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.

Integrating scFv into xMAP Assays for the Detection of Marine Toxins.

Marine toxins, such as saxitoxin and domoic acid are associated with algae blooms and can bioaccumulate in shell fish which present both health and economic concerns. The ability to detect the presence of toxin is paramount for the administration of the correct supportive care in case of intoxication; environmental monitoring to detect the presence of toxin is also important for prevention of intoxication. Immunoassays are one tool that has successfully been applied to the detection of marine toxins. Herein, we had the variable regions of two saxitoxin binding monoclonal antibodies sequenced and used the information to produce recombinant constructs that consist of linked heavy and light variable domains that make up the binding domains of the antibodies (scFv). Recombinantly produced binding elements such as scFv provide an alternative to traditional antibodies and serve to "preserve" monoclonal antibodies as they can be easily recreated from their sequence data. In this paper, we combined the anti-saxitoxin scFv developed here with a previously developed anti-domoic acid scFv and demonstrated their utility in a microsphere-based competitive immunoassay format. In addition to detection in buffer, we demonstrated equivalent sensitivity in oyster and scallop matrices. The potential for multiplexed detection using scFvs in this immunoassay format is demonstrated.

Negative tail fusions can improve ruggedness of single domain antibodies.

Single-domain antibodies (sdAbs), the recombinantly expressed binding domains derived from the heavy-chain-only antibodies found in camelids and sharks, are valued for their ability to refold after heat denaturation. However, some sdAbs are prone to aggregation on extended heating at high concentration. Additionally, sdAbs prepared cytoplasmically often lack the conserved disulfide bond found in variable heavy domains, which both decreases their melting point and can decrease their ability to refold. Genetic fusions of sdAbs with the acid tail of α-synuclein (ATS) resulted in constructs that had enhanced ability to resist aggregation. In addition, almost complete refolding was observed even in the absence of the disulfide bond. These sdAb-ATS fusions expand the utility of sdAbs. They provide sdAbs that are resistant to aggregation, and enable the production of re-foldable sdAbs in the reducing environment of the cytoplasm.

Comparison of single domain antibody immobilization strategies evaluated by surface plasmon resonance.

The use of single domain antibodies (sdAbs) in place of conventional antibodies for both therapeutic and diagnostic applications continues to grow. SdAbs offer a number of advantages when compared to conventional antibodies such as their small size and low structural complexity which allows them to readily be produced as fusions in a variety formats. In this work we compared the utility of various C-terminal fusions and immobilization strategies for two sdAbs; one which recognizes ricin and the other EA1, an S-layer protein, of Bacillus anthracis. Comparisons were made between direct covalent attachment and affinity immobilization using a biotin-streptavidin interaction for the standard sdAb monomers, randomly and site-specifically biotinylated monomers, and fusion constructs of alkaline phosphatase dimers and streptavidin core tetramers. The sdAb binding and regeneration was evaluated by surface plasmon resonance in a multiplexed format. The construct that provided the highest density of active molecules by at least a factor of two was the sdAb-streptavidin core tetramer, followed by the sdAb-alkaline phosphatase and then the site-specifically biotinylated monomer. The poorest performing immobilization methods were the two most common, direct covalent attachment and the randomly biotinylated sdAb attached via NeutrAvidin. These improvements directly correlated to antigen capture in SPR assays. Similarly, the oriented immobilization method also translated to improvements in limit of detection assays using a bead-based system. The sdAb-streptavidin core provided more than a 100-fold improvement in the limit of detection of EA1, from ~200 ng/mL to to 1.6 ng/mL, while improvement for ricin detection was less but still a significant 5-fold decrease, going from 1.6 ng/mL down to 0.32 ng/mL. This demonstrated improvement in limits of detection is an advantage that should be transferable to most assay formats.

Rugged single domain antibody detection elements for Bacillus anthracis spores and vegetative cells.

Significant efforts to develop both laboratory and field-based detection assays for an array of potential biological threats started well before the anthrax attacks of 2001 and have continued with renewed urgency following. While numerous assays and methods have been explored that are suitable for laboratory utilization, detection in the field is often complicated by requirements for functionality in austere environments, where limited cold-chain facilities exist. In an effort to overcome these assay limitations for Bacillus anthracis, one of the most recognizable threats, a series of single domain antibodies (sdAbs) were isolated from a phage display library prepared from immunized llamas. Characterization of target specificity, affinity, and thermal stability was conducted for six sdAb families isolated from rounds of selection against the bacterial spore. The protein target for all six sdAb families was determined to be the S-layer protein EA1, which is present in both vegetative cells and bacterial spores. All of the sdAbs examined exhibited a high degree of specificity for the target bacterium and its spore, with affinities in the nanomolar range, and the ability to refold into functional antigen-binding molecules following several rounds of thermal denaturation and refolding. This research demonstrates the capabilities of these sdAbs and their potential for integration into current and developing assays and biosensors.