Department of Chemistry, Tufts University, Medford, MA, 02155, USA.
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, 01605, USA.
Anal Bioanal Chem. 2021 Jun;413(14):3695-3706. doi: 10.1007/s00216-021-03317-4. Epub 2021 Apr 14.
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 (K) and on rate (k) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best K (3-25 nM) exhibited the lowest limits of detection (ca. μg mL), while mAbs with K > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to k. 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.
在流行病期间，例如历史上困扰非洲部分地区的频繁且具有破坏性的埃博拉病毒爆发，血清学监测对于病毒控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的人群监测，以更全面地估计总感染人数。埃博拉监测工作依赖于酶联免疫吸附测定（ELISA），这限制了检测只能在实验室中进行，并且不能适应资源有限的环境。在本文中，我们描述了一种基于纸张的免疫测定法，该方法能够使用埃博拉病毒包膜糖蛋白外域（GP）作为亲和试剂检测抗埃博拉 IgG。我们评估了针对 GP-KZ52、13C6、4G7、2G4、c6D8、13F6 和 4F3 的七种单克隆抗体（mAb），以阐明结合亲和力和结合表位对测定性能的影响，最终对结果的解释。我们使用生物层干涉测量法在评估其在基于纸张的设备中的性能之前，对每种抗体与 GP 的结合进行了表征。结合亲和力（K）和结合速率（k）是影响基于纸张的免疫测定法灵敏度的主要因素。具有最佳 K（3-25 nM）的 mAb 表现出最低的检测限（约μg mL），而 K>25 nM 的 mAb 在我们的设备中无法检测到。此外，最令人惊讶的是，我们确定纸张设备中观察到的信号与 k 直接成正比。这些结果强调了确保识别试剂的质量足以支持所需的测定性能的重要性，并表明个体免疫反应的强度可能会影响测定结果的解释。
