关键词: 间接免疫荧光, 鸭病毒性肿头出血症病毒, 检测, 抗原定位

Abstract: 【Objective】To establish an Indirect Immunofluorescence assay (IFA) technique for the detection of Duck Swollen Head Haemorrhagic Disease Virus (DSHDV) in paraffin-embedded tissue,and provide an effective examination method for the diagnosis, subcellular antigen localization and dynamic distribution in vivo of DSHDV.【Method】Through differential centrifugation purified DSHDV,DSHDV-specofic antibodies were obtained from rabbits vaccinated with purified virus, and then purified through DEAE-SephadexA-50 columns. An IFA was established to detect the DSHDV antigen in paraffin-embedded tissue. then DSHDV-antigen was detected in each organ tissues of 28-day-old ducks which were experimentally infected with DSHDV.【Result】The optimum conditions of this IFA were as follows: Antigen was retrieved by microwave in 0.01 mmol/L citrate buffer solution(PH6.0) for twenty minutes, and blocked with 10% horse serum for 30min at 37℃.Slices were incubated overnight at 4℃ with diluted primary antibody(1:50) ,and then incubated for 30 min at 37℃ with diluted FITC-labelled-secondary antibody(1:100)which contain 0.01% Evans blue. The IFA was applied in detecting the DSHDV antigen in liver tissues of dead ducks, and the results were positive in DSHDV infected dead ducks and negative in dead ducks infected by Duck Plague Virus, Duck viral hepatitis Virus and Riemerella Anatipestifer. The IFA was also applied in detecting the virulent DSHDV antigen in different organs of artificially infected dead 28 old days ducks, and the viral antigen was detected in the heart、liver、spleen、pancreas、lung、kidney、bursa of fabricius、esophagus、trachea、muscle、thymus、duodenum、jejunum、ileum、cecum and rectum, while viral antigen was not detected in the harderian gland、brain、skin and proventriculus. The antigen distributes mainly in the cytoplasm of the infected cells. 【Conclusion】The establishment of IFA is a sensitive and specific method in detecting DSHDV antigen in paraffin-embedded tissue, and it’s a good method in diagnosis and antigen location of DSHDV. IFA can application in the experimentally diagnosis and study on the antigen distribution dynamics in duck tissue cells.

Key words: Indirect Immunofluorescence assay, DSHDV, detection, antigen localization