Wang D, Zheng Y, Kang X, Zhang X, Hao H, Chen W, Liu L, Li X, Li L, Yuan Q, Chen F, Yang Y, Jiang Y, Jiang H
State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, 100071, China.
Eur J Clin Microbiol Infect Dis. 2015 Jul;34(7):1327-36. doi: 10.1007/s10096-015-2353-6. Epub 2015 Mar 22.
Assays with the ability to detect multiple antibodies in parallel have a wide range of potential applications in epidemiologic research. Here, a multiplex enzyme-linked immunosorbent assay-based protein array (ELISA-array) was developed to simultaneously detect five Flaviviridae infections. The platform was based on an indirect ELISA and 15 antigens were constructed for specific antibody detection against five Flaviviridae viruses (Japanese B, tick-borne encephalitis, West Nile, dengue, and yellow fever viruses) and four serotypes of dengue virus. The specificity was evaluated by calculating the signal value cross-reacting with serum immunized with other viruses, and the sensitivity of antigens was compared with conventional ELISAs using immunized rabbit polyclonal antisera. IgG and IgM calibration curves were constructed to evaluate the reproducibility of the platform. Finally, 24 dengue fever (DF) infection and 15 tick-borne encephalitis (TBE) infection clinical sera were used to compare the advantage of ELISA-array to ELISA. After initial screening, 9 out of 15 antigens were chosen for ELISA-array printing. By using different virus-immunized rabbit antiserum, 7 out of 9 antigens showed good specificity in the ELISA-array. Eight out of 9 antigens showed four-fold greater sensitivity in ELISA-array compared to that in conventional ELISAs. The coefficients of determination (r (2)) close to 1 showed high reproducibility, and clinical sera test showed that ELISA-array had higher specificity and sensitivity than traditional ELISA. ELISA-array was a good platform for antigen screening and this multiplexed assay might be a useful and convenient tool for multiple immunological detection of infectious viral antibodies.
能够同时检测多种抗体的检测方法在流行病学研究中具有广泛的潜在应用。在此，开发了一种基于多重酶联免疫吸附测定的蛋白质阵列（ELISA阵列），用于同时检测五种黄病毒科感染。该平台基于间接ELISA，构建了15种抗原，用于特异性检测针对五种黄病毒科病毒（日本乙型脑炎病毒、蜱传脑炎病毒、西尼罗河病毒、登革病毒和黄热病毒）以及登革病毒四种血清型的抗体。通过计算与用其他病毒免疫的血清交叉反应的信号值来评估特异性，并使用免疫兔多克隆抗血清将抗原的敏感性与传统ELISA进行比较。构建IgG和IgM校准曲线以评估该平台的重现性。最后，使用24份登革热（DF）感染临床血清和15份蜱传脑炎（TBE）感染临床血清来比较ELISA阵列与ELISA的优势。经过初步筛选，选择了15种抗原中的9种用于ELISA阵列打印。通过使用不同病毒免疫的兔抗血清，9种抗原中的7种在ELISA阵列中显示出良好的特异性。与传统ELISA相比，9种抗原中的8种在ELISA阵列中的敏感性高四倍。决定系数（r (2)）接近1表明重现性高，临床血清检测表明ELISA阵列比传统ELISA具有更高的特异性和敏感性。ELISA阵列是抗原筛选的良好平台，这种多重检测方法可能是用于感染性病毒抗体多重免疫检测的有用且便捷的工具。
