H5亚型禽流感病毒间接免疫荧光快速诊断方法的建立

Establishment of indirect fluorescent antibody test to the rapid diagnosis of H5 subtype highly pathogenecity avian influenza virus

本研究以当前严重威胁我国养禽业的高致病性禽流感H5亚型病毒为研究对象,病毒在犬肾细胞(MDCK)上培养增殖,经蔗糖梯度离心对病毒进行纯化,免疫清洁级的新西兰公兔,高免血清经辛酸-硫酸铵法和葡聚糖G50柱纯化,制得第一抗体。以FITC标记的山羊抗兔IgG为第二抗体,通过反应条件的优化,建立了间接免疫荧光快速诊断方法。本法的最佳检测组织为心肌和胰腺,检测时间只需3小时,本法可检出人工感染后36小时尚未表现出临床症状鸡只中的病毒,对禽流感H7亚型、H9亚型病、新城疫、传染性支气管炎和传染性喉气管炎禽出败等病料进行特异性检验结果均为阴性。运用本方法对69个禽场的临床病料进行了检测,检测结果与鸡胚分离法进行比对,9个阳性场(广东省2004年9个原疫点的病料)的9份病料中,检出8份阳性;而鸡胚分离法阴性样品,本法检测结果与之完全相符。本法用于禽流感H5亚型病毒的快速诊断具有快速、简便、敏感、特异、费用低廉和不存在交叉污染等优点,在当前流行的H5亚型高致病性禽流感快速诊断中具有良好的应用前景。

An indirect fluorescent antibody （IFA） test was established with special polyclonal antibody for the detection of highly pathogenic avian influenza （HPAI） H5 viral antigens. Avian influenza HSN1 virus was propagated in Madin-Darby canine kidney cell （MDCK）, and purified by sucrose velocity gradient technique. Two clean New Zealand rabbits were vaccinated with this oil-emulsion vaccine. The rabbit anti-avian influenza virus HSN1 specific IgG was extracted by caprylic-ammonium sulphate method, purified by Sephadex GSO chromatography after dialyzing. An IFA test was developed to the rapid diagnosis of HPAI by using the anti-AIV H5 lgG as the primary antibody and the fluorescein isothiocyanated（FITC） conjugate goat anti-rabbit lgG as the second antibody, cardiac muscle and pancreas were the most suitable tissues to choose in diagnosis of HPAIV when using this fluorescent antibody test. Fluorescence was observed in artificial infection chickens as early as 36h after AIV H5 inoculation, when clinic symptoms were not seen at this period of time. Apply this method to detect AIV H7, AIV H9, infectious bronchitis viruse and infectious laryngotracheitis virus infected chickens tissues, all showed negative results. These results indicated the indirect fluorescent antibody test was specific to AIV HS. A total of 104 clinical specimens from 69 farms were detected by this method, 8 of 9 virus isolated positively specimens were positive, while the other 60 isolated negatively specimens were negative either. Obviously, IFA and virus isolation method had a good correlation. The IFA established in this study had been demonstrated to be a rapid, sensitive, specific and economical test for the diagnosis of HPAI H5 subtype.