Measuring the Variability of Treated Bacillus Anthracis Delta Stern Spores

The development of biological detectors for the military and homeland defense 26 relies on the ability to test hardware with biological materials that can be produced reliably 27 and reproducibly. To ensure occupational safety during the development of new devices, 28 the production and testing with biological materials necessitates compromises that include 29 selection of a virulent strains and inactivation using various techniques. The materials and 30 methods selected for production and post-production treatment can greatly influence 31 detection outcomes using sensitive DNA or antibody based systems. The optimal situation 32 would be to reproducibly create biological testing materials that accurately simulate the 33 true pathogen yet carry minimal risk to the scientists and engineers developing the 34 biosensors. In this study, avirulent Bacillus anthracis spores were used to demonstrate the 35 varied outcomes that can be induced by density gradient purification and post-growth 36 inactivation using gamma irradiation. The responses using Quantitative Polymerase Chain 37 Reaction, Electrochemiluminescence, Enzyme-Linked Immunosorbent Assay, 38 Bicinchoninic Acid assay, and protein electrophoresis varied significantly with treatment. 39 Gradient purification removed proteins that interfered with monoclonal and polyclonal 40 antibodies- abilities to form immunocomplexes in Electrochemiluminescent and ELISA 41 assays. Irradiation had a varied effect on qPCR depending on the sample preparation 42 employed and produced a reduced response in Electrochemiluminescent and ELISA 43 assays. Varied results from enumeration techniques show the importance of measuring the 44 dead cellular material and cellular debris contribution to any sample. Production and 45 material treatments need to conform to a yet to be established guideline that can bring 46 uniformity to available biological agent reference materials. The original document contains color images.