Diagnosis of Early Anthrax Infection and the Quantification of Anthrax Toxins using Mass Spectrometry
Wed 3:30 PM - Track 3: Emergency Response
John R. Barr
John R. Barr, Anne E. Boyer, Conrad P. Quinn, Maribel Gallegos, Renato Lins, Susan Kuklenyik, James L. Pirkle.

Centers for Disease Control and Prevention
Anthrax is a zoonotic disease caused by Bacillus anthracis. The toxins of B. anthracis are composed of three proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF). PA combines with EF and LF to form, respectively, the binary toxins edema toxin (ETx) and lethal toxin (LTx). LTx inactivates members of the mitogen-activated protein kinase kinase (MAPKK) family which are important to the eukaryotic cell signal transduction pathways. PA targets and internalizes the toxin via host cell receptors and LF is a zinc-dependent endoproteinase that cleaves at least 5 members the (MAPKK) family of response regulators. LTx is lethal in a range of animal species.

We developed a sensitive mass spectrometry-based assay for quantifying total LF (LF + LTx) which can be measured early in infection. The mass spectrometry based assay uses Anti-LF and anti-PA monoclonal antibodies immobilized on magnetic beads. These beads were each used to capture and concentrate total LF or PA-LF (the LTx complex) from plasma samples and captured LF is exposed to a MAPKK-based peptide substrate for a cleavage reaction. The reaction mixture is then analyzed by mass spectrometry for the detection and quantification of LF cleaved product peptides as compared to standard curve reactions from recombinant LF and LF-PA. The limits of detection are 0.005 ng/mL.

We have used the mass spectrometry based anthrax lethal factor method to better understand toxemia in rhesus macaques and on human clinical samples. The mass spectrometry based anthrax lethal factor method is rapid (96 samples in 4 hours with one analyst). This method can diagnose inhalation anthrax in rhesus macaques as early as 12 hours after exposure to B. anthracis spores. This method can diagnose anthrax at an earlier stage of infection than any other method described.

The data indicates that anthrax has triphasic kinetics of toxemia and that toxin levels, both with and without antibiotic treatment appear to correlate with clinical outcome in rhesus macaques.