A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.

人感染甲型H7N9禽流感病毒（avian influenza A H7N9 virus）于2013年3月首次被发现，至今仍对公共卫生构成持续威胁。针对这一新型流感病毒，亟需优化其血清学检测手段。本研究针对甲型（H7N9）禽流感病毒的人源抗体检测，比较了血凝抑制试验（hemagglutinin inhibition, HI）、微量中和试验（microneutralization, MN）与蛋白质印迹试验（Western blot, WB）的灵敏度与特异性。采用马红细胞（horse erythrocytes, hRBCs）的血凝抑制试验，以及经改良的微量中和试验，其灵敏度分别优于使用火鸡红细胞的血凝抑制试验与标准微量中和试验。利用上述检测方法，确诊H7N9病例的受检血清中，有80%在感染后21天可检出H7N9特异性抗体。为兼顾灵敏度与特异性，本研究发现，当血清效价≥20（微量中和试验）或≥160（血凝抑制试验）时，可最为有效地判定样本为H7N9病毒血清学阳性。对于血凝抑制效价介于20~80之间，或微量中和效价为10的单份血清样本，可通过两种试验交叉验证或辅以蛋白质印迹试验进行确认。与成人或老年人群的血清样本不同，轻症儿童患者的抗体应答水平较低，甚至无法检出。上述检测方法的联合应用，可为人感染病例的临床诊断与血清学调查提供有力支持。