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 plasma. This presentation focuses on the evaluation
of a fluorescently labeled peptide substrate named MAPKKide® Plus* for both specific cleavage by anthrax lethal
factor (LF), one of the anthrax virulence factors present in blood early in infection, and resistance to cleavage by
nonspecific proteases found in plasma.
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 plasma after 2 hours of incubation.
The limit of detection using the simpler microplate method is 1 ng LF/ml of plasma after 5 hours of digestion.
CONCLUSIONS: Significantly, this newly designed substrate is highly sensitive to LF and resistant to cleavage
by plasma proteases making it ideal for detection of early infections with Bacillus anthracis.
*Patent Pending