摘要
以超速离心浓缩结合蔗糖密度梯度离心法提纯的鸭瘟病毒(DPV)作为抗原免疫家兔制备兔抗DPV血清,用饱和硫酸铵沉淀结合DEAE-Sephedax-A-50离子交换柱层析提纯兔抗DPV IgG,建立了检测甲醛固定鸭体组织石蜡切片上DPV抗原的间接酶免疫组化法。该法能特异地检测出DPV感染鸭组织中的DPV而对鸭疫里默氏菌、鸭源多杀性巴氏杆菌(5∶A)、鸭源大肠杆菌(O1)感染致死的鸭组织以及正常鸭组织呈阴性反应,检测DPV感染死亡鸭的肝、十二指肠、脑、脾和胸腺的结果与PCR检测结果相同。其敏感性高于原位杂交。对DPV CH强毒株人工感染致死鸭的检测结果表明,该法可特异检测到肝、肺、肾、脑、十二指肠、空肠、回肠、直肠、法氏囊、脾脏、腺胃以及食管中的DPV。DPV主要分布于这些器官的上皮细胞和巨噬细胞。对1992—2004年经10%福尔马林保存的鸭瘟临床病例的肝脏检测结果均为阳性。该法可用于DPV感染鸭的诊断和定位检测,也可用于对甲醛固定组织进行回顾性诊断检测。
The duck plague viruses purified by the ultracentrifugation and sucrose density gradient process were used to immune the domestic rabbit for preparing the rabbit anfi-DPV serum. The sera were deposited by the saturated ammonium sulfate precipitation method and purified by the DEAE-Sephedax-A-50 ionic exchange column chromatography. Then the indirect imrnunoperoxidase technique was established to detect DPV antigen in formaldehyde fixed tissues from DPV infected duck on the paraffin section. This method specially detected DPV antigen in DPV infected duck tissues but the duck Riemerella anatipestifer, Pasteurella multocida（5 ：A）, E. coli（ 01 ） infected duck tissues and the normal duck tissues revealed the negative result. The examination results of liver, dodecadactylon, brain, spleen and thymus from died ducks infected with DPV were in consistent with those by PCR. Thus its sensitivity was better than that in-situ hybridization. The positive results can be obtained in liver, lung,kidney, brain, dodecadactylon,jejunum, ileum rectum, bursa of Fabricius, spleen, stomach glandularis and esophagus from died ducks experimentally infected with DPV, and DPV antigen are located mainly in their epithelial cells and macrophages. All of the liver samples from 1992 to 2004 conserved in 10% formalin from the died ducks diagnosed as DP in clinical outbreak were detected and revealed the positive results. The method was proved to be useful for the DPV antigen localization and its retrospective diagnosis.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2007年第5期640-644,共5页
Chinese Journal of Veterinary Science
基金
国家科技攻关重大资助项目(2004BA901A03)
四川省杰出青年学科带头人基金后续资助项目(03ZQ026-28)
四川省重点建设学科资助项目(SZD0418)
关键词
间接酶免疫组化法
鸭瘟病毒
抗原定位
鸭
indirect immunoperoxidase
duck plague virus
antigen localization
duck
