The accurate detection of an aerosol of weaponized anthrax spores, using antibody-based biosensor technology, is centrally dependent upon antibodies of singular specificity for anthrax spore-surface antigens. As an initial step toward this objective, we propose the identification of a pool of genes encoding such antigens, using a combination of genetic (RT-PCR), and immunological (immunoscreening) techniques. Genes isolated using this combination strategy, will then be identified by a BLAST search against sequences contained in the National Center for Biotechnology Information (NCBI) non-redundant database, and against the B. anthracis genome sequence available at The Institute for Genomic Research website (www.TIGR.orq). Owing to time and budgetary constraints, in this study, only a few (one or two) of the identified genes will be selected for polyclonal antibody development, which will be generated via genetic immunization (GI). Selection will be based on whether the identified genes contain the consensus promoter sequence for the binding of sigma factor, (K, which directs transcription of several genes during late sporulation, and also whether such genes are unique to B. anthracis as determined by BLAST and Southern blotting experiments. A rational approach will be used in the selection of DNA sequences for GI in order to prevent or minimize cross reactivity of the polyclonal antibodies with spores of close relatives. To accomplish this, protein sequences predicted by genes that meet the above criteria, will be extensively analyzed using protein analytical tools. Regions of such genes that encode antigenic domains not part of motifs, patterns, signatures, active enzyme sites or other sequences, common to sequenced members of the genus Bacillus, will then be amplified, cloned into a suitable plasmid DNA (pDNA) vaccine vector, and used to elicit polyclonal antibodies via GI. Gene sequences encoding domains, which elicit polyclonal antibodies that strongly react with anthrax spores in an intact spore-ELISA, and exhibit little or no cross-reactivity with those of close relatives, will be used to generate monoclonal antibodies (MAbs) via GI. Following evaluation of MAbs in the spore- ELISA, highly specific polyclonal and MAbs will be evaluated further in a micro electrical and mechanical (MEMS) sensor device in the future, with the long-term goal of eveloping a portable, antibody-based sensor for the detection of aerosolized anthrax spores.
| Kudva, Indira T; Griffin, Robert W; Garren, Jeonifer M et al. (2005) Identification of a protein subset of the anthrax spore immunome in humans immunized with the anthrax vaccine adsorbed preparation. Infect Immun 73:5685-96 |
