【作者】 谢联香；

【导师】 陆承平； 徐汉坤；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 犬细小病毒病是具有高度接触性的烈性传染病，既严重威胁着家养宠物的身体健康，又是对当前我国养犬业、毛皮动物养殖业和野生动物保护业危害较大的疫病。在犬细小病毒病诊断方法中，ELISA检测方法方便快捷、费用低廉，适于在基层推广采用F81细胞大量扩增犬细小病毒CPV-GN株，利用聚已二醇(PEG6000)浓缩后，测得其蛋白浓度的基础上。采用倍比稀释法获得一系列的蛋白浓度值，同时将阳性血清也倍比稀释，利用方阵滴定法进行酶联免疫吸附试验(ELISA)检测确定了抗原最佳的包被浓度为9μg／ml；阳性对照的血清稀释度为1：160。最适的封闭液为1.75％的明胶，封闭条件为37℃1h；待检样品与包被抗原的反应时间为37℃2h：酶标二抗的反应时间为37℃1h。经过一系列的验证试验，证明建立的间接ELISA方法具有较强的稳定性和特异性。取50份免疫犬血清，用该方法与常规的HI试验分别进行抗体检测，两者的共同符合率为94％。其中，明显阳性和阴性的样品，共同符合率能达到100％。说明建立的间接ELISA方法具有简便、快速、特异的优点。为了评估犬细小病毒的免疫情况，将建立的间接ELISA方法应用于140份犬细小病毒血清抗体的检测，而且一年后，对其中的60只犬再次采集血清进行检测。结果证明：现在采取的免疫程序，完全可以提供足够的保护力抵制犬细小病毒的感染。而且疫苗的免疫效果与犬的性别和品种没有太大的相关性，而犬的年龄在很大程度上影响着疫苗的免疫效果。以纯化的犬细小病毒(Canine parvovirus，CPV)CPV-GN毒株为免疫原，接种6周龄的Balb／c小鼠。最后一次加强免疫后3天，取小鼠脾细胞与骨髓瘤细胞(SP2／0)融合。以纯化的CPV建立的间接酶联免疫吸附试验(ELISA)和中和试验(HT)筛选阳性克隆，并用有限稀释法进行亚克隆，用F81细胞包被板子进行ELISA检测，剔除与细胞蛋白交叉反应的阳性克隆，共获得3株CPV特异性杂交瘤细胞株A7、C5、E9。它们的中和效价分别为1；160、1；80和1；160。经过一系列鉴定，这3株单抗株(McAbS)均有很好的特异性和稳定性。利用A7单抗株制备单抗的腹水，间接ELISA和血凝抑制试验(HI)检测单抗腹水的效价分别为1：1280和1；640。在已经制备犬细小病毒单克隆抗体的基础上，将单抗加以辣根过氧化物酶标记，建立了双抗夹心酶联免疫吸附试验(ELISA)检测犬细小病毒的方法。结果表明：单抗包被的最佳浓度为7ug／ml；最适的封闭液为1.75％的明胶，封闭条件为37℃1h：待检样品与包被单抗的反应时间为37℃2h：酶标单抗的反应时间为37℃1h；检测样品直接用生理盐水稀释后，即可检测。经过一系列的验证试验，证明建立的夹心ELJSA方法具有较强的稳定性和特异性。将建立的夹心ELISA方法应用于40份临床样品的检测，并且与血凝试验相比较，两者的共同符合率为92.5％。其中，明显阳性和阴性的样品，共同符合率能达到100％。对临床收集的20份粪便样品，利用建立的夹心ELISA方法进行检测，并且与PCR方法进行比较，对于检测结果不符合的样品接种F81细胞，观察病变情况，加以验证。更多还原

【Abstract】 The disease of canine parvovirus, caused by canine parvovirus(CPV), is acute andhighly contagious disease in canine and other carnivores, and is one of the most severeinfectious diseases in canine farming, far cultivation and wildlife conservation. In latestyears. The research about diagnosis of CPV has a lot of development. Among thesemethods, The Enzyme-linked immunosorbent assay(ELISA) has the excellence ofconvenience. Shortcut. Inexpensive and suitably spread in grass roots.At first, The canine parvovirus (CPV-GN) strain was largely cultivate in cell of F81,purified by PEG6000, mensurated the consistence of protein, in succession, CPV-GN strainwas used as antigen for developing indirect enzyme-linked immunosorbent assay (ELISA).The conditions of indirect ELISA were determined as follows: 9ug/ml of CPV-GN strainwas used to coat ELISA plate, the positive sera need 1:160 diluted. The optimal closedliquid is 1.75% glutin and its optimal woking time is 37℃1h; the woking time of thesample and antigen is 37℃2h. A series of test proved this method possessed upstandingstability and distinctness. 50 samples were detected by this method and hemagglutinationinhibitory(HI) test respectively, the concident rate was 94%. For strong positive andnegative samples, the concident rate was 100%. The indirect ELISA proved to be morespecific, rapid and easier to perform than the HI test. For evaluating the vaccinal effect,This indirect ELISA was used to detect sera collected from 160 dogs; after one year, 60dogs among these dogs was also collected sera for detection in this indirect ELISA. Theresult revealed that the immune procedure can offer enough safeguard to resisting CPVinfection. In simultaneity, The vaccinal effect was not signilicantly associated with sex andbreed, but, was signilicantly associated with age.The purified canine parvovirus (CPV-GN) strain was inoculated Balb/C mouse. Afterthe last immunity, spleen cells from Balb/C mouse was fused with SP2/0 in the fourth day,Positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA)and neutralization test (NT) and were subclonized with limited dilution. The false positiveclones were rejected by F81 cell coating ELISA plates. Three Monoclonal Antibodies (McAbs) were gained. They are named A7.C5.E9. The titer of viral neutralization isrespective 1:160.1:80 and 1:160. A series of test proved all of these three McAbs possessedupstanding stability and distinctness. The titer of ascites,which was produced from A7, wasdetermined by indiect ELISA and hemagglutination inhibitory(HI). The result is respective1:1280 and 1:640.A double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) kitfor detection of canine parvovirus (CPV) in fecal was estabilished with the monoclonalantibodies against CPV. The result revealed that the optimal concentration of monoclonalantibody is 7ug/ml; the optimal closed liquid is 1.75% glutin and its optimal woking time is37~Clh; the woking time of the sample and McAb is 37℃2h; the reaction time ofHRP-labeled McAb is 37℃1h; the sample was direct diluted by physiological brine, aseries of test proved this method possessed upstanding stability and distinctness. 40samples were detected by the DAS-ELISA kit and hemagglutination (HA) test respectively,The results showed that the total coincident rate was 92.5%. For strong positive andnegative samples, the concident rate was 100%. The DAS ELISA kit and PCR were used todetect 20 samples, The sample which has the different result by the two method wasinoculated FS1 cell.更多还原