H7亚型禽流感病毒cELISA抗体检测方法的建立

doi:10.1016/S2095-3119(21)63645-6

摘要/Abstract

摘要：

【目的】H7亚型禽流感病毒(Avian influenza virus, AIV)和H5亚型一样，多数属于高致病性毒株，通常在禽类间传播，引起家禽出现严重的临床症状和高死亡率。从1996年至2012年，加拿大、意大利、墨西哥、荷兰、英国和美国出现了人感染H7亚型流感病毒(H7N2、H7N3和H7N7)的病例。2013年，中国报告了人感染H7N9流感病毒的事件，此后，该病毒持续在人和禽类中传播。因此，开展H7亚型禽流感病毒抗体检测工作成为一个重要问题。【方法】该研究用纯化的H7-AIV作为包被抗原，辣根过氧化物酶（Horseradish peroxidase, HRP）标记的单克隆抗体1H9（HRP-1H9）作为竞争抗体，采用棋盘滴定法建立了一种检测H7亚型AIV抗体的竞争性酶联免疫吸附实验(Competitive enzyme-linked immunosorbent assay, cELISA)。【结果】抗原包被的最佳浓度为5 μg mL^-1，血清稀释度为1/10，酶标抗体为1/3000。用ROC曲线分析法测定178份AIV阴性和368份AIV阳性血清（n=546）的cELISA临界值，当PI为40%时，cELISA的特异性和敏感性分别为99.15%和98.12%。该方法可检测H7Nx（N1--N4、N7--N9）禽流感病毒抗体，与H1 -- H6、H8 -- H15亚型禽流感病毒及鸡新城疫病毒(Newcastle disease virus, NDV)、传染性支气管炎病毒(Infectious bronchitis virus, IBV)和传染性法氏囊病病毒(Infectious bursal disease virus, IBDV)等常见禽类病毒无反应，具有良好的特异性。该方法与血凝素抑制试验的符合率为98.56%。重复性实验表明，批内和批间重复的变异系数均小于12%。【结论、创新性】综上所述，本研究利用H7亚型特异性酶标单克隆抗体作为竞争抗体，建立了cELISA抗体检测方法，其优势是酶标抗体与待检血清同时加入反应孔中进行竞争反应，与先加入待检血清，再加竞争抗体反应的常规方法比较，简化了操作步骤，缩短反应时间，为开展H7-AIV抗体的大量检测提供了一种简单、便捷的技术手段。

Abstract: H7 avian influenza viruses (AIVs) normally circulated among birds before. From 1996 to 2012, human infections with H7 AIVs (H7N2, H7N3, and H7N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA. Until March 2013, human infections with H7N9 AIVs were reported in China. Since then, H7N9 AIVs have continued to circulate in both humans and birds. Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic. In this study, a competitive enzyme-linked immunosorbent assay (cELISA) method for the detection of antibody against H7 AIVs was established. The optimal concentration of antigen coating was 5 μg mL^–1, serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000. To determine the cut-off value of cELISA, percent inhibition (PI) was determined by using receiver operating characteristic (ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples (n=546). When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively. This method could detect the antibodies against H7Nx (N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) and Infectious bursal disease virus (IBDV), exhibiting good specificity. This method showed a coincidence rate of 98.56% with hemagglutinin inhibition (HI) test. And the repeatability experiment revealed that the coefficients of variation (CV) of intra- and inter-batch repetition were all less than 12%. The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.

Key words: H7 subtype , influenza , monoclonal antibody , cELISA

. H7亚型禽流感病毒cELISA抗体检测方法的建立 [J]. Journal of Integrative Agriculture, 2022, 21(1): 199-207.

WANG Cong-cong, WANG Si-wen, ZHANG Ying, SHI Jian-zhong, YIN Xin, LI Cheng-jun, WANG Xiu-rong. Development of a cELISA for effective detection of the antibody against H7 subtype of avian influenza virus[J]. Journal of Integrative Agriculture, 2022, 21(1): 199-207.

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