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犬细小病毒单克隆抗体的制备和Abstract : Canine parvovirus（CPV）is a potent infectious diseases, found in dogs in recent years, It causes acute haemorrhagic diarrhoea and sometimes fatal myocarditis , and to kill a large number of white blood cells, the morbidity and mortality are very highin the puppy, it has become an important canine infectious diseases. By using the CPV as antigen,Monoclonal antibody(McAb)was prepared,and two positive clones,designated as 4E4 and 5C8 respectively,were obtained from hybridoma cell lines.The BALB/c mice were immunized subcutaneously with CPV,which was purified with polyethylene glycol.After being immunized three times,the mice were immunized intraveinally with equal the dose for the final immunization.Three days later,spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay(iELISA)was used to screen hybridoma cells,and limiting dilution method was applied to subclone positive hybridoma cells three times,and two positive clones,designated as 4E4 and 5C8 respectively,were obtained from hybridoma cell lines.4E4 was subtyped to be IgM,and 5C8 was subtyped to be IgG.1. Two hybridoma cell lines secreting McAb against CPV have been established with the characteristic of counteraction can apply to treating CPV-infected dogs. Key words : Canine parvovirus;isolation;PCR;Expression;Monoclonal antibody;Application 【摘要】犬细小病毒(Canine Parvirus,CPV)感染是近年来发现的犬的一种烈性传染病,可引起犬出血性肠炎和心肌炎,并使白细胞大量死亡,在幼犬中的发病率和死亡率均很高,目前已成为犬的一种重要传染病,。 本研究利用收集到抗原制备了针对该病毒的单克隆抗体(Monoclonal antibody,McAb)。McAb的方法如下：用PEG沉淀法浓缩CPV的细胞培养液，依此为抗原免疫8周龄LB/c小鼠，经过3次皮下注射免疫后，再进行尾静脉加强免疫，3d后取小鼠脾和骨髓瘤细胞 SP2/0-Ag-14进行融合。通过间接ELISA试验对杂交瘤细胞进行，阳性株按有限稀释法进行3次亚克隆后，共获得了2株能稳定分泌单克隆抗杂交瘤细胞，分别命名为4E4和5C8，细胞上清效价为1:103和1:104，腹水效价:107和1:108，中和效价都为1:320，亚类鉴定结果表明4E4属于IgM、5C8属于IgG1。应用这两株McAb的中和特性，可以对CPV感染犬进行临床治疗试验。 【关键词】犬细小病毒；分离；PCR； 表达；单克隆抗体；应用 犬细小病毒（Canine Parvovirus，CPV）属细小病毒科，细小病毒属。自1982年以来，犬细小病毒病已在我国广泛流行，成为一种犬的急性烈性传染病，严重影响了我国的养犬业。为了诊断