Integrating Single Domain Antibodies into Field-Deployable Rapid Assays.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19.

Modifications of Epitaxial Graphene on SiC for the Electrochemical Detection and Identification of Heavy Metal Salts in Seawater.

The electrochemical detection of heavy metal ions is reported using an inexpensive portable in-house built potentiostat and epitaxial graphene. Monolayer, hydrogen-intercalated quasi-freestanding bilayer, and multilayer epitaxial graphene were each tested as working electrodes before and after modification with an oxygen plasma etch to introduce oxygen chemical groups to the surface. The graphene samples were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, Raman spectroscopy, and van der Pauw Hall measurements. Dose-response curves in seawater were evaluated with added trace levels of four heavy metal salts (CdCl2, CuSO4, HgCl2, and PbCl2), along with detection algorithms based on machine learning and library development for each form of graphene and its oxygen plasma modification. Oxygen plasma-modified, hydrogen-intercalated quasi-freestanding bilayer epitaxial graphene was found to perform best for correctly identifying heavy metals in seawater.

Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis.

Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein.

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents.

Multilayer Epitaxial Graphene on Silicon Carbide: A Stable Working Electrode for Seawater Samples Spiked with Environmental Contaminants.

The electrochemical response of multilayer epitaxial graphene electrodes on silicon carbide substrates was studied for use as an electrochemical sensor for seawater samples spiked with environmental contaminants using cyclic square wave voltammetry. Results indicate that these graphene working electrodes are more robust and have lower background current than either screen-printed carbon or edge-plane graphite in seawater. Identification algorithms developed using machine learning techniques are described for several heavy metals, herbicides, pesticides, and industrial compounds. Dose-response curves provide a basis for quantitative analysis.

Lipid-tagged single domain antibodies for improved enzyme-linked immunosorbent assays.

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged single domain antibodies gave a 3-fold improvement in the observed limit of detection. This increase was likely due to an increased amount of single domain antibody adsorbed to the microtiter plate, which translated to improved limits of detection of Staphylococcal Enterotoxin B over using the same single domain antibody sans lipid-tag. However, improved orientation may also play a role. Regardless of the mechanism, the biosynthetic lipid-tagging of single domain antibodies represent a facile modality that can enhance their ability to be utilized as immunoassay capture reagent as well as facilitate their incorporation into liposome targeting applications in the future.

Microbial Nanocellulose Printed Circuit Boards for Medical Sensing.

We demonstrate the viability of using ultra-thin sheets of microbially grown nanocellulose to build functional medical sensors. Microbially grown nanocellulose is an interesting alternative to plastics, as it is hydrophilic, biocompatible, porous, and hydrogen bonding, thereby allowing the potential development of new application routes. Exploiting the distinguishing properties of this material enables us to develop solution-based processes to create nanocellulose printed circuit boards, allowing a variety of electronics to be mounted onto our nanocellulose. As proofs of concept, we have demonstrated applications in medical sensing such as heart rate monitoring and temperature sensing-potential applications fitting the wide-ranging paradigm of a future where the Internet of Things is dominant.

Experimental evaluation of single-domain antibodies predicted by molecular dynamics simulations to have elevated thermal stability.

Recently Bekker et al. [Bekker G-J et al. Protein Sci. 2019;28:429-438.] described a computational strategy of applying molecular-dynamics simulations to estimate the relative stabilities of single-domain antibodies, and utilized their method to design changes with the aim of increasing the stability of a single-domain antibody with a known crystal structure. The structure from which they generated potentially stabilizing mutations is an anti-cholera toxin single domain antibody selected from a naïve library which has relatively low thermal stability, reflected by a melting point of 48°C. Their work was purely theoretical, so to examine their predictions, we prepared the parental and predicted stabilizing mutant single domain antibodies and examined their thermal stability, ability to refold and affinity. We found that the mutation that improved stability the most (~7°C) was one which changed an amino acid in CDR1 from an asparagine to an aspartic acid. This change unfortunately was also accompanied by a reduction in affinity. Thus, while their modeling did appear to successfully predict stabilizing mutations, introducing mutations in the binding regions is problematic. Of further interest, the mutations selected via their high temperature simulations, did improve refolding, suggesting that they were successful in stabilizing the structure at high temperatures and thereby decrease aggregation. Our result should permit them to reassess and refine their model and may one day lead to a usefulin silico approach to protein stabilization.

Oriented Immobilization of Single-Domain Antibodies Using SpyTag/SpyCatcher Yields Improved Limits of Detection.

Single-domain antibodies (sdAb), recombinantly produced variable heavy domains derived from the unconventional heavy chain antibodies found in camelids, provide stable, well-expressed binding elements with excellent affinity that can be tailored for specific applications through protein engineering. Complex matrices, such as plasma and serum, can dramatically reduce assay sensitivity. Thus, to achieve highly sensitive detection in complex matrices a highly efficient assay is essential. We produced sdAb as genetically linked dimers, and trimers, each including SpyTag at their C-terminus. The constructs were immobilized onto dyed magnetic microspheres to which SpyCatcher had been coupled and characterized in terms of their performance as capture reagents in sandwich assays. Initial tests on the ability of oriented monomer, dimer, and trimer captures to improve detection versus unoriented constructs in an assay for staphylococcal enterotoxin B spiked into buffer showed the oriented dimer format provided the best sensitivity while offering robust protein production. Thus, this format was utilized to improve a sdAb-based assay for the detection of dengue virus (DENV) nonstructural protein 1 (NS1) in serum. Detection of NS1 from each of the four DENV serotypes spiked into 50% normal human serum was increased by at least a factor of 5 when using the oriented dimer capture. We then demonstrated the potential of using the oriented dimer capture to improve detection of NS1 in clinical samples. This general method should enhance the utility of sdAb incorporated into any diagnostic assay, including those for high consequence pathogens.

A Fully-Flexible Solution-Processed Autonomous Glucose Indicator.

We present the first demonstration of a fully-flexible, self-powered glucose indicator system that synergizes two flexible electronic technologies: a flexible self-powering unit in the form of a biofuel cell, with a flexible electronic device - a circuit-board decal fabricated with biocompatible microbial nanocellulose. Our proof-of-concept device, comprising an enzymatic glucose fuel cell, glucose sensor and a LED indicator, does not require additional electronic equipment for detection or verification; and the entire structure collapses into a microns-thin, self-adhering, single-centimeter-square decal, weighing less than 40 mg. The flexible glucose indicator system continuously operates a light emitting diode (LED) through a capacitive charge/discharge cycle, which is directly correlated to the glucose concentration. Our indicator was shown to operate at high sensitivity within a linear glucose concentration range of 1 mM-45 mM glucose continuously, achieving a 1.8 VDC output from a flexible indicator system that deliver sufficient power to drive an LED circuit. Importantly, the results presented provide a basis upon which further development of indicator systems with biocompatible diffusing polymers to act as buffering diffusion barriers, thereby allowing them to be potentially useful for low-cost, direct-line-of-sight applications in medicine, husbandry, agriculture, and the food and beverage industries.

Machine Learning Techniques for Chemical Identification Using Cyclic Square Wave Voltammetry.

Electroanalytical techniques are useful for detection and identification because the instrumentation is simple and can support a wide variety of assays. One example is cyclic square wave voltammetry (CSWV), a practical detection technique for different classes of compounds including explosives, herbicides/pesticides, industrial compounds, and heavy metals. A key barrier to the widespread application of CSWV for chemical identification is the necessity of a high performance, generalizable classification algorithm. Here, machine and deep learning models were developed for classifying samples based on voltammograms alone. The highest performing models were Long Short-Term Memory (LSTM) and Fully Convolutional Networks (FCNs), depending on the dataset against which performance was assessed. When compared to other algorithms, previously used for classification of CSWV and other similar data, our LSTM and FCN-based neural networks achieve higher sensitivity and specificity with the area under the curve values from receiver operating characteristic (ROC) analyses greater than 0.99 for several datasets. Class activation maps were paired with CSWV scans to assist in understanding the decision-making process of the networks, and their ability to utilize this information was examined. The best-performing models were then successfully applied to new or holdout experimental data. An automated method for processing CSWV data, training machine learning models, and evaluating their prediction performance is described, and the tools generated provide support for the identification of compounds using CSWV from samples in the field.

Selection and characterization of protective anti-chikungunya virus single domain antibodies.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes an arthralgia febrile illness that has affected millions of people on three continents. Previously, neutralizing monoclonal antibodies that have prophylactic and therapeutic activity were found to remove virus in joint tissues, thereby reducing the severity of symptoms in mice and non-human primates. In this study, we sought to develop thermostable small recombinant antibodies against CHIKV for future diagnostic, prophylactic and therapeutic applications. To develop these single domain antibodies (sdAb) a CHIKV immune library was constructed by displaying the consortium of variable heavy domains (VHH) amplified from peripheral white blood cells isolated from llamas immunized with CHIKV virus-like particles (VLPs). Five anti-CHIKV sdAb isolated using bio-panning were evaluated for their affinity and thermal stability. Their ability to detect CHIKV VLPs was demonstrated in both MagPlex- and ELISA- based assays. Finally, the ability of two sdAb, CC3 and CA6, to inhibit CHIKV infection were tested using a plaque reduction and neutralization test (PRNT), yielding PRNT50 values of 0.6 and 45.6 nM, respectively.

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies.

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

Characterization of longitudinal canal tissue in the acorn barnacle Amphibalanus amphitrite.

The morphology and composition of tissue located within parietal shell canals of the barnacle Amphibalanus amphitrite are described. Longitudinal canal tissue nearly spans the length of side shell plates, terminating near the leading edge of the specimen basis in proximity to female reproductive tissue located throughout the peripheral sub-mantle region, i.e. mantle parenchyma. Microscopic examination of stained longitudinal canal sections reveal the presence of cell nuclei as well as an abundance of micron-sized spheroids staining positive for basic residues and lipids. Spheroids with the same staining profile are present extensively in ovarioles, particularly within oocytes which are readily identifiable at various developmental stages. Mass spectrometry analysis of longitudinal canal tissue compared to tissue collected from the mantle parenchyma reveals a nearly 50% overlap of the protein profile with the greatest number of sequence matches to vitellogenin, a glycolipoprotein playing a key role in vitellogenesis-yolk formation in developing oocytes. The morphological similarity and proximity to female reproductive tissue, combined with mass spectrometry of the two tissues, provides compelling evidence that one of several possible functions of longitudinal canal tissue is supporting the female reproductive system of A. amphitrite, thus expanding the understanding of the growth and development of this sessile marine organism.

Orthogonal Synthetic Zippers as Protein Scaffolds.

Protein scaffolds have proven useful for co-localization of enzymes, providing control over stoichiometry and leading to higher local enzyme concentrations, which have led to improved product formation. To broaden their usefulness, it is necessary to have a wide choice of building blocks to mix and match for scaffold generation. Ideally, the scaffold building blocks should function at any location within the scaffold and have high affinity interactions with their binding partners. We examined the utility of orthogonal synthetic coiled coils (zippers) as scaffold components. The orthogonal zippers are coiled coil domains that form heterodimers only with their specific partner and not with other zipper domains. Focusing on two orthogonal zipper pairs, we demonstrated that they are able to function on either end or in the middle of a multiblock assembly. Surface plasmon resonance was employed to assess the binding kinetics of zipper pairs placed at the start, middle, or end of a construct. Size-exclusion chromatography was used to demonstrate the ability of a scaffold with two zipper domains to bind their partners simultaneously. We then expanded the study to examine the binding kinetics and cross-reactivities of three additional zipper pairs. By validating the affinities and specificities of synthetic zipper pairs, we demonstrated the potential for zipper domains to provide an expanded library of scaffolding parts for tethering enzymes in complex pathways for synthetic biology applications.

Paper-Based Electrochemical Detection of Chlorate.

We describe the use of a paper-based probe impregnated with a vanadium-containing polyoxometalate anion, [PMo11VO40]5-, on screen-printed carbon electrodes for the electrochemical determination of chlorate. Cyclic voltammetry (CV) and chronocoulometry were used to characterize the ClO3- response in a pH = 2.5 solution of 100 mM sodium acetate. A linear CV current response was observed between 0.156 and 1.25 mg/mL with a detection limit of 0.083 mg/mL (S/N > 3). This performance was reproducible using [PMo11VO40]5--impregnated filter paper stored under ambient conditions for as long as 8 months prior to use. At high concentration of chlorate, an additional catalytic cathodic peak was seen in the reverse scan of the CVs, which was digitally simulated using a simple model. For chronocoulometry, the charge measured after 5 min gave a linear response from 0.625 to 2.5 mg/mL with a detection limit of 0.31 mg/mL (S/N > 3). In addition, the slope of charge vs. time also gave a linear response. In this case the linear range was from 0.312 to 2.5 mg/mL with a detection limit of 0.15 mg/mL (S/N > 3). Simple assays were conducted using three types of soil, and recovery measurements reported.

Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase.

The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (ß-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with ß-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.

Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus.

In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

Selection, characterization, and thermal stabilization of llama single domain antibodies towards Ebola virus glycoprotein.

BACKGROUND: A key advantage of recombinant antibody technology is the ability to optimize and tailor reagents. Single domain antibodies (sdAbs), the recombinantly produced variable domains derived from camelid and shark heavy chain antibodies, provide advantages of stability and solubility and can be further engineered to enhance their properties. In this study, we generated sdAbs specific for Ebola virus envelope glycoprotein (GP) and increased their stability to expand their utility for use in austere locals. Ebola virus is extremely virulent and causes fatal hemorrhagic fever in ~ 50 percent of the cases. The viral GP binds to host cell receptors to facilitate viral entry and thus plays a critical role in pathogenicity. RESULTS: An immune phage display library containing more than 107 unique clones was developed from a llama immunized with a combination of killed Ebola virus and recombinantly produced GP. We panned the library to obtain GP binding sdAbs and isolated sdAbs from 5 distinct sequence families. Three GP binders with dissociation constants ranging from ~ 2 to 20 nM, and melting temperatures from ~ 57 to 72 °C were selected for protein engineering in order to increase their stability through a combination of consensus sequence mutagenesis and the addition of a non-canonical disulfide bond. These changes served to increase the melting temperatures of the sdAbs by 15-17 °C. In addition, fusion of a short positively charged tail to the C-terminus which provided ideal sites for the chemical modification of these sdAbs resulted in improved limits of detection of GP and Ebola virus like particles while serving as tracer antibodies. CONCLUSIONS: SdAbs specific for Ebola GP were selected and their stability and functionality were improved utilizing protein engineering. Thermal stability of antibody reagents may be of particular importance when operating in austere locations that lack reliable refrigeration. Future efforts can evaluate the potential of these isolated sdAbs as candidates for diagnostic or therapeutic applications for Ebola.

A Simple and Inexpensive Electrochemical Assay for the Identification of Nitrogen Containing Explosives in the Field.

We report a simple and inexpensive electrochemical assay using a custom built hand-held potentiostat for the identification of explosives. The assay is based on a wipe test and is specifically designed for use in the field. The prototype instrument designed to run the assay is capable of performing time-resolved electrochemical measurements including cyclic square wave voltammetry using an embedded microcontroller with parts costing roughly $250 USD. We generated an example library of cyclic square wave voltammograms of 12 compounds including 10 nitroaromatics, a nitramine (RDX), and a nitrate ester (nitroglycine), and designed a simple discrimination algorithm based on this library data for identification.