qualitative immunoassay, chicken sera samples taken from different farms were used for the anti-IBDV titre assessment. The results of DICT strips showed very high sensitivity and specificity as compared to that analyzed by FlockChek enzyme linked immunosorbent assay (F-ELISA) kits. For quantitative immunoassay, it was found that the color intensity measured with DICT was linearly correlated to that of F-ELISA titre (r(2)=0.9687). Therefore, DICT was further applied to the detection of chicken anti-IBDV sera under vaccination in the farms. The average titres of the sampling groups exhibited a strong agreement to that of F-ELISA. Accordingly, the DICT method developed in this study, shown to be reliable, cheap and simple in both qualitative and quantitative immunoassays, is particularly suitable for point-of-need testing (PONT) in agricultural applications.     

山羊痘琼脂凝胶扩散(AGID)抗原的制备及初步应用

筛选所分离到的山羊痘云南省地方流行毒株KM株,应用牛睾丸原代及次代细胞进行病毒增殖、提纯及浓缩,制备山羊痘琼脂凝胶扩散试验(AGID)抗原。同时对该抗原进行抗原效价、判定标准、特异性、符合率及稳定性测定。测定结果显示该抗原效价为1∶4,且特异性及符合率高、稳定性好。并应用所制备抗原及参考阳性血清对采集自非疫点山羊血清186份,疫点390份,自然发病康复山羊血清46份,山羊痘免疫血清216份进行AGID初步应用。检测结果与流行病学相符合,证明我们所制备的山羊痘AGID抗原完全能够用于山羊痘抗体的检测及监测,且经济、简单、易操作,适于在基层单位推广应用。     

鸭瘟病毒抗体间接ELISA检测试剂盒的研制

将鸭瘟病毒（DPV）CHv株经过鸭胚成纤维细胞增殖、反复冻融、差速离心和蔗糖密度介质离心制备纯化抗原,建立了用于检测鸭血清中抗DPV特异性IgG抗体的间接ELISA方法,并对其进行了标准化研究,确定了最佳工作条件,研制了相应的检测试剂盒并对盒内各组分的保存条件、质量控制以及试剂盒的特异性、重复性进行了研究。结果显示,4.75μg/孔的纯化DPV抗原包被反应板可获得良好的特异性和敏感性,1∶100稀释的待检血清样品反应后D405 nm值换算为抗体效价的标准直线方程为y=1.573＋3.552x（r=0.953,n=40）。试剂盒在4℃保存3个月或-20℃保存10个月后各项性能都很好。应用该试剂盒对皮下注射、口服和滴鼻免疫DPV弱毒的20日龄樱桃谷鸭血液中抗DPV特异性IgG抗体效价进行了测定,皮下免疫鸭于第6 d,口服和滴鼻免疫鸭于第9 d可在血清中检测到DPV特异性抗体,且至第60 d时依然可检测到高效价抗体。证实,该试剂盒可用于鸭瘟的血清流行病学调查和鸭场免疫抗体水平的监测。     

Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against duck swollen head hemorrhagic disease virus

An indirect enzyme-linked immunosorbent assay (iELISA) was developed for detecting antibody to duck swollen head hemorrhagic disease virus (DSHDV) using purified whole virus by sucrose density gradient ultracentrifugation as a coating antigen. Antiserum against DSHDV strains HY-99 (hyperimmume serum) was prepared in 30-day-old ducks by vaccination with inactivated DSHDV and used as positive sera. The iELISA test was optimized with different reagents or dilutions. The validation results showed that this assay was only specific for antibodies against duck viral swollen head hemorrhagic disease. The OD450 value for positive serum diluted 1:800 was also determined to be greater than the positive threshold. The highest coefficient of variation value was 3.66% for the intra-assay and 5.79% for the interassay, which were all less than 10%. This assay has been successfully applied to the examination of the duck sera clinically. These results in this study indicate that the newly-developed iELISA offers a precise, specific, sensitive, and reproducible means of measuring DSHDV antibodies in duck sera.     

牛口蹄疫病毒VP2结构蛋白抗体间接ELISA方法的建立

为建立牛口蹄疫(FMD)抗体的检测方法,本研究将口蹄疫病毒(FMDV)的VP2基因,通过pPROEXTM HTb表达载体在大肠杆菌DH5α中表达,获得大小为35ku的重组VP2蛋白(rVP2),western blot证实rVP2可与FMDV5种血清型的牛阳性血清发生特异性反应。以纯化复性的rVP2为抗原建立了FMDVrVP2间接ELISA方法。重复性试验证实批内、批间变异系数均小于10%。特异性交叉试验表明,该抗原不与常见的其他7种牛病阳性血清发生交叉反应。检测非免疫无口蹄疫国家牛阴性血清的特异性为100%;检测感染血清敏感性为97.3%;检测O-AsiaⅠ的二价苗免疫牛血清,与4种商品化试剂盒比较,其符合率分别为69.0%、95.0%、90.4%和86.8%。实验结果表明建立的ELISA方法可以用于口蹄疫感染和免疫抗体检测。     

茨城病毒VP7蛋白基因的克隆表达及间接ELISA方法的建立

采用RT-PCR方法扩增出了茨城病毒（IBAV）No．2株编码VP7蛋白的S7全长基因，并克隆入原核表达载体pET-28a（＋）中，在E．coli BI.21中表达。经Western-blot检测，表达的重组VP7蛋白具有良好的反应原性。以纯化的重组蛋白作为包被抗原，初步建立了检测IBAV血清抗体的间接ELISA方法。