Detection and quantification of anthrax lethal factor in serum by mass spectrometry.
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.
Boyer
Anne E
AE
Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Highway, NE, Atlanta, Georgia 30341, USA.
Quinn
Conrad P
CP
Woolfitt
Adrian R
AR
Pirkle
James L
JL
McWilliams
Lisa G
LG
Stamey
Karen L
KL
Bagarozzi
Dennis A
DA
Hart
John C
JC
Barr
John R
JR
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
D000906:Antibodies; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D010455:Peptides; C030325:anthrax toxin; D020929:Mitogen-Activated Protein Kinase Kinases