用免疫荧光技术检测细胞培养物中兔出血症病毒

Detection of Rabbit Hemorrhagic Disease Viruses (RHDV) in Cell Culture Materials by Fluorescent Antibody Technique

用本所实验室制备的兔出血症病毒(rabbit hemorrhagic diseasevirus,RHDV)高免血清,按常规方法提纯出抗体(IgG),用异硫氰酸荧光素(FITC)标记IgG。在细胞培养时,培养瓶中放入玻片,当细胞长成单层时,按常规方法接种病毒液,培养24、48、96、120h时取出玻片,用荧光抗体染色,在荧光显微镜下观察不同代次的细胞毒。经观察:兔肾上皮细胞(RK)毒培养到36～48h、羊睾丸细胞(ST)毒培养到72～96h时可观察到特异性荧光,随着培养时间的延长,荧光亮度增强,胞浆内充满特异性荧光。用肝组织强毒病料触片,呈特异性荧光,对照细胞培养48h无荧光出现,证实了两株细胞培养物中有大量的兔出血症病毒存在,从而成功的分离培养出了兔出血症病毒细胞毒。

Rabbit hemorrhagic disease viruses （RHDV） IgG antibodies were purified from the highimmunized serum prepared according to conventional methods, and were labeled with fluorescein isothiocyanate （FITC）. Sheep testis cells and rabbit kidney epithelial cells were cultured and were inoculated with RHDV after the cells developed into monolayer on microscope glass slide respectively, and were stained with fluorescent antibodies at 24h,48h,96h, 120h after inoculation and observed under fluorescence microscope .The results showed that there were specific fluorescence in rabbit kidney epithelial cells between 36h and 48h and in sheep testis cells between 72h and 96h, and also the brightness of the fluorescence increased with the passage of time and the cytoplasm was full of florescence. The smears of liver tissue infected with virulent RHDV appeared specific fluorescence, while the control cells had no florescence at 48h. It indicated that there were RHDV in the two lines of cells culture materials.