By: Nancy Shine, PhD, Director of R&D, List Labs
Bacillus anthracis is regarded as a major biological warfare threat. The inhalation form of Bacillus anthracis infection can kill quickly. While antibiotic treatment can clear the bacterium from the host, if diagnosis is delayed, the toxin, which is rapidly produced, may already be present in lethal amounts. There is a critical need for a rapid, accurate, sensitive and simple assay to determine whether infection has occurred thereby allowing immediate treatment.
Anthrax Detection Method
Anthrax lethal factor (LF), an endopeptidase, is present in blood in the early stages of the infection. The use of peptidic substrates in plasma is problematic due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. A fluorescently labeled peptide substrate, MAPKKide Plus, Prod #532, which is not cleaved by plasma proteases and thus is specific for LF has been designed. 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. Concentration of the LF using the antibody-coated plates allows for the detection of 5 pg LF/ml of neat plasma after 2 hours of incubation. Alternately, the MAPKKide Plus may be added directly to diluted plasma and cleavage monitored by an increase in fluorescence as a function of time using a fluorescent microplate reader. The limit of detection by this simpler method is 1 ng LF/ml of plasma after 5 hours of digestion. Both methods can be confirmed by analysis of the reaction as a function of time. These methods are described in the poster Sensitive Detection of Anthrax Lethal Factor in Plasma Using a Specific Biotinylated Fluorogenic Substrate.
What’s Next for Anthrax Detection Method
We are currently working with a biotinylated form of MAPKKide Plus to enhance the sensitivity of the simpler method using the fluorescent plate reader rather than HPLC.
Interested in learning more about this List Labs patented peptide substrate? Contact us!