Development and study of phage-derived detection probes.

摘要

The threat of bioterrorism has created a demand for continuous monitoring of the environment for threat agents. Existing monitoring systems use antibody-derived detection probes which are not hardy enough to withstand long-term use in harsh environments. Alternative phage-derived probes have been shown to function as robust and stable substitutes for antibodies in a variety of platforms. I describe here the study and development of phage-derived probes for potential use in detection of the threat agent Bacillus anthracis.; Naturally occurring phages are resistant to heat, organic solvents, and proteases. We hypothesized that landscape phage probes inherit the thermostability of their parental phage. The stability of a phage probe and a monoclonal antibody that bind to beta-galactosidase was examined at 25°C to 76°C. The phage probe was found to be more stable than the antibody at 37°C and above, and had a half life of ∼2.5 years at 37°C. The activation energy (the amount of energy required to ensure that the reaction occurs) of phage degradation was found to be 31,987 cal/mol.; Feasibility of identifying landscape phage probes that could be used to detect threat agents was demonstrated using B. anthracis Sterne spores as a model. It was hypothesized that landscape phage clones that bind to B. anthracis spores could be selected from a landscape phage display library in biased and non-biased selection procedures. Several phage clones that bound to B. anthracis spores in a highly specific and moderately selective manner were identified in the non-biased procedure; the most selective spore-binding phage displayed several thousand copies of the peptide EPRLSPHS, and bound from 3.5- to 70-fold better to B. anthracis than to spores of other Bacillus species. The use of biased selection procedures to identify new probes, or the modification of the surface of existing probes may allow the development of highly specific and selective robust probes suitable for long term use in continuous monitoring devices and biosorbents.; The evolution of the landscape phage display library f8/8 was examined during biased selection procedures. It was hypothesized that examining the evolution of the library during selection might reveal predictors of successful isolation of target-binding clones, and offer clues about which clones are the strongest target binders. No reliable predictor of target binding ability was found, but characteristics of a successful selection procedure were identified.