Topographical depth reveals contact guidance mechanism distinct from focal adhesion confinement.

Cellular response to the topography of their environment, known as contact guidance, is a crucial aspect to many biological processes yet remains poorly understood. A prevailing model to describe cellular contact guidance involves the lateral confinement of focal adhesions (FA) by topography as an underlying mechanism governing how cells can respond to topographical cues. However, it is not clear how this model is consistent with the well-documented depth-dependent contact guidance responses in the literature.

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 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.

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.

The SARS-CoV-2 SSHHPS Recognized by the Papain-like Protease.

Viral proteases are highly specific and recognize conserved cleavage site sequences of ∼6-8 amino acids. Short stretches of homologous host-pathogen sequences (SSHHPS) can be found spanning the viral protease cleavage sites. We hypothesized that these sequences corresponded to specific host protein targets since >40 host proteins have been shown to be cleaved by Group IV viral proteases and one Group VI viral protease.

Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families.

Stabilization of a Broadly Neutralizing Anti-Chikungunya Virus Single Domain Antibody.

A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses.

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.

Multi-Enzyme Assembly on T4 Phage Scaffold.

Over the past two decades, various scaffolds have been designed and synthesized to organize enzyme cascades spatially for enhanced enzyme activity based on the concepts of substrate channeling and enhanced stability. The most bio-compatible synthetic scaffolds known for enzyme immobilization are protein and DNA nanostructures. Herein, we examined the utility of the T4 phage capsid to serve as a naturally occurring protein scaffold for the immobilization of a three-enzyme cascade: Amylase, Maltase, and Glucokinase.

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.

Problem of Diminished cRGD Surface Activity and What Can Be Done about It.

RGD peptides play a pivotal role in growing and diverse areas of biological research, ranging from experiments probing fundamental molecular mechanisms of cell adhesion to more applied strategies in medical imaging and cancer therapeutics. To better understand the outcomes of RGD-based approaches, we quantified the degree to which cyclic RGD (cRGD) activity is blocked by nonspecific binding of commonly used medium constituents. First, we show that recombinant αβ integrins can be used as a highly sensitive cell-free sensor to quantitatively and reliably characterize the activity of cRGD-functionalized surfaces surface plasmon resonance (SPR).

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).

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

The Bacillus collagen-like protein of anthracis (BclA), found in spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to 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.

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.

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.

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.

Nanoplasmonic pillars engineered for single exosome detection.

Exosomes are secreted nanovesicles which incorporate proteins and nucleic acids, thereby enabling multifunctional pathways for intercellular communication. There is an increasing appreciation of the critical role they play in fundamental processes such as development, wound healing and disease progression, yet because of their heterogeneous molecular content and low concentrations in vivo, their detection and characterization remains a challenge. In this work we combine nano- and microfabrication techniques for the creation of nanosensing arrays tailored toward single exosome detection.

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.

Improving biosensing activity to carcinoembryonic antigen with orientated single domain antibodies.

Carcinoembryonic antigen (CEA), also referred as CEACAM5, is integral to the adhesion process during cancer invasion and metastasis and is one of the most widely used tumor markers for assisting the diagnosis of cancer recurrence and cancer metastasis. Antibodies against CEA molecules have been developed for detection and diagnostic applications following tumor removal. Single domain antibodies (sdAbs) against CEA isolated from dromedary and llama exhibited high specificity in binding to tumor cells.

Bglbrick strategy for the construction of single domain antibody fusions.

Single domain antibodies, recombinantly expressed variable domains derived from camelid heavy chain antibodies, are often expressed as multimers for detection and therapeutic applications. Constructs in which several single domain antibodies are genetically fused serially, as well as those in which single domain antibodies are genetically linked with domains that naturally form multimers, yield improvement in apparent binding affinity due to avidity. Here, using a single domain antibody that binds envelope protein from the Dengue virus, we demonstrated the construction of single domain antibody dimers using the Bglbrick cloning strategy.

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.

Enhancing Stability of Camelid and Shark Single Domain Antibodies: An Overview.

Single domain antibodies (sdAbs) are gaining a reputation as superior recognition elements as they combine the advantages of the specificity and affinity found in conventional antibodies with high stability and solubility. Melting temperatures (s) of sdAbs cover a wide range from below 50 to over 80°C. Many sdAbs have been engineered to increase their , making them stable until exposed to extreme temperatures.

Conjugation of biotin-coated luminescent quantum dots with single domain antibody-rhizavidin fusions.

Straightforward and effective methods are required for the bioconjugation of proteins to surfaces and particles. Previously we demonstrated that the fusion of a single domain antibody with the biotin binding molecule rhizavidin provided a facile method to coat biotin-modified surfaces with a highly active and oriented antibody. Here, we constructed similar single domain antibody-rhizavidin fusions as well as unfused rhizavidin with a His-tag.

Thermal stabilization of anti-α-cobratoxin single domain antibodies.

There is an unmet need for snake antivenoms that can be stored ready to use near the point of care. To address that need we have taken two anti-α-cobratoxin single domain antibodies and increased their thermal stability to improve their ambient temperature shelf-life. The anti-α-cobratoxin single domain antibodies C2 and C20 were first isolated, and demonstrated to be toxin neutralizing by Richard et al.

Pairing Alpaca and Llama-Derived Single Domain Antibodies to Enhance Immunoassays for Ricin.

Previously, our group isolated and evaluated anti-ricin single domain antibodies (sdAbs) derived from llamas, engineered them to further increase their thermal stability, and utilized them for the development of sensitive immunoassays. In work focused on the development of therapeutics, Vance et al. 2013 described anti-ricin sdAbs derived from alpacas.

Label free checkerboard assay to determine overlapping epitopes of Ebola virus VP-40 antibodies using surface plasmon resonance.

Immunoassay formats, in which antibodies provide sensitivity and specificity, are often utilized to provide rapid and simple diagnostic tests. Surface plasmon resonance is frequently used to evaluate the suitability of antibodies by determining binding kinetics to agents or surrogate antigens. We used SPR to evaluate a number of commercial monoclonal antibodies as well as single domain antibodies produced in-house.