| abstract |
A targeted approach is described for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. Single chain antibodies (scFvs) are produced to EA1, a Bacillus anthracis S-layer protein that is also present, although is not identical, in related Bacillus species. These antibodies detect Bacillus anthracis EA1 protein and intact spores with a high degree of specificity, but do not detect other Bacillus species. Recombinant anti-EA1 scFvs were isolated from an B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection are disclosed; standard (non-competitive) panning, and competitive panning. The non-competitive bio-panning strategy isolated scFvs that recognised EA1 from B. anthracis , but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to compete out any cross reacting antibodies, generating scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. The specific, real time detection of B. anthracis spores was demonstrated with these scFvs by using an evanescent wave biosensor, the Resonant Mirror. The approach described here can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents. |