Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.
Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.
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抗体亲和力作为埃博拉病毒监测纸基免疫测定中信号产生的驱动因素
DOI：
10.1007/s00216-021-03317-4
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发表时间：
2021-06
影响因子：
4.3
通讯作者：
Mace CR
中科院分区：
文献类型：
--
作者：
Murray LP;Govindan R;Mora AC;Munro JB;Mace CR
在流行病期间，例如历来困扰非洲地区的频繁和破坏性的埃博拉病毒爆发，血清学监测工作对于病毒遏制和开发有效的抗病毒疗法至关重要。抗体血清学也可用于回顾性人群水平监测，以提供更完整的感染总数估计。埃博拉监测工作依赖于酶联免疫吸附测定法（elisa），这种方法只能在实验室进行检测，不适合在资源有限的环境中使用。在这篇论文中，我们描述了一种基于纸张的免疫分析法，能够检测抗埃博拉病毒IgG，使用埃博拉病毒包膜糖蛋白外结构域（GP）作为亲和试剂。我们评估了7种针对GP-KZ52、13C6、4G7、2G4、c6D8、13F6和4f3的单克隆抗体（mab），以阐明结合亲和力和结合表位对检测性能的影响，并最终解释结果。我们使用生物层干涉法来表征每个抗体与GP的结合，然后评估它们在我们的纸质设备中的性能。结合亲和率（KD）和on rate （kon）是影响纸基免疫测定灵敏度的主要因素。最佳KD值（3 ~ 25 nM）的单克隆抗体的检出限最低（约为1 μg mL−1），而KD值为bb0 ~ 25 nM的单克隆抗体在我们的装置中检测不到。此外，最令人惊讶的是，我们确定在纸质设备中观察到的信号与kon成正比。这些结果强调了确保识别试剂的质量足以支持所需的分析性能的重要性，并表明个体免疫反应的强度会影响分析结果的解释。在线版本包含补充材料，可在10.1007/s00216-021-03317-4获得。
During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual’s immune response can impact the interpretation of assay results. The online version contains supplementary material available at 10.1007/s00216-021-03317-4.
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影响因子：
5.4
作者：
Maruyama, T;Rodriguez, LL;Burton, DR
通讯作者：
Burton, DR
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