Abstract
Inhalation anthrax has a rapid progression and high fatality rate. Pathology and death from inhalation of Bacillus anthracis spores are attributed to the actions of secreted protein toxins. Protective antigen (PA) binds and imports the catalytic component lethal factor (LF), a zinc endoprotease, and edema factor (EF), an adenylyl cyclase, into susceptible cells. PA-LF is termed lethal toxin (LTx) and PA-EF, edema toxin. As the universal transporter for both toxins, PA is an important target for vaccination and immunotherapeutic intervention. However, its quantification has been limited to methods of relatively low analytic sensitivity. Quantification of LTx may be more clinically relevant than LF or PA alone because LTx is the toxic form that acts on cells. A method was developed for LTx-specific quantification in plasma using anti-PA IgG magnetic immunoprecipitation of PA and quantification of LF activity that co-purified with PA. The method was fast (<4 h total time to detection), sensitive at 0.033 ng/mL LTx in plasma for the fast analysis (0.0075 ng/mL LTx in plasma for an 18 h reaction), precise (6.3–9.9 % coefficient of variation), and accurate (0.1–12.7 %error; n ≥ 25). Diagnostic sensitivity was 100 % (n = 27 animal/clinical cases). Diagnostic specificity was 100 % (n = 141). LTx was detected post-antibiotic treatment in 6/6 treated rhesus macaques and 3/3 clinical cases of inhalation anthrax and as long as 8 days post-treatment. Over the course of infection in two rhesus macaques, LTx was first detected at 0.101 and 0.237 ng/mL at 36 h post-exposure and increased to 1147 and 12,107 ng/mL in late-stage anthrax. This demonstrated the importance of LTx as a diagnostic and therapeutic target. This method provides a sensitive, accurate tool for anthrax toxin detection and evaluation of PA-directed therapeutics. Graphical Method schematic for analysis of anthrax lethal toxin activity by ID-MALDI-TOF MS
Highlights
Bacillus anthracis produces two binary toxins associated with the pathogenesis of anthrax [1]
edema factor (EF) bound to Protective antigen (PA) is described as edema toxin and lethal factor (LF) bound to PA as lethal toxin (LTx)
Since the measurement of LTx relies on detection of LF activity in LTx complex, the aim was to create standards with a minimum of free LF
Summary
Bacillus anthracis produces two binary toxins associated with the pathogenesis of anthrax [1]. Protective antigen (PA) is an 83-kDa protein responsible for cell binding and target cell translocation of catalytic toxin components lethal factor (LF) and edema factor (EF). Methods described for detecting PA include ELISA, europium nanoparticle-based immunoassay, time-resolved fluorescence immunoassay [2,3,4,5], electro-chemiluminescence, metal-enhanced fluorescence, AlphaLISA, and surface plasmon resonance [6,7,8,9] These methods can be lacking in precision, sensitivity, and quantitative accuracy, and in some, their utility has not been verified by matrix testing or application to infection samples. PA63 forms heptameric and octameric complexes [12, 13], which are capable of binding up to four molecules of the catalytic toxin components, edema factor (EF), and lethal factor (LF) [13, 14]. EF, an adenylate cyclase and LF, a zincdependent endoproteinase cause irreversible changes in their known substrates, adenosine tri-phosphate and mitogenactivated protein kinase (MAPKK), respectively [16, 17]
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