The Bacillus collagen-like protein of anthracis (BclA), found inBacillus anthracisspores, is anattractive target for immunoassays. Previously, using phage display we had selected llama-derivedsingle-domain antibodies that bound toB. anthracisspore proteins including BclA. Single-domainantibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-onlyantibodies found in camelids, provide stable and well-expressed binding elements with excellentaffinity. In addition, sdAbs offer the important advantage that they can be tailored for specificapplications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Betagalactosidase (β-gal) would enable highly sensiti... More
The Bacillus collagen-like protein of anthracis (BclA), found inBacillus anthracisspores, is anattractive target for immunoassays. Previously, using phage display we had selected llama-derivedsingle-domain antibodies that bound toB. anthracisspore proteins including BclA. Single-domainantibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-onlyantibodies found in camelids, provide stable and well-expressed binding elements with excellentaffinity. In addition, sdAbs offer the important advantage that they can be tailored for specificapplications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Betagalactosidase (β-gal) would enable highly sensitive immunoassays with no need for a secondaryreagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identifiedbut 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 withβ-gal and shownto enable sensitive detection. This fusion has the potential to be incorporated into highly sensitiveassays for the detection of anthrax spores.
