Diagnostic Probes for Bacillus anthracis Spores Selected from a Landscape Phage Library

Abstract

Background: Recent use of Bacillus anthracis spores as a bioweapon has highlighted the need for a continuous monitoring system. Current monitoring systems rely on antibody-derived probes, which are not hardy enough to withstand long-term use under extreme conditions. We describe new, phage-derived probes that can be used as robust substitutes for antibodies. Methods: From a landscape phage library with random octapeptides displayed on all copies of the major phage coat protein of the phage fd-tet, we selected clones that bound to spores of B. anthracis (Sterne strain). ELISA, micropanning, and coprecipitation assays were used to evaluate the specificity and selectivity with which these phage bound to B. anthracis spores. Results: Peptides on the selected clones directed binding of the phage to B. anthracis spores. Most clones exhibited little or no binding to spores of distantly related Bacillus species, but some binding was observed with spores of closely related species. Our most specific spore-binding phage displayed a peptide EPRLSPHS (several thousand peptides per phage) and bound 3.5-to 70-fold better to spores of B. anthracis Sterne than to spores of other Bacillus species. Conclusions: The selected phage probes bound preferentially to B. anthracis Sterne spores compared with other Bacillus species. These phage could possibly be further developed into highly specific and robust

Landscape phage probes for Salmonella typhimurium

“…Landscape phages have been shown to serve as substitutes for antibodies against various soluble and cell-displayed antigens and receptors (Legendre and Fastrez, 2002;Mount et al, 2004;Petrenko and Smith, 2000;Petrenko et al, 2002;Romanov et al, 2001;Samoylova et al, 2003;Samoylova et.al., 2004) including bacterial cells and spores (Brigati et al, 2004;Petrenko and Sorokulova, 2004). The phage probes have been used in ELISA and thickness shear mode quartz sensors to detect bacterial and mammalian antigens (Petrenko and Smith, 2000;Petrenko and Vodyanoy, 2003).…”

Landscape phage as a molecular recognition interface for detection devices

“…It is interesting to note that representative landscape phage selected with B. anthracis spores in a non-biased selection scheme can bind the selector strain at a higher level than other species of Bacillus spores [18]. Similarly, the phage selected with S. typhimurium demonstrated higher affinity to the selector strain in comparison with nine other gram-negative bacteria, predominately Enterobacteriaceae [20].…”

“…Landscape phages have been shown to serve as substitutes for antibodies and detection probes in enzyme-linked immuno assay (ELISA) and biosensors [13][14][15][16][17][18][19][20][21][22][23], as immunogens [24], gene-delivery vehicles [25] and affinity matrices [30,32].…”

“…Phage probes against biological threat agents, such as Bacillus anthracis spores and Salmonella typhimurium were isolated in a nonbiased multistage selection procedure using immobilized spores or bacteria as a selector [18][19][20]. Binding of the selected phages to their respective targets was characterized by a precipitation test, fluorescence-activated cell sorting, enzyme-linked immunosorbent assay (ELISA), and fluorescent, optical and electron microscopy.…”

“…Environmental monitoring requires thermostable probes, such as preselected phages that are superior to antibodies and can operate in non-controlled conditions. For example, when thermostability of a landscape phage probe and a monoclonal antibody specific for β-galactosidase was examined in parallel in the ELISA format they were both stable for greater than 6 months at room temperature, but at higher temperatures the antibody degraded more rapidly than the phage probe [18]. At 37 °C, phage degraded only slightly (half-life of phage as a probe at this temperature was 950 days), while monoclonal antibodies lost virtually all of their activity for 30-week study (half-life 107 days).…”