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Due to their portability, versatility for supporting multiple assay formats, and potential for resulting in low-cost assays, paper-based analytical devices (PADs) are an increasingly popular format as a platform for the development of point-of-care tests. However, very few PADs have been translated successfully to their intended environments outside of academic settings. Often overlooked as a factor that inhibits translation, usability is a vital characteristic of any successful point-of-care test. Recent advancements in PAD design have demonstrated improved usability by simplifying various aspects of user operation, including sample collection, sample processing, device operation, detection, and readout/interpretation. Field testing at various stages of device design can offer critical feedback about device usability, especially when it involves the proposed end-user or other stakeholders. By highlighting advances in usability, we aim to encourage thoughtful and rigorous design at the academic prototyping stage to address one outstanding hurdle that limits the number of PADs that make it from the benchtop to the point-of-care. Image 1 • Paper-based devices have potential for use at the point-of-care. • Devices that are user-friendly are more likely to be translated to the field. • Additional user steps create additional opportunities for user error. • We discuss ways to streamline user operation of paper-based devices. • We highlight three case studies for devices deployed successfully to the field. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]