Detection and Quantification of Anthrax Lethal Factor in Serum by Mass Spectrometry

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• 作者：Anne E. Boyer ; Conrad P. Quinn ; Adrian R. Woolfitt ; James L. Pirkle ; Lisa G. McWilliams ; Karen L. Stamey ; Dennis A. Bagarozzi ; John C. Hart ; Jr. ; John R. Barr
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：November 15, 2007
• 年：2007
• 卷：79
• 期：22
• 页码：8463 - 8470
• 全文大小：227K
• 年卷期：v.79,no.22(November 15, 2007)
• ISSN：1520-6882

文摘

The lethal toxin produced during Bacillus anthracisinfection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF),a zinc-dependent endoproteinase whose known targetsinclude five members of the mitogen-activated proteinkinase kinase (MAPKK) family of response regulators. Wehave developed a method for detecting functional LF inserum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to captureand concentrate the LF from serum. The captured LF wasexposed to an optimized MAPKK-based peptide substrate,which it hydrolyzed into two smaller peptides. The LFcleavage products were then analyzed by matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. Theentire analytical method can be performed in less than4 h with detection of LF levels as low as 0.05 ng/mL. Themethod was used to quantify LF levels in serum fromrhesus macaques infected with B. anthracis. Serumsamples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detectLF earlier in the infection cycle. This method representsa highly specific and rapid diagnostic tool for early anthraxand has a potential additional role as a research tool forunderstanding toxemia and effects of medical countermeasures for anthrax.