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Substrate For Specific and Quantitative Detection of Anthrax Lethal Factor in Plasma

Shine, N., Suryadi, K.

Bacillus anthracis is regarded as a major biological warfare threat. The inhalation form of Bacillus anthracis infection can rapidly lead to a blood infection and kill quickly. Antibiotic treatment can clear the bacterium from the host, but this treatment must be initiated rapidly. By the time clinical symptoms are observed, 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 permitting immediate treatment. The presence of one of the virulence factors, anthrax lethal factor (LF), in the blood, early in an infection, offers the opportunity for detection prior to catastrophic decline of the patient. This report describes a fast, sensitive, specific and accurate detection method to determine active infection by Bacillus anthracis in plasma. LF is an endopeptidase, however, 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. Fluorescently labeled peptide substrates which are not cleaved by plasma proteases and thus are specific for LF are described here. 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 less than 5 pg LF/ml of neat plasma after 2 hours of incubation. Alternately the substrate 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 is1 ng LF/ml of plasma after 5 hours of digestion.