title: Detection and quantification of anthrax lethal factor in serum by mass spectrometry.
abstract: The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.
authors.affiliation: Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Highway, NE, Atlanta, Georgia 30341, USA.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Boyer
authors.forename: Conrad P
authors.initials: CP
authors.lastname: Quinn
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Woolfitt
authors.forename: James L
authors.initials: JL
authors.lastname: Pirkle
authors.forename: Lisa G
authors.initials: LG
authors.lastname: McWilliams
authors.forename: Karen L
authors.initials: KL
authors.lastname: Stamey
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Bagarozzi
authors.forename: John C
authors.initials: JC
authors.lastname: Hart
authors.forename: John R
authors.initials: JR
authors.lastname: Barr
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000942:Antigens, Bacterial / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D001427:Bacterial Toxins / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D008253:Macaca mulatta / Q000097:blood; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry / Q000145:classification; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D010455:Peptides; C030325:anthrax toxin; D020929:Mitogen-Activated Protein Kinase Kinases