检测H6亚型禽流感病毒的双抗体夹心ELISA方法的建立及初步应用  

Developing a Double Antibody Sandwich ELISA for Detecting H6 Subtype Avian Influenza Virus and Its Preliminary Application

作　　者：万志敏 龚简汐 赵喆泓 汤婷 李亚锋 谢泉[1,2,3,4] 李拓凡 邵红霞 秦爱建[1,2,3] 叶建强 WAN Zhimin;GONG Jianxi;ZHAO Zhehong;TANG Ting;LI Yafeng;XIE Quan;LI Tuofan;SHAO Hongxia;QIN Aijian;YE Jianqiang(Key Laboratory of Jiangsu Preventive Veterinary Medicine,Key Laboratory for Avian Preventive Medicine,Ministry of Education,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu 225009;Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou,Jiangsu 225009;Joint International Research Laboratory of Agriculture and Agri-Product Safety,Ministry of Education,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学兽医学院,禽类预防医学教育部重点实验室,江苏省动物预防医学重点实验室,江苏扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]扬州大学农业科技发展研究院(国际联合实验室),江苏扬州225009 [4]教育部农业与农产品安全国际合作联合实验室,江苏扬州225009

出　　处：《中国家禽》2024年第11期34-39,共6页China Poultry

基　　金：江苏省基础研究计划自然科学基金项目(BK20200922);国家自然科学基金项目(32002262);江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：为建立快速检测H6亚型禽流感病毒的血清学方法,试验利用前期制备的两株抗H6亚型禽流感病毒血凝素单克隆抗体,建立检测H6亚型禽流感病毒的双抗体夹心ELISA方法,并进行特异性、灵敏度、稳定性试验以及对活禽市场采集的48份咽拭子样品的检测效果。结果显示:该ELISA方法只与H6亚型禽流感病毒反应,而与H1、H3、H4、H5、H7、H9、H10、H12等其他亚型禽流感病毒,血清4型禽腺病毒、血清8b型禽腺病毒、血清3型鸭腺病毒、传染性支气管炎病毒、新城疫病毒、鹅星状病毒、小鹅瘟病毒、传染性法氏囊病病毒以及禽白血病病毒均无交叉反应;该ELSIA方法可检测2.32×10^(4)TCID50/mLH6亚型禽流感病毒,批间和批内重复试验变异系数均小于10%;该ELISA方法和RT-PCR方法对活禽市场采集的48份咽拭子样品检测结果一致,进一步检测攻毒鸡的咽拭子显示该方法可有效检测咽拭子中H6亚型禽流感病毒。研究表明,基于两株单克隆抗体建立的检测H6亚型禽流感病毒的双抗体夹心ELISA方法具有特异性强、灵敏度高的特点,适用于H6亚型禽流感病毒感染的临床检测。To develop a serological method for the detection of H6 subtype avian influenza virus(AIV),a double antibody sandwich ELISA based on monoclonal antibodies against hemagglutinin of H6 subtype AIV was established in this study,and the specificty,sensitivity and stability of which were evaluated.The 48 throat swab samples collected from a live poultry market were detected with the established ELISA.The results showed that the established ELISA only reacted with H6 subtype AIV,did not react with other viruses,such as H1,H3,H4,H5,H7,H9,H10,H12 subtypes of AIVs,serotype 4 fowl adenovirus,serotype 8b fowl adenovirus,serotype 3 duck adenovirus,infectious bronchitis virus,Newcastle disease virus,goose astrovirus,gosling plague virus,infectious bursal virus and avian leukemia virus.The established ELISA could detect 2.32×10^(4) TCID50/mL of H6 subtype AIV,the inter-and intra-batch coefficients of variation of which were all less than 10%.The detection result of 48 throat swab samples by the established ELISA and RT-PCR were consistent,and the established ELISA could effectively detect the virus in throat swabs from challenged chickens.The results indicated that the double antibody sandwich ELISA based on two monoclonal antibodies with high specificity and sensitivity was established for detecting H6 subtype AIV,which was suitable for clinical detection of H6 subtype AIV.

关 键 词：H6亚型禽流感病毒 单克隆抗体 血凝素蛋白 双抗体夹心ELISA 

分 类 号：S855.3[农业科学—临床兽医学]