Vertical Flow Cellulose-Based Assays for SARS-CoV-2 Antibody Detection in Human Serum

Abstract

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

Epitope-Functionalized Gold Nanoparticles for Rapid and Selective Detection of SARS-CoV-2 IgG Antibodies

“…However, this strategy intrinsically reduces the assay sensitivity because non-SARS-CoV-2-specific antibodies are abundant in blood (IgG: 7–16 g/L; IgM: 0.4–2.3 g/L), and they may compete with target SARS-CoV-2 antibodies for binding with antihuman antibody reagents. 37 , 38 …”

Exponential Amplification Using Photoredox Autocatalysis

“…The tube was illuminated for 10 minutes under 5 mW/cm 2 green light (λmax = 530 nm) from LED (M530L4, Thorlabs, Newton, NJ, USA). Then, the irradiated solution was applied to a wax-printed paper test zone 5 with absorbent pad underneath it before imaging (X-E2S, Fujifilm, Tokyo, Japan). For non-amplified samples, the suspension with amplification reagents were applied to the paper test zone before irradiation.…”

Nanobody-Functionalized Cellulose for Capturing and Containing SARS-CoV-2

“…Having validated the increased capture efficiency of fusion proteins immobilized on filter paper, we further sought to enhance the neutralization efficiency by incorporating the fusion proteins into regenerated amorphous cellulose (RAC). Because RAC has been shown to exhibit higher surface area per unit mass than filter paper (Yang et al, 2020), we reasoned that using RAC can increase the immobilization density of Ty1-CBD on cellulose, which in turn improves the rate and the degree of target capture based on the theoretical modeling by others (S. Kim et al, 2021). Moreover, this strategy can potentially expand the utility of Ty1-CBD by packing Ty1-CBD-functionalized RAC in a column, which allows for an affinity chromatography system to reduce viral load from contaminated fluids (e.g., blood and saliva) in a continuous mode.…”

“…Specifically, we designed a bifunctional fusion protein that comprises of a cellulose-binding domain (CBD) and Ty1 for cellulose immobilization and SARS-CoV-2 capturing, respectively. (S. Kim et al, 2021) The CBD originating from thecipA gene in the bacterium C. thermocellum has been shown to be resistant to heat denaturation (Tm ≥ 70 ºC) due to the thermophilic nature of C. thermocellum (Voutilainen et al, 2014). Additionally, Nbs are generally more thermostable and easier to manufacture (e.g., E. coli fermentation) than conventional human immunoglobin (IgG) based antibodies, the latter of which require mammalian cell hosts for production (Harmsen & De Haard, 2007).…”