Citation

PURPOSE OF STUDY: The goal of this study was to develop a fast, sensitive, specific and accurate detection method
to determine active infection by Bacillus anthracis in complex matrices. This presentation focuses on the evaluation of a
fluorescently labeled peptide substrate, MAPKKide® Plus*.
METHODS USED: Two methods for the detection of LF in plasma using this fluorogenic substrate are described: one
HPLC-based and one a microplate assay.
For the HPLC-based method, the LF is enriched by capture from plasma using an LF antibody-coated microtiter plate,
and the captured LF is then exposed to the fluorescent substrate. The amount of cleaved peptide substrate is
determined by HPLC with fluorescence detection.
Alternately the substrate may be added directly to diluted plasma in a microplate and cleavage monitored by an increase
in fluorescence as a function of time using a fluorescent microplate reader.
SUMMARY OF RESULTS: Using the HPLC-based method, concentration of the LF using the antibody-coated plate
allows for the detection of less than 5 pg LF/ml of neat bovine plasma after 2 hours of incubation. Aliquots of sheep
plasma and 2% milk were also spiked with LF to determine the level of LF detectable using MAPKKide® Plus*.
The limit of detection using the simpler microplate method is 1 ng LF/ml of bovine plasma after 5 hours of digestion.
CONCLUSIONS: Significantly, this newly designed substrate, MAPKKide® Plus*, is highly sensitive to LF and resistant
to cleavage by plasma proteases making it ideal for detection of early infections with Bacillus anthracis. MAPKKide
® Plus*
can also be used to detect low levels of LF in milk.
MAPKKide® Plus* allows for both specific cleavage by anthrax lethal factor (LF), one of the anthrax virulence factors
present in blood early in infection and, most importantly, resistance to cleavage by nonspecific proteases found in
complex matrices including plasma.
*Patent Pending