应用跨膜区置换的血凝素蛋白建立H7N9禽流感病毒抗体间接ELISA检测方法

【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r0.5,P0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。

Establishment of an indirect ELISA for detecting H7N9 subtype avian influenza virus antibodies applied with hemagglutinin containing a replaced transmembrane domain

Objective] H7N9 avian influenza virus can infect chickens, and high pathogenic avian influenza (HPAI) H7N9 strains have appeared after natural mutation. Thus, H7N9 vaccines immunization in chickens would be a tendency, and developing an antibody detection method for immunization is a need. To establish a sensitive, rapid, high-throughput enzyme-linked immunosorbent assay (ELISA) to detect H7N9 subtype avian influenza virus antibodies in chickens. Methods] Three wild-type hemagglutinin (HA) proteins belonging to W1, W2-A and W2-B clades were expressed by an insect cell-baculovirus expression system, and one recombinant HA (H7-53TM) containing a replaced H3 HA transmembrane domain (TM) was expressed as well. Four HA proteins were purified by ion-exchange chromatography and used as ELISA antigens to detect H7N9 avian influenza virus antibodies. Results] The results of specificity, sensitivity and repeatability assays showed TM replacement mainly affected the repeatability of the HA antigen. The intra- and inter-coefficient of variables of H7-53TM were less than 10%, showing better repeatability; whereas those of 3 wild-type HA proteins were more than 10%, showing worse repeatability. Therefore, H7-53TM was chosen as ELISA antigen. The results of the Receiver operating characteristic curve (ROC curve) analysis show that the established ELISA could accurately discriminate between H7N9 subtype avian influenza virus-positive and -negative serum specimens. Based on correlation, the established ELISA had significantly strong correlation with HI assay to test 134 chicken serum specimens (r=0.854 6, P<0.000 1), and the established ELISA had significantly correlation with HI assay to test sera collected from chickens vaccinated with vaccine strains belonging to three different clades (r>0.5, P<0.05). Conclusion] TM replacement can increase the repeatability of the HA protein to detect H7N9 avian influenza virus antibodies, and establishes an indirect ELISA for detecting specific antibodies against H7N9 subtype avian influenza viruses belonging to different clades applied with HA containing a replaced transmembrane domain.