Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance

Abstract

Graphical abstract 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 D ) and on rate (k on ) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best K D (3–25 nM) exhibited the lowest limits of detection (ca. μg mL −1 ), while mAbs with K D > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to k on . 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. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03317-4.

Low-Cost Microfluidic Systems for Detection of Neglected Tropical Diseases

“…This device can also be used for POC clinical diagnosis of other viruses by changing the complementary CRISPR RNA (crRNA) spacer sequence programming for different analytes. Using the glycoprotein ectodomain of the EBOV envelope as an affinity reagent, Murray and coauthors (104) proposed a μPAD to diagnose EBOV based on a single assay to detect anti-Ebola IgG found in patients during the second week of acute illness (Figure 3c). The authors observed that the affinity and binding rate of antibodies for the glycoprotein ectodomain influenced the analytical sensitivity.…”

Paper-Based Cytometer for the Detection and Enumeration of White Blood Cells According to Their Immunophenotype

“…24 The inclusion of lateral, directed flow in patterned paper devices allows for many more opportunities in paper-based device design, including the ability to incorporate complex sample-processing (e.g., selective lysis), multiplexing, and to allow for proper mixing of any affinity-based labeling reagents in situ. 45,46 We overcame these limitations through the identification of a porous material that allows for the unimpeded transport of WBCs by wicking in both the vertical and lateral directions: a cof fee f ilter. This discovery has allowed us to create a new class of paper-based devices capable of the controlled transport, labeling, and detection of intact WBCs.…”

“…The inclusion of lateral, directed flow in patterned paper devices allows for many more opportunities in paper-based device design, including the ability to incorporate complex sample-processing ( e.g. , selective lysis), multiplexing, and to allow for proper mixing of any affinity-based labeling reagents in situ . , …”

A Novel Paper-Based Cytometer for the Detection and Enumeration of White Blood Cells According to their Immunophenotype

“…24 The inclusion of lateral, directed flow in patterned paper devices allows for many more opportunities in paper-based device design, including the ability to incorporate complex sample-processing (e.g., selective lysis), multiplexing, and to allow for proper mixing of any affinity-based labeling reagents in situ. 44,45 We overcome these limitations through the identification of a porous material that allows for the unimpeded transport of WBCs by wicking in both the vertical and lateral directions: a coffee filter. This discovery has allowed us to create a new class of paper-based devices, capable of the controlled transport, labeling, and detection of intact WBCs.…”