抗番鸭呼肠孤病毒(MDRV)单克隆抗体的制备及应用

【目的】制备抗番鸭呼肠孤病毒（MDRV）的单克隆抗体，建立番鸭呼肠孤病毒病的间接免疫荧光（IFA）快速诊断方法。【方法】制备MDRV抗原，用之免疫Babl/c小鼠，取免疫小鼠脾细胞和SP2/0骨髓瘤细胞进行融合，采用IFA和ELISA筛选阳性杂交瘤细胞株，体内诱生腹水法制备单克隆抗体，检测其生物学特性，应用制备的单克隆抗体建立MDRV IFA快速诊断方法。【结果】筛选到237-11，45-12和3-H3 3株特异性好并能稳定分泌抗MDRV单克隆抗体的杂交瘤细胞株，其中45-12株单抗为IgM亚型，237-11和3-H3株单抗为IgG3亚型。45-12和3-H3 2株单抗具有ELISA特性，效价分别为10-4和10-5；237-11株单抗具有IFA特性，效价达10³。特异性测定结果显示，3株单抗仅与MDRV反应，而与正常细胞培养物（MDEF）、番鸭细小病毒（MPV）、鹅细小病毒(GPV)、禽呼肠孤病毒(ARV) 、鸭副黏病毒（PMV）和鸭肝炎病毒（DHV）均无交叉反应。应用抗MDRV单抗建立的IFA方法与病毒分离法（VI）的符合率为91%。【结论】筛选到2株具有ELISA特性、1株具有IFA特性且能分泌抗MDRV单克隆抗体的杂交瘤细胞株，建立了MDRV IFA快速诊断方法，为番鸭呼肠孤病毒病的快速诊断奠定了基础。

Preparation and application of monoclonal antibody against muscovy duck reovirus

【Objective】 The research was done to prepare anti-musovy duck reovirus(MDRV) monoclonal antibody(McAb) and build up immunological assay to detect MDRV.【Method】 After Babl/c mice were immunized with muscovy duck reovirus(MDRV) antigen,the spleen cells were fused with SP2/0 myeloma cell lines.Hybridoma cell lines secreting McAb against MDRV were screened by indirect ELISA and IFA and biological characteristic of these McAb were determined.Then IFA was built up.【Result】 Three hybridoma cell lines could steadily secrete specific monoclonal antibodies(McAbs) against MDRV,named 237-11,45-12 and 3-H3.Isotyping analysis showed that 45-12 belonged to IgM,237-11 and 3-H3 belonged to IgG3.45-12 and 3-H3 had chracteristic of ELISA but 237-11 had characteristic of IFA.These McAbs had high specificity with MDRV and had no cross-reactivity with muscovy duck embryo-fibroblast(MDEF),muscovy duck parpovirus(MPV),goose parpovirus(GPV),avian reovirus(ARV),duck paramyxovirus(PMV) and duck hepatis virus(DHV).Indirect fluorescence assay could detect MDRV and was good concidence(91%) with virus isolation.【Conclusion】 Preparing monoclonal antibody against MDRV,two strains had chracteristic of ELISA and one strain had chracteristic of IFA.Detecting MDRV by IFA laid foundation for further research on rapidly clinical diagnosis.