Citation

PURPOSE OF STUDY: A fast, sensitive, specific and accurate detection method to determine active infection
by Bacillus anthracis in complex matrices has been developed using a fluorescently labeled peptide
substrate, MAPKKide® Plus*. Two methods for the detection of lethal factor (LF) in plasma using this
fluorogenic substrate were demonstrated: one HPLC-based and the other a microplate assay. The goal
of this study was to improve the sensitivity of the microplate assay.
METHODS: The MAPKKide® Plus* substrate was biotinylated on the N-terminal. Several strategies were
evaluated to determine the sensitivity that could be achieved.
1. The biotinylated MAPKKide® Plus* substrate was attached to streptavidin-coated beads prior to
exposure to a solution of LF in plasma or to LF enriched by antibody capture from plasma.
2. The biotinylated MAPKKide® Plus* substrate was added directly to a solution of LF in plasma,
or to LF enriched by capture from plasma. Streptavidin-coated beads were then used to remove
uncleaved substrate from the solution to minimize background fluorescence.
RESULTS: The results obtained from the strategies outlined above are compared to those obtained using
both the HPLC-based method which allows for the detection of less than 5 pg LF/ml of neat bovine
plasma after 2 hours of incubation; and the simpler microplate assay where the limit of detection is 1 ng
LF/ml of bovine plasma after 5 hours of digestion.
CONCLUSIONS: MAPKKide® Plus*, is highly sensitive to cleavage by LF and resistant to cleavage by plasma
proteases making it ideal for detection of early infections with Bacillus anthracis.
*Patent Pending