Citation

Bacillus anthracis is regarded as a major biological warfare threat. Inhalation of Bacillus anthracis spores can quickly lead to a lethal blood infection. Antibiotic treatment can clear the bacterium from the host; however, this treatment must be initiated rapidly. By the time clinical symptoms are observed, toxin may already be present in lethal amounts. There is a critical need for an accurate, sensitive and simple assay to determine that infection has occurred, permitting immediate treatment. The presence early in infection of one of the virulence factors, anthrax lethal factor (LF), offers the opportunity for detection prior to decline of the patient. This presentation will describe a fast, sensitive, specific and accurate detection method to determine active infection by Bacillus anthracis in plasma. LF is detected using a fluorescently labeled peptide substrate, MAPKKide® Plus (US Patent No. US 9,932,570 B2), which is not cleaved by plasma proteases and thus is specific for LF. Several detection strategies have been evaluated. In the simplest method, the substrate is added directly to diluted plasma, and cleavage is monitored by the increase in fluorescence as a function of time. The limit of detection by this method is 25 ng lethal factor/ml of plasma in 15 minutes, 5 ng/ml after 45 minutes, and less than 1 ng lethal factor/ml of plasma after 5 hours. In recent developments the simplest method has been optimized to increase the sensitivity by including a sample preparation step. The most sensitive method involves the concentration of lethal factor using antibody-coated plates and HPLC with fluorescence detection. This method allows for detection of less than 5 pg lethal factor/ml of neat plasma after 2 hours of incubation. In conclusion, data is presented demonstrating that 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. This detection method has the potential to be used as a point-of-care method to monitor both the initial level of severity of the intoxication as well as the success of therapeutic interventions