Carly A. Holstein

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant “flu binders” for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

Statistical method for determining and comparing limits of detection of bioassays.

The current bioassay development literature lacks the use of statistically robust methods for calculating the limit of detection of a given assay. Instead, researchers often employ simple methods that provide a rough estimate of the limit of detection, often without a measure of the confidence in the estimate. This scarcity of robust methods is likely due to a realistic preference for simple and accessible methods and to a lack of such methods that have reduced the concepts of limit of detection theory to practice for the specific application of bioassays. Here, we have developed a method for determining limits of detection for bioassays that is statistically robust and reduced to practice in a clear and accessible manner geared at researchers, not statisticians. This method utilizes a four-parameter logistic curve fit to translate signal intensity to analyte concentration, which is a curve that is commonly employed in quantitative bioassays. This method generates a 95% confidence interval of the limit of detection estimate to provide a measure of uncertainty and a means by which to compare the analytical sensitivities of different assays statistically. We have demonstrated this method using real data from the development of a paper-based influenza assay in our laboratory to illustrate the steps and features of the method. Using this method, assay developers can calculate statistically valid limits of detection and compare these values for different assays to determine when a change to the assay design results in a statistically significant improvement in analytical sensitivity.

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 107 and 1.34 × 107 CEID50/mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Chapter 8:Microfluidic Diagnostics for Low-resource Settings: Improving Global Health without a Power Cord

The ability to diagnose a patient quickly and accurately is of paramount importance in the management of most diseases, as the appropriate treatment cannot be administered until the cause has been identified. In the developed world, hospitals and large clinics often employ sophisticated equipment and trained laboratory staff to enable an accurate diagnosis. Performing this sophisticated laboratory testing is not possible in many areas of the developing world that lack these resources and infrastructure, however, leaving patients untreated even when medication is available. The goal of this chapter is to provide the reader with an assessment of the need for and use of microfluidic diagnostics in low-resource settings, highlighting the successes of and opportunities for microfluidic diagnostics in global health. Included is a section emphasizing paper-based microfluidics, which we view as an important and rapidly growing component of the microfluidics field with significant potential to revolutionize diagnostic testing in low-resource settings. Most importantly, we aim to provide a useful context with which to think about the development of microfluidic diagnostics for global health applications.

Development of a paper-based diagnostic for influenza detection

The development of novel paper-based diagnostic tests has surged in recent years, due to the suitability of these tests for use at the point of care. These emerging paper-based tests retain the low cost and ease of use of traditional lateral flow tests, while offering increased sophistication and capabilities that approach those of traditional microfluidic devices. Here, we report on the development of a novel paper-based test for the diagnosis of influenza, commonly known as the flu. Influenza is a ubiquitously occurring infection, affecting 5-20% of Americans and resulting in an average of 23,000 deaths in the U.S., and up to 500,000 deaths globally, each year. Despite its prevalence, the diagnosis of influenza remains unsatisfactory, especially at the point of care. In particular, lateral flow tests for influenza suffer from poor sensitivity and provide only limited information about the infecting flu virus. Point-of-care testing of influenza therefore stands to benefit substantially from improved technology. To this end, we have developed two different versions of a paper-based flu assay, both using computationally designed affinity proteins, or “binders,” that bind to the influenza hemagglutinin (HA) protein. One version of the assay utilizes an HA stem-region binder and the other an HA head-region binder. With these assays, we demonstrate the detection of clinically relevant concentrations of recombinant HA and intact influenza virus, as well as the translation of this paper-based system to a two-dimensional paper network (2DPN) folding card device.