Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10 min, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 and 80 pM limits of detection in 1× phosphate-buffered saline (mock swab) and saliva matrices spiked with cell-culture-generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way toward the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

Developing a SARS-CoV-2 Antigen Test Using Engineered Affinity Proteins.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10-minute, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 pM and 80 pM limits of detection in 1×PBS (mock swab) and saliva matrices spiked with cell-culture generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way towards the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.

Functional Comparison of Bioactive Cellulose Materials Incorporating Engineered Binding Proteins.

Whatman No. 1 chromatography paper is widely used as a substrate for cellulose-based immunoassays. The immobilized proteins are used to capture target biomarkers for detection. However, alternative paper substrates may facilitate mass production of immunoassays as diagnostic tests. Here, we assessed the physical characteristics and protein immobilization capabilities of different commercial papers. Some substrates fulfilled our design criteria, including adequate flow rate and sufficient protein immobilization for efficient target capture. This study demonstrates that a variety of paper substrates can be bioactivated and used to capture target biomarkers, enabling development of affordable diagnostic tests from a range of starting materials.

Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins.

Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.

Beyond Epitope Binning: Directed in Vitro Selection of Complementary Pairs of Binding Proteins.

Many biotechnological applications require the simultaneous binding of affinity reagents to nonoverlapping target epitopes, the most prominent example being sandwich immunoassays. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Here, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process, which enables the efficient identification of affinity reagents that function together as complementary pairs, from in vitro libraries of ∼109 variants. We used RAPIDS to develop highly specific affinity pairs against biomarkers of tuberculosis, Zika virus, and sepsis. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or nontargets, yielding diagnostic assays that are sensitive and specific by design.

Sensitivity and binding kinetics of an ultra-sensitive chemiluminescent enzyme-linked immunosorbent assay at arrays of antibodies.

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 µL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-µm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays-mostly high signal to noise ratio of extended chemiluminescent imaging-are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.

Emulsion Agglutination Assay for the Detection of Protein-Protein Interactions: An Optical Sensor for Zika Virus.

A new class of Janus emulsion agglutination assay is reported for the detection of interfacial protein-protein interactions. Janus emulsion droplets are functionalized with a thermally stable, antigen binding protein rcSso7d variant (rcSso7d-ZNS1) for the detection of Zika NS1 protein. The emulsion droplets containing fluorescent dyes in their hydrocarbon and fluorocarbon phases intensify the intrinsic optical signal with the emission intensity ratio, which can be detected by a simple optical fiber. This assay provides an opportunity for the in-field detection of Zika virus and other pathogens with a stable, inexpensive, and convenient device.

Bead mediated separation of microparticles in droplets.

Exchange of components such as particles and cells in droplets is important and highly desired in droplet microfluidic assays, and many current technologies use electrical or magnetic fields to accomplish this process. Bead-based microfluidic techniques offer an alternative approach that uses the bead's solid surface to immobilize targets like particles or biological material. In this paper, we demonstrate a bead-based technique for exchanging droplet content by separating fluorescent microparticles in a microfluidic device. The device uses posts to filter surface-functionalized beads from a droplet and re-capture the filtered beads in a new droplet. With post spacing of 7 µm, beads above 10 µm had 100% capture efficiency. We demonstrate the efficacy of this system using targeted particles that bind onto the functionalized beads and are, therefore, transferred from one solution to another in the device. Binding capacity tests performed in the bulk phase showed an average binding capacity of 5 particles to each bead. The microfluidic device successfully separated the targeted particles from the non-targeted particles with up to 98% purity and 100% yield.

Effects of solution conditions on virus retention by the Viresolve® NFP filter.

Virus filtration can provide a robust method for removal of adventitious parvoviruses in the production of biotherapeutics. Although virus filtration is typically thought to function by a purely size-based removal mechanism, there is limited data in the literature indicating that virus retention is a function of solution conditions. The objective of this work was to examine the effect of solution pH and ionic strength on virus retention by the Viresolve(®) NFP membrane. Data were obtained using the bacteriophage ϕX174 as a model virus, with retention data complemented by the use of confocal microscopy to directly visualize capture of fluorescently labeled ϕX174 within the filter. Virus retention was greatest at low pH and low ionic strength, conditions under which there was an attractive electrostatic interaction between the negatively charged membrane and the positively charged phage. In addition, the transient increase in virus transmission seen in response to a pressure disruption at pH 7.8 and 10 was completely absent at pH 4.9, suggesting that the trapped virus are unable to overcome the electrostatic attraction and diffuse out of the pores when the pressure is released. Further confirmation of this physical picture was provided by confocal microscopy. Images obtained at pH 10 showed the migration of previously captured phage; this phenomenon was absent at pH 4.9. These results provide important new insights into the factors governing virus retention using virus filtration membranes.